ipj=R= ifsb =L=ipj=R= ifsb = aìçpÅ~å = i~ëÉê=pÅ~ååáåÖ=jáÅêçëÅçéÉ= oÉäÉ~ëÉ=QKM= j~êÅÜ=OMMS= INTRODUCTION Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Knowledge of this manual is required for the operation of the instrument. Would you therefore please make yourself familiar with the contents of this manual and pay special attention to hints concerning the safe operation of the instrument. The specifications are subject to change; the manual is not covered by an update service. © Unless expressly authorized, forwarding and duplication of this document, and the utilization and communication of its contents are not permitted. Violations will entail an obligation to pay compensation. All rights reserved in the event of granting of patents or registration of a utility model. Issued by Carl Zeiss MicroImaging GmbH 07740 Jena, Germany Phone: +49 (0) 3641 64-3400 Fax: +49 (0) 3641 64-3144 E-mail: [email protected] www.zeiss.de/lsm II B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan INTRODUCTION Carl Zeiss How to make best use of the LSM 5 LIVE operating instructions A few symbols in these operating instructions will help you to recognize the nature and purpose of information immediately: The WARNING symbol warns against hazards for the user that might arise when operating the laser. This WARNING symbol warns against hazards from dangerously high voltages. The CAUTION symbol warns against faults and hazards that might arise during operation and which might cause damage to the unit. The NOTE symbol will help you to optimally solve your work problem. It represents a practical tip which will help you to find out which settings and methods are capable of improving or accelerating a procedure. The HOT SURFACE symbol warns against hazards for the user that might arise when touching the lamp housing during operation. The MAINS PLUG symbol remembers service personal to pull the mains plug before opening the device housing. Depending on the problem, these operating instructions will supply you with various possibilities: • If you want to know where to find certain general areas of information, refer to the following outline of sections to get a general overview. • You will find a detailed table of contents at the start of every chapter. There you will see at a glance what topics are dealt with in detail. Always remember: 03/06 The time you invest in getting acquainted with the product will pay for itself many times over in your application task. B 45-0019 e III INTRODUCTION Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Contents 1 Notes on Device Safety This section contains general notes on device safety, safe operation, and possible hazards caused by failure to observe the instructions. 2 LSM 5 LIVE - Setup Requirements The Setup Requirements section outlines the installation and supply requirements of the LSM 5 LIVE Microscope Systems, together with the relevant specifications. 3 Introduction to Laser Scanning Microscopy Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging. The section also outlines the ways to present LSM image series in three dimensions, and introduces you to the performance features of your LSM 5 LIVE. 4 Operation In the Operation section you will find the most important steps and procedures of the LSM menu structure. The step-by-step description how to get an image will be shown by typical application examples including the WINDOWS XP graphic user environment. 5 Tools This section contains a description of the use of the tools for setting the microscope. 6 3D for LSM This section contains a description of the LSM 3D software package (basic program and addons). At the same time, all functions and settings are presented in a systematic form and in the order in which they can be reached from the basic menu via sub-menus and dialog boxes. 7 Annex The annex contains the Application-specific Configurations, special notes and information for using the LSM microscope and the brochure The confocal Laser Scanning Microscopy. 8 Certification 9 Brief Operating Manual This section contains the brief instructions of the LSM 5 LIVE Microscope Systems. 10 IV Laser safety warning labels B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 1 NOTES ON DEVICE SAFETY Contents Carl Zeiss NOTES ON DEVICE SAFETY CONTENTS Page 1 NOTES ON DEVICE SAFETY .............................................................................................1-2 1.1 General..............................................................................................................................1-2 1.2 Warning and Information Labels ........................................................................................1-3 1.3 Regulations ......................................................................................................................1-10 1.4 Protection against diffuse laser excitation emitted around the objective and sample .........1-12 1.5 Protection against white light emitted around the objective and sample by an X-cite fluorescence illumination..................................................................................................1-13 1.6 Notes on Setting up the Microscope System .....................................................................1-13 1.7 Power Requirements ........................................................................................................1-15 1.8 Notes on Handling the Laser Components........................................................................1-16 1.9 Notes on Handling HBO and X-Cite Light Sources ............................................................1-18 1.10 Physical Dimensions .........................................................................................................1-18 1.11 Environmental Requirements............................................................................................1-18 1.12 Notes on Handling the Computer and Data Media ...........................................................1-19 1.13 Notes on Care, Maintenance and Service .........................................................................1-20 1.14 1.14.1 1.14.2 User Interface...................................................................................................................1-21 Mounting and Dismounting Lamps, TPMT and Switching Mirror ......................................1-21 Mounting and Dismounting the Scan Heads.....................................................................1-23 03/06 B 45-0019 e 1-1 NOTES ON DEVICE SAFETY General Carl Zeiss 1 NOTES ON DEVICE SAFETY 1.1 General LSM 5 LIVE LSM 5 LIVE DuoScan The LSM 5 LIVE laser scanning microscope, including its original accessories and compatible accessories from other manufacturers, may only be used for the purpose of microscopic techniques. Laser Scanning Microscopes (LSM) are intended for high resolution imaging of biological or material samples, whereby in contrast to wide field microscopy the specimen is illuminated raster-fashion with a focused laser beam and the optical arrangement prevents light from out-of-focus regions of the specimen contributing to image formation. Installation and commissioning of the LSM 5 LIVE system must be performed by authorized Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss representative. The manufacturer will not assume liability for any malfunction or damage caused by any thing other than the intended use of the LSM 5 LIVE or individual modules or parts of it, nor by any repair or other service operation performed or attempted by persons other than duly authorized service staff. Any such action will invalidate any claim under warranty, including parts not directly affected by such action. This also includes the modification of the system computer with new cards, etc. by the user. The use of a camera at the base port of Axiovert 200 M Combi stands is not allowed for reasons of laser safety. Any manipulation will result in the loss of warranty of laser safety. Please read also the notes on device safety and manuals of the microscope, the HBO, the HAL and additional optional devices if ordered as the UV Laser, the piezo focusing device and heating inserts. As the system is largely operated via menus on a computer, you should be acquainted with the principles of the operating system and its WINDOWS, WINDOWS 2000 or Windows XP graphical user interface. The respective manuals are supplied together with the programs. The LSM 5 LIVE is a device that belongs to laser hazard class 3B. The system is equipped with safety interlocks that comply with laser hazard class 3B and 4. WHO recommendations concerning health and industrial protection when handling laser devices must be observed. The operator of the unit must also observe all and any relevant statutory accident prevention regulations. The user is referred to the safety data sheet provided together with the manual. 1-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 1.2 NOTES ON DEVICE SAFETY Warning and Information Labels Carl Zeiss Warning and Information Labels The warning and information labels attached on the LSM 5 LIVE must be observed. Check whether all of the labels shown below are provided on your instrument, and contact Carl Zeiss Germany or one of the service agencies if you should discover that any of the labels should be missing. You will receive a free replacement. Description of labels Caution: Faults and hazards that might arise during operation which might cause damage to the unit or injury to the user. Attention: Laser irradiation hazards possible when operating the system. Attention: High voltage. Pull the mains plug before opening the device housing. Caution: Hot surface. Caution: UV radiation. Caution: Fingers can be caught. The arrow points to the opening where laser light comes out during operation of the system. Other labels on the system include one of the above depicted symbols and a detailed description of the handling instructions. See also the following drawings of the system parts. 03/06 B 45-0019 e 1-3 Carl Zeiss NOTES ON DEVICE SAFETY Warning and Information Labels LSM 5 LIVE LSM 5 LIVE DuoScan 1:1 E TIV JEC RO OB ZE OR CT FLE RE S CU FO Fig. 1-1 1-4 Warning and information labels on the Axiovert 200 M microscope with the LSM 5 LIVE scanning module B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 1-2 03/06 NOTES ON DEVICE SAFETY Warning and Information Labels Carl Zeiss Warning and information labels on the Axio Imager.Z1 microscope with scanning module B 45-0019 e 1-5 Carl Zeiss NOTES ON DEVICE SAFETY Warning and Information Labels LSM 5 LIVE LSM 5 LIVE DuoScan 04 01 07 02 06 03 05 08 0 10 90 Fig. 1-3 1-6 Warning and information labels on the Axioskop 2 FS MOT microscope with scanning module B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 1-4 03/06 NOTES ON DEVICE SAFETY Warning and Information Labels Carl Zeiss Warning and information labels on LSM DuoScan (systems LSM 5 LIVE DuoScan only) B 45-0019 e 1-7 Carl Zeiss Fig. 1-4 1-8 NOTES ON DEVICE SAFETY Warning and Information Labels LSM 5 LIVE LSM 5 LIVE DuoScan Warning and information labels on laser components B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan NOTES ON DEVICE SAFETY Warning and Information Labels CO HE RE Carl Zeiss nT En TE RP RIS E AK PE CH AR SE E4 En TE RP RIS E E5 CO HE RE nT Fig. 1-5 03/06 um xim ma ter wa ure ss pre p.s.I. 60 T OU ter wa 7 J3 E4 J6 Warning and information labels on UV laser components (LSM DuoScan only) B 45-0019 e 1-9 NOTES ON DEVICE SAFETY Regulations Carl Zeiss 1.3 LSM 5 LIVE LSM 5 LIVE DuoScan Regulations Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM 5 LIVE. Read these operating instructions and all device publications belonging to the system conscientiously before operating the LSM 5 LIVE! You can obtain additional information on the hardware configuration delivered and on optional system extensions from the manufacturer or via the service hotline. ⇒ The LSM 5 LIVE has been designed, built and tested in conformity with the following regulations and guidelines: DIN EN 61010-1 (IEC 601010-1) "Safety requirements for electrical equipment for measurement, control and laboratory use" DIN EN 60825-1 (IEC publication 60825-1) "Safety of laser equipment", taking relevant CSA and UL specifications into account DIN EN 61326: “Electrical equipment for control technology and laboratory use – EMC-requirements” Low voltage directive: 73/23/EWG EMC directive: 89/336/EWG ⇒ The company works according to a certified Environment Management System according to ISO 14001. The Product was developed, tested and produced in accordance with the valid regulations and guidelines for environmental law of the European Union. The product and its accessories have been classified as instrument category 9 (laboratory equipment or comparable standard). The product and its accessories agree with the EU-regulations 2002/95/EG (RoHS) and 2002/96/EG (WEEE), if applicable for the product. Carl Zeiss has installed a process for taking back and recycling the instruments within the member states of the European Union, which takes care of the appropriate utilization according to the said EU guidelines. For details on the disposal and recycling please refer to your relevant Carl Zeiss sales or service organization. The product must not be disposed in the household waste or through the municipal disposal organizations. In case of resale the seller is obliged to inform the buyer, that the product has to be disposed according to the said regulations. 1-10 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 1.4 NOTES ON DEVICE SAFETY Protection against diffuse laser excitation … Carl Zeiss Protection against diffuse laser excitation emitted around the objective and sample Though the radiation levels around the objective and sample is very low (comparable to a common laser pointer used for e.g. talks), avoid to approach this area closer than 10 cm with your eye. Also limit the time of skin exposure of your hands to the time needed to place the sample and to mechanically set up the acquisition. Switch off the scanning while doing such manipulation around the objective and sample! In case of scanning, don’t stare into the beam! To further reduce the radiation level of fluorescent emission and excitation light around the microscope, Carl Zeiss delivers antiglare screens for all microscopes. Axiovert 200M: 000000-1109-741 Axio Imager.Z1/M1: 452163-0000-000 Axioskop 2 FS mot: 452163-0000-000 Fig. 1-6 Antiglare screens for Axiovert 200M (left) and Axio Imager and Axioskop 2 FS (right) Use these screens for improved working ergonomy! 03/06 B 45-0019 e 1-11 Carl Zeiss 1.5 NOTES ON DEVICE SAFETY Protection against white light … LSM 5 LIVE LSM 5 LIVE DuoScan Protection against white light emitted around the objective and sample by an X-cite fluorescence illumination Follow the guidelines in the X-Cite manual provided by EXFO Inc.. Don’t use a neutral beamsplitter (e.g. “NT80/20” position in LSM DuoScan or LSM 5 DUO systems) in the P&C modules while looking through the eyepieces. Strong white light might project into your eyes. In case such strong light is projected into your eye, the natural lid closure effect will protect you from any damage. Nonetheless such exposure is unpleasant and should absolutely be avoided. 1.6 Notes on Setting up the Microscope System Installation and commissioning of the LSM 5 LIVE system must be performed by authorized Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss representative. The LSM 5 LIVE laser scanning microscope is delivered in several crates: The LSM 5 LIVE must be set up so as to ensure that the minimum clearance between the wall and the rear of the system is no less than 0.5 m. This clearance is needed for adjustment and maintenance operations. Do not set up the unit in the proximity of heat sources such as radiators or direct sunlight. To avoid heat build-ups, the ventilation slots on the microscope system must not be covered up. The system must not be set up in areas with potential danger by explosives. The unit must be connected to a properly installed socket outlet with earthing contact by means of the mains cables supplied. Continuity of PE connection must not be affected by the use of extension leads. The system contains components with dangerous voltage. The system must not be opened by anybody else than authorized Carl Zeiss Service staff. Before opening the main plug has to be disconnected. Before connecting the mains cables, please check whether your mains voltage corresponds to the voltage specified on the rating plate of the laser module. For reasons of laser safety, all ports must either be equipped with the corresponding device (scan head, camera, HBO lamp etc.) or covered with the counterpart of the laser safety kit provided. 1-12 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan NOTES ON DEVICE SAFETY Protection against white light … Carl Zeiss Maintenance, repair, modification, addition, removal or exchange of components, or other interference with the equipment beyond the operations described in this manual may only be carried out by the manufacturer Carl Zeiss or by persons expressly authorized by Carl Zeiss to do so. This applies especially to the microscope system, the laser scanning module, lasers, the PC system, the power supply units, cable connections, safety interlock devices and other system components. Please note that the LSM 5 LIVE are high-precision opto-electronic instruments. Inexpert handling may easily impair their function or even damage them. The openings for ventilation must not be covered. There are hot surfaces on the HBO and HAL lamp. When sliding the compact Laser module in and out of the System electronic rack take care not to catch your fingers. The HBO 50 and 100 W, XBO 75 W and X-cite 120 lamps used on the light microscopes incident light path emit UV light which is harmful to the human eye and skin when observed without appropriate filters. Never remove the lamp and look direct into the emitted light. After installation or conversion of the LSM system, authorized specialized staff must carefully check that it is in a proper condition and, particularly, that covers protecting against laser radiation are provided. Tube openings or other unused mounts should always be protected against dust and moisture with the corresponding device components or with termination covers/blind plugs. By establishing a corresponding workplace environment, please ensure that the formation of electrostatic charges of electronic components is avoided. To avoid vibrations during operation, the LSM 5 LIVE should only be operated in conjunction with the system table (vibration damping). 03/06 B 45-0019 e 1-13 NOTES ON DEVICE SAFETY Power Requirements Carl Zeiss 1.7 LSM 5 LIVE LSM 5 LIVE DuoScan Power Requirements The LSM 5 LIVE comes with a mains power supply cord and plug, either CEE red (3/N/PE 400/230 V/16 A), or NEMA L 14-30P (2/N/Ground 120/240 V/30 A), and with the matching mains socket outlet. A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system. The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter. A suitable grounding point must be installed in the room. For systems (220 ... 240 V AC) equipped with X-Cite 120 the mains socket outlet must be equipped with a fuse having minimum tripping characteristic C according to IEC/EN 60898. Line voltage 220 … 240 V AC (±10 %) 100 … 125 V AC (±10 %) Line frequency 50...60 Hz 50...60 Hz − Max. current 3 phases at 16 A 2 phases at 25 A − Power Phase 1 = 1.9 kVA max. Phase 1 = 3.2 kVA max. Phase 2 = 1.5 kVA max. Phase 2 = 2.8 kVA max. LSM incl. VIS laser Phase 3 = 2.6 kVA max. − Power Consumption 5000 VA max. 5000 VA max. 208...240 V AC 208...240 V AC (±10 %) 50 / 60 Hz (±10 %) 50 / 60 Hz 1 phase at 63 A 1 phase at: Argon UV laser − − Line Voltage Max. current 208 V: 34 Amps Note: For Line Voltage 220 V the connector and power plug are rated for 63 Amps, However wiring and fuse should be rated for 32 Amps. 240 V: 29 Amps 7000 VA max. 7000 VA max. Class of protection I I Type of protection IP 20 IP 20 Overvoltage category II II Pollution degree 2 2 − Power consumption 230 V: 31 Amps 1-14 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 1.8 NOTES ON DEVICE SAFETY Notes on Handling the Laser Components Carl Zeiss Notes on Handling the Laser Components The LSM 5 LIVE are laser hazard class 3B instruments. This moderate and high-risk class embrace medium-power and high power lasers. You must take care not to expose yourself to the radiation of such lasers. In particular, never look into the laser beam! Only personnel which has been instructed on laser safety is allowed to operate the system. The following laser types are currently intended for use in the LSM 5 LIVE. The use of any other lasers as the ones listed below is not authorized. Laser Class Power 1 Diode laser 405 nm 3B 50 mW 2 Diode laser 440 nm 3B 16 mW 3 Diode laser 488 nm 3B 100 mW 4 DPSS laser 532 nm 3B 75 mW 5 Diode laser 635 nm 3B 35 mW 6 Ar laser 351/364 nm (UV, for DuoScan only) 4* 80 mW * Laser type class 4, if mounted on laser module with fiber output class 3B. Please note that for the maintenance of the UV Laser it is recommended to run the laser at maximum power once a day if the laser is not used frequently or only at low power levels. This enables the Autofill process which keeps up the correct tube gas pressure. This operation prolongs the life time of the tube and prevents complete tube failure if the laser is not used for a prolonged period of time. For details please refer to the Operator’s Manual of the UV laser. Please contact Carl Zeiss if you intend to use a different laser other than the ones above. If used properly, the LSM 5 LIVE will not pose any laser radiation risks for operating staff. Nevertheless, you should observe the following warnings: 03/06 B 45-0019 e 1-15 Carl Zeiss NOTES ON DEVICE SAFETY Notes on Handling the Laser Components LSM 5 LIVE LSM 5 LIVE DuoScan • If necessary - insofar as specified by law - inform the laser protection officer before commissioning the laser. • The laser modules are equipped with a key-interlock. • Always store keys for laser key switches and, if applicable, keys for further laser power supply units, where they are inaccessible to persons not authorized to operate the laser. • A red LED on the front of the scan head lights up when one or all of the lasers are switched on. • Do not place any reflecting objects into the beam path. • Never open any covers or panels. • Never look into the laser beam, not even to simply view the specimen, whether with the aid of optical instruments or without. Otherwise you risk going blind! • Do not leave any empty objective positions of the nosepiece uncovered. Suitable protective measures must be taken if gases, dust or vapors hazardous to health, secondary radiation or explosive objects should arise on the specimen as a result of laser radiation. 1-16 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 1.9 NOTES ON DEVICE SAFETY Notes on Handling HBO and X-Cite Light Sources Carl Zeiss Notes on Handling HBO and X-Cite Light Sources For LIVE systems equipped with a rearport mounted scan head be aware that the mirror cube in the reflector turret leads to a strong back reflection of HBO light into the specimen plane and the ocular lens. When observing the specimen through the ocular lens the use of the mirror cube position should be avoided. The light flash is not harmful but unpleasant. The reflex of closing the eyelid is sufficiently protective. To avoid this situation an additional filter LP 397 (from filter cube 01, # 48 80 01) can be mounted into the mirror cube which prevents the back reflection of the HBO UV light in the ocular plane. 1.10 Physical Dimensions Length (cm) Passively damped anti-vibration table Width (cm) Height (cm) Weight (kg) 130 100 75 137 Scanning Module LSM 5 LIVE 55 17 30 19,5 Scanning Module LSM DuoScan 39 15 13 7 Microscope 50 35 50 20 Laser Module LIVE 66 52 22 58 Laser Module, UV (for DuoScan only) 140 20 20 60 System Electronic Rack 110 70 58 90 Plug-in unit external laser (UV) 66 52 22 9 Power supply for Ar (UV) 50 50 30 30 Cooling unit for Ar (UV) 80 45 50 30 1.11 Environmental Requirements 1. Operation, specified performance T = 22 °C ± 3 °C without interruption (24 h a day independently whether system is operated or switched-off) 2. Operation, reduced performance T = 10 °C to 35 °C, any conditions different from 1. and 5. 3. Storage, less than 16 h T = -40 °C to 55 °C 4. Storage, less than 6 h T = -55 °C to 70 °C 5. Temperature gradient ± 0.5 °C/h 6. Warm up time 1 h, for high-precision and/or long-term measurements ≥ 3 h 7. Relative humidity < 65 % at 30 °C 8. Operation altitude max. 2000 m 9. Loss of heat 4 kW 03/06 B 45-0019 e 1-17 Carl Zeiss 1.12 NOTES ON DEVICE SAFETY Notes on Handling the Computer and Data Media LSM 5 LIVE LSM 5 LIVE DuoScan Notes on Handling the Computer and Data Media The computer used as standard in your LSM system is an IBM-compatible high-end Pentium computer with WINDOWS XP operating system. Do make sure, though, that you receive your LSM system with the operating system installed, with initialization and start files set up and with the LSM program also installed. When working with the hard disk, it is important to know that the more data it contains, the slower its operation will become. Therefore, data that you do not need permanently should be stored on other external devices. When handling diskettes, avoid data losses by protecting them against extreme temperatures, moisture and magnetic fields. The data on a diskette is stored in the form of magnetic signals. To some extent, monitors, telephones or even lamps generate magnetic fields that might destroy this data. Also, never open the metal cover on diskette cases. A diskette´s surface can also be destroyed by touching it. When handling CDs, CD ROMs or DVDs, do not touch the data side of the disc (the side of the disc with no label or printing). Do not apply paper labels or write on any part of the disc, data side or label side. If dust or fingerprints get on the disc, wipe it with a soft cloth from the center to the edge, but do not use benzine, paint thinner, record cleaner, or static repellent. This can damage the disc. Do not place the disc in any place where it is exposed to direct sunlight or high temperatures. Backup your data on a regular basis. Do not install any other software without talking to your Carl Zeiss representative. Never turn your computer off before you have terminated the LSM program and run down the WINDOWS XP operating system. Otherwise, the program and/or data files may get lost. 1-18 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 1.13 NOTES ON DEVICE SAFETY Notes on Care, Maintenance and Service Carl Zeiss Notes on Care, Maintenance and Service The manufacturer of the unit cannot be held liable for damage resulting from operating errors, negligence or unauthorized tampering with the device system, particularly as the result of removal or replacement of parts of the unit or as the result of the use of unsuitable accessories from other manufacturers. Any such action will also render all warranty claims null and void and laser safety might no longer be warranted. You are well advised to arrange a service agreement with your nearest Carl Zeiss representative to guarantee perfect functioning of the microscope system in the long term. Modifications and conversion work on the components of the system must only be carried out by the manufacturer, by the service agency or by persons authorized and trained for this purpose by the manufacturer. Damaged units or parts may only be repaired or maintained by the responsible service agency. During maintenance or repair carried out by the service personnel the customer is requested to stand aside and wear a pair of laser safety goggles if needed. Before opening the housing of the halogen lamp switch off all laser units. Care operations that may be carried out by operating staff are limited to cleaning painted and glass surfaces. • Before cleaning the instrument make sure the main power supply is disconnected. • Cleaning painted surfaces To do this, use a clean cloth that has been moistened in a mixture of water and some detergent; do not use any solvent, however. Dry with a lint-free cloth. • Cleaning glass surfaces Glass surfaces that have become soiled or which are marked with fingerprints may be rubbed with a clean optical cleaning cloth. If soiling is persistent, dip the optical cleaning cloth into a mixture of distilled water and a small quantity of detergent. To complete cleaning, lightly breathe on the glass surface and rub it dry with a clean cloth. Lint or dust is best removed with a clean brush. • Make sure that no cleaning liquid penetrates into the system. • Dust filters in the ventilation entries of the system electronic rack have to be replaced every 6 month. For replacement please contact your local service representative. 03/06 B 45-0019 e 1-19 NOTES ON DEVICE SAFETY User Interface Carl Zeiss 1.14 LSM 5 LIVE LSM 5 LIVE DuoScan User Interface All user interface ports are equipped with a safety interlock system which warrants laser safety. These interlock devices must not be manipulated. Other interfaces which are not described here are service interfaces and are only to be operated by authorized Carl Zeiss service personnel. The following devices can be mounted and dismounted by the user: 1.14.1 − Halogen and HBO lamp − Scan head Mounting and Dismounting Lamps, TPMT and Switching Mirror The ports of the lamps, the switching mirror and the transmission PMT are equipped with hardware interlock devices. At most ports (all ports of the Axioskop 2 FS MOT and the Axio Imager.Z1 as well as the HBO port of the Axiovert 200 M) the following interlock devices are present and have to be operated in the following way: Interlock with sensor ring and contact ring: Fig. 1-7 Sensor ring mounted to the interface ports on the microscope side Fig. 1-8 Contact ring mounted to the lamp, TPMT or switching mirror The interlock is working when the sensors of the sensor ring (Fig. 1-7/1) are depressed by the pins on the contact ring (Fig. 1-8/1). Whenever this is not the case, for example if the distance between the two devices is too large, the laser will be blocked and the system cannot be used. In case the system is not operating following the removal or attachment of any device on a port with safety interlock check again the connection of the Contact ring to the Sensor ring. 1-20 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan NOTES ON DEVICE SAFETY User Interface Carl Zeiss For dismounting the lamps, TPMT or switching mirror slightly unscrew the contact ring first (Fig. 1-8/2 or Fig. 1-9/2) so it can be moved away from the sensor ring (Fig. 1-9/1). Then unscrew the lamp, TPMT or switching mirror (main screw Fig. 1-9/3) which is in one of the recesses of the sensor ring (Fig. 1-7/2). Hold the device to be dismounted with one hand while unscrewing to keep it from dropping. The now empty port has to be closed with the blind cap to restore the functionality of the system. Use the main screw of the port to fix the cap. Make sure the pins of the cap depress the sensors of the sensor ring. Do not remove the sensor ring from the microscope. This might result in failure of laser safety and a non operating system. Fig. 1-9 HBO lamp mounted to Axiovert 200 M with sensor ring and contact ring for laser safety Fig. 1-10 Blind cap for closing any port equipped with an interlock device For mounting any lamp, TPMT or the switching mirror back onto the microscope reverse the steps for dismounting the device. Be careful not to bend the pins on the contact ring when screwing the device onto the microscope port. For the Axiovert 200 M transmission port and the two ports available on the motorized switching mirror no sensor ring is present. Instead the sensors are directly installed at the Axiovert 200 M transmission port or the two ports of the motorized switching mirror. 03/06 B 45-0019 e 1-21 Carl Zeiss NOTES ON DEVICE SAFETY User Interface 1.14.2 LSM 5 LIVE LSM 5 LIVE DuoScan Mounting and Dismounting the Scan Heads The scan heads are connected to the microscope via an integrated safety interlock. They can be moved between two microscopes. Make sure the system is shut off completely before starting the following procedure: Be aware that the LSM 5 LIVE scan head weights up to 19,5 kg. Moving the scan heads between Axiovert 200 M, Axioskop 2FS MOT and Axio Imager.Z1: Fig. 1-11 Fig. 1-12 Electronic connection port of the LSM 5 LIVE scan head • Loosen the three screws on LSM 5 LIVE or LSM DuoScan scan head (Fig. 1-12/1 or Fig. 1-13/1). Port connection between LSM 5 LIVE and Axiovert 200 M Fig. 1-13 Fastening screws of the scan head at the front of the tube on Axioskop 2 FS MOT and Axio Imager.Z1 • Slowly pull the scan head away from the microscope port or the tube. For mounting the scan head onto a microscope, make sure the pins and the electronic connections of the safety interface match closely. Fasten the three screws on the LSM 5 LIVE or LSM DuoScan scan head (Fig. 1-12/1 or Fig. 1-13/1). 1-22 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan NOTES ON DEVICE SAFETY User Interface Carl Zeiss • Use the three fastening screws (Fig. 1-14/1 and 2) to attach or detach the LSM DuoScan to or from the microscope stand. Fig. 1-14 Fastening screws and connections on LSM DuoScan To ensure functioning of the system and laser safety the following connections have to be changed: 1. The connection of the microscope to the safety interface of the system is located either on the additional Safety-Box (Axioskop 2 FS MOT, Fig. 1-15/1) or on the rear side of the microscope (Axiovert 200 M or Axio Imager.Z1; Fig. 1-16/1 and Fig. 1-17/1). This connection has to be unplugged from the microscope which is not in use and plugged into the microscope to be used following the exchange of the scan head. Fig. 1-15 03/06 Safety-Box of Axioskop 2 FS MOT with main connection to safety interface (1) B 45-0019 e Fig. 1-16 Connection of Axio Imager.Z1 to safety interface (1) and electronics (2) 1-23 Carl Zeiss NOTES ON DEVICE SAFETY User Interface LSM 5 LIVE LSM 5 LIVE DuoScan 1 Fig. 1-17 Fig. 1-19 1-24 Connection of Axiovert 200 M to safety interface (1) Fig. 1-18 Four CAN connections (1) are available on the rear side of the electronics module. The microscope in use has to be connected to one of them 2. The plug for the main connection of the microscope to the electronics is situated on the rear side of the electronics rack. It is either of the four CAN connections shown in Fig. 1-18. Only ONE microscope can be connected to the electronics at a time. The connection has to be plugged in and unplugged at the electronics rack. The corresponding connections on the microscopes are shown in Fig. 1-16/2, Fig. 1-19/1 and Fig. 1-20/1. Remove the connection of the microscope no longer in use at the electronics rack and plug in the connection of the microscope which should be used instead. B 45-0019 e 03/06 CAN connections on the Axiovert 200 M (1). One of the plugs is occupied with the connection to the electronics LSM 5 LIVE LSM 5 LIVE DuoScan 3. NOTES ON DEVICE SAFETY User Interface After the connections of the non used microscope are disconnected and the ones of the used microscope are connected, the system can be switched on again. Before initializing the system with the LSM Software make sure to use the right database according to the microscope in use. It can be chosen via the icon Stand Select. Fig. 1-20 03/06 Carl Zeiss B 45-0019 e The Axioskop 2 FS MOT is connected to the electronics via the Interface Control. The CAN connection is fixed (1) to the control box 1-25 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 2 LSM 5 LIVE - SETUP REQUIREMENTS Carl Zeiss Contents LSM 5 LIVE/LSM 5 LIVE DuoScan SETUP REQUIREMENTS CONTENTS Page 2 LSM 5 LIVE - SETUP REQUIREMENTS ..............................................................................2-2 2.1 General Remarks................................................................................................................2-2 2.2 System Components and Recommended Setup..................................................................2-2 2.3 Room Requirements...........................................................................................................2-2 2.4 Minimum Space Requirements of the System .....................................................................2-3 2.5 Dimensions (Overview) .......................................................................................................2-3 2.6 Schematic Outline of a System ...........................................................................................2-4 2.7 2.7.1 Space Requirements...........................................................................................................2-4 LSM DuoScan with Ar UV Laser ..........................................................................................2-5 2.8 Power Requirements ..........................................................................................................2-6 2.9 Physical Dimensions ...........................................................................................................2-9 2.10 Dimension of Shipment Crates ...........................................................................................2-9 2.11 Environmental Requirements............................................................................................2-10 2.12 Vibrations ........................................................................................................................2-10 2.13 Microscopes.....................................................................................................................2-11 2.14 Scanning Module LSM 5 LIVE ...........................................................................................2-12 2.15 Laser Module LIVE............................................................................................................2-12 2.16 Laser Module UV for LSM DuoScan UV ............................................................................2-12 2.17 System Overview LSM 5 LIVE ............................................................................................2-13 2.18 System Overview LSM 5 LIVE DuoScan .............................................................................2-15 03/06 B 45-0019 e 2-1 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss 2 LSM 5 LIVE - SETUP REQUIREMENTS 2.1 General Remarks LSM 5 LIVE LSM 5 LIVE DuoScan The LSM 5 LIVE is a user friendly, high performance laser scanning system with unique technological performance. It is made of high precision components representing latest technological developments and a design that was guided by ergonomics, best possible performance and every day usability. The system is highly modular and flexible, but also easy to service and open for upgrades. 2.2 System Components and Recommended Setup The core of the system is a vibration isolating, air damped microscope table with a breadboard for fixation of the system and possible accessories. We strongly recommend the use of our Science Desk tables, since these have the most compact size that allows the setup of the various system configurations and future upgrades. A wide range of special accessories for physiological, molecular or cellbiological, or technical experiments (e.g. posts, shelves, grounding, faraday cages, cabinets etc.) is available worldwide by our partner Melles Griot. The science desk is a professional, open system platform for your experiments. A wide and a narrow version of the science desk table is available, matching the side- or rear port configurations of the microscope stands. The microscope table is complemented by the laser and electronic system rack, which can be positioned at the side or at the back of the microscope table. This rack contains all electronic and laser components the LSM 5 LIVE needs, and is again designed with best performance, usability and future upgrades in mind. For setup and service purposes, free access is needed to the long side of the rack. Hence approx. 50 cm space should be provided to the next wall. Use of the LSM 5 LIVE is mainly done by operating the software. Consequently, we provide an ergonomical computer workplace that fits the microscope table and should best be positioned on the right side of it. We recommend the use of the 45° triangle extension to get the best ergonomical setup of the complete system, but also a 0° or a 90° orientation to the microscope table is possible. The best overall system design depends on the shape of your room (see below). 2.3 Room Requirements The LSM 5 LIVE needs approx. an area of 7 m². The room for the LSM 5 LIVE should therefore offer a minimum size of 10 m². Due to the modular design, both square shaped rooms (approx. 3,2 m x 3 m) or rectangular shaped rooms (approx. 2.6 m x 3.9 m) match the setup requirements of the LSM 5 LIVE. Due to the modern diode technology of the lasers, the system will produce much less heat and noise than older confocals. Nonetheless an lab typical air condition or ventilation should be provided. A constant temperature of 23 °C would be ideal for constant and best system performance. Details should be discussed with your local Zeiss LSM specialist. 2-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 2.4 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss Minimum Space Requirements of the System For a list of the components dimensions see below; in general, a space of 1.8 m x 3.6 m for systems with a 0° or 45° oriented computer workplace, or 2.8 m x 2.6 m for systems with a 90° oriented computer workplace is required. As mentioned, free access to the long side of the electronic and laser system rack is required for service and setup of the system. If possible, we also recommend to have a free space of 30 cm to the next wall all around the system. 2.5 Dimensions (Overview) Component Overall dimensions Remarks Science desk microscope table (wide) 125 cm x 100 cm Side port systems Science desk microscope table (narrow) 100 cm x 125 cm Side- and Rear port systems Electronic and laser system rack 75 cm x 115 cm Long side must be accessible Computer workplace (0° or 90°) 120 cm x 80 cm Computer workplace diagonal (45°) 130 cm x 160 cm Recommended for ergonomics Complete system in ideal configuration, wide 180 cm x 360 cm table, 45° computer workplace Complete system in ideal configuration, 180 cm x 340 cm narrow table, 45° computer workplace Most often used setup Complete system square setup, 90° computer 280 cm x 260 cm workplace orientation 03/06 B 45-0019 e 2-3 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss 2.6 Schematic Outline of a System Fig. 2-1 2.7 Schematic outline of a system Space Requirements Fig. 2-2 2-4 LSM 5 LIVE LSM 5 LIVE DuoScan LSM 5 LIVE with microscope table and PC table (dim. in mm) The System rack contains all electronics and laser unit. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 2.7.1 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss LSM DuoScan with Ar UV Laser We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat accumulation and vibration. Length of the water hose: 400 cm Fig. 2-3 Space requirements for LSM DuoScan using an AR UV Laser (measurements in mm) Fig. 2-4 Lab container for UV laser setup The lab container cart 000000-0465-515 is recommended to provide space for the setup of the external UV-laser, the UV laser power supply and the plug in unit for external laser. 03/06 B 45-0019 e 2-5 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss 2.8 LSM 5 LIVE LSM 5 LIVE DuoScan Power Requirements The LSM 5 LIVE comes with a mains power supply cord and plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A), and with the matching mains socket outlet. A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system. The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter. A suitable grounding point must be installed in the room. Line voltage 220 … 240 V AC (±10 %) 100 … 125 V AC (±10 %) Line frequency 50...60 Hz 50...60 Hz − Max. current 3 phases at 16 A 2 phases at 25 A − Power Phase 1 = 1.9 kVA max. Phase 1 = 3.2 kVA max. Phase 2 = 1.5 kVA max. Phase 2 = 2.8 kVA max. LSM incl. VIS laser Phase 3 = 2.6 kVA max. − Power Consumption 5000 VA max. 5000 VA max. 208...240 V AC 208...240 V AC (±10 %) 50 / 60 Hz (±10 %) 50 / 60 Hz 1 phase at 63 A 1 phase at: Argon UV laser (LSM DuoScan only) − Line Voltage − Max. current 208 V: 34 Amps Note: For Line Voltage 220 V the connector and power plug are rated for 63 Amps, However wiring and fuse should be rated for 32 Amps. − Power Consumption 230 V: 31 Amps 240 V: 29 Amps 7000 VA max. 7000 VA max. Class of protection I I Type of protection IP 20 IP 20 Overvoltage category II II Pollution degree 2 2 2-6 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 2-5 03/06 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss Multipoint connectors B 45-0019 e 2-7 LSM 5 LIVE - SETUP REQUIREMENTS Power Requirements Carl Zeiss 1 2 LSM 5 LIVE LSM 5 LIVE DuoScan Key-interlock Laser ON/OFF Door interlock interface Fig. 2-6 Key-interlock Laser ON/OFF and interface for connection of door interlock The door interlock interface is covered with a green plug to bypass a door interlock. • To use the interface remove the top of the green plug and the bypass wire. • Then connect the wires of the door interlock at the same position. Two door interlocks can be connected. 2-8 B 45-0019 e 03/06 LSM 5 LIVE - SETUP REQUIREMENTS Physical Dimensions / Dimension of Shipment Crates LSM 5 LIVE 2.9 Carl Zeiss Physical Dimensions Length (cm) Passively damped anti-vibration table Width (cm) Height (cm) Weight (kg) 130 100 75 137 Scanning Module LSM 5 LIVE 55 17 30 19.5 Scanning Module LSM DuoScan 39 15 13 7 Microscope 50 35 50 20 Laser Module LIVE 66 52 22 58 140 20 20 60 66 52 22 9 110 70 58 90 Power supply for Ar (UV)* 50 50 30 30 Cooling unit for Ar (UV)* 80 45 50 30 Water hose for Ar (UV)* 700 Fiber optic cable, UV* 300 Fiber optic cable, VIS(ible) 300 Cables 350 Laser Module UV *(DuoScan only) Plug-in unit external laser (UV)* System Electronic Rack 2.10 Dimension of Shipment Crates Crate containing Length (cm) Width (cm) Height (cm) Weight (kg) Passively damped antivibration table 145 115 115 150 System Electronic Rack and Laser module 135 90 100 300 LSM, Microscope, Computer 135 90 100 150 Additional Hardware Components 135 90 61 100 UV laser unit 125 55 50 100 UV cooling unit 120 60 90 50 03/06 B 45-0019 e 2-9 LSM 5 LIVE - SETUP REQUIREMENTS Microscopes Carl Zeiss 2.11 LSM 5 LIVE LSM 5 LIVE DuoScan Environmental Requirements 1. Operation, specified performance T = 22 °C ± 3 °C without interruption (24 h a day independently whether system is operated or switched-off) 2. Operation, reduced performance T = 10 °C to 35 °C, any conditions different from 1. and 5. 3. Storage, less than 16 h T = -40 °C to 55 °C 4. Storage, less than 6 h T = -55 °C to 70 °C 5. Temperature gradient ± 0.5 °C/h 6. Warm up time 1 h, for high-precision and/or long-term measurements ≥ 2 h 7. Relative humidity < 65 % at 30 °C 8. Operation altitude max. 2000 m 9. Loss of heat 4 kW 2.12 Vibrations Vibrations under operation conditions (with system table) Shipping shock (LSM 5 box) 5 µm pp at 5 Hz 3g 10 µm pp at 10 Hz 10 µm pp at 20 Hz 2-10 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 2.13 LSM 5 LIVE - SETUP REQUIREMENTS Microscopes Carl Zeiss Microscopes Inverted Axiovert 200 M SP Upright Axioskop 2 FS MOT Upright Axio Imager.Z1 All ICS objectives from Carl Zeiss and their accessories can be accommodated. Z motor DC servomotor, opto-electronically coded Least Z interval: Piezo Objective focus 50 nm (Axiovert 200 M SP) 100 nm (Axioskop 2 FS MOT) 10 nm (Axio Imager.Z1) Piezo-driven single objective drive Max. travel 250 µm; resolution 5-10 nm In the unlikely case of extreme fluctuations of the external power net or electromagnetical radiation, the piezo crystal will vary and disturbance in the image is visible. Note that this is not a defect and the piezo drive will not be damaged. 03/06 B 45-0019 e 2-11 LSM 5 LIVE - SETUP REQUIREMENTS Scanning Module / Laser Module LIVE Carl Zeiss 2.14 LSM 5 LIVE Scanning Module LSM 5 LIVE Scanners 2 individually driven galvanometric scanners Scanning speed Up to 120 frames/sec (512 × 512 pixels) Field resolution Max. 512 × 2048 pixels (optional adjustable for each axis) Field of view 10 × 10 mm² with a 1.25× objective Zoom 0.5× ... 2×, continuous control Channels Up to 2 channels simultaneously: Optional transmitted light detection mode Dynamic range 12-bit DAC for each detection channel Apertures 1 or 2 individual variable aperture Computer controlled automatic alignment 2.15 Laser Module LIVE Single-mode polarization preserving fiber Laser beam attenuation (AOTF) for all lasers Diode laser (405 nm, 50 mW) Diode laser (440 nm, 16 mW) OPSS laser (488 nm, 100 mW) DPSS laser (532 nm, 75 mW) Diode laser (635 nm, 35 mW) In the unlikely case of extreme fluctuations of the external power net, the laser diode will switch off or the laser power might vary. Note that this is not a defect and the laser will not be damaged. 2.16 Laser Module UV for LSM DuoScan UV Single-mode polarization preserving fiber Laser beam attenuation (UV-AOTF) Ar UV Laser (351/364 nm, 80 mW) 2-12 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 2.17 03/06 LSM 5 LIVE - SETUP REQUIREMENTS System Overview LSM 5 LIVE - Material Carl Zeiss System Overview LSM 5 LIVE B 45-0019 e 2-13 Carl Zeiss LSM 5 LIVE - SETUP REQUIREMENTS System Overview LSM 5 LIVE - Material LSM 5 LIVE LSM 5 LIVE DuoScan 04 08 01 07 02 06 03 05 100 90 2-14 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 2.18 LSM 5 LIVE - SETUP REQUIREMENTS System Overview LSM 5 LIVE - Material Carl Zeiss System Overview LSM 5 LIVE DuoScan 04 08 01 07 02 06 03 05 100 90 03/06 B 45-0019 e 2-15 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 3 INTRODUCTION TO LASER SCANNING MICROSCOPY Contents Carl Zeiss INTRODUCTION TO LASER SCANNING MICROSCOPY CONTENTS Page 3 INTRODUCTION TO LASER SCANNING MICROSCOPY ...................................................3-2 3.1 Principle of Laser Scanning Microscopy...............................................................................3-2 3.2 Three-Dimensional Presentations of LSM Image Stacks .......................................................3-3 3.3 Optical Diagram of the LSM 5 LIVE (Schematic) ..................................................................3-4 3.4 3.4.1 3.4.2 3.4.3 Performance Features of the LSM 5 LIVE ............................................................................3-5 Optical and Mechanical Aspects .........................................................................................3-5 Microscope Equipment of the LSM 5 LIVE System...............................................................3-6 Computer Hardware and Software.....................................................................................3-8 03/06 B 45-0019 e 3-1 Carl Zeiss INTRODUCTION TO LASER SCANNING MICROSCOPY Principle of Laser Scanning Microscopy 3 INTRODUCTION TO LASER SCANNING MICROSCOPY 3.1 Principle of Laser Scanning Microscopy LSM 5 LIVE LSM 5 LIVE DuoScan To yield information on their inner structure by conventional transmitted-light microscopy, specimens have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a problem to satisfy these requirements. The essential considerations have led to trailblazing changes in conventional microscopy and supplied a successful solution to the above problem. • Unlike the practice of even illumination in conventional microscopy, the LSM technique projects the light of a point light source (a laser) through a high-NA objective onto a certain object plane of interest as a nearly diffraction-limited focus. However, if not for another "trick", the stray light produced outside the object plane, or the fluorescence of fluorescent specimens, would disturb the infocus image of object point of interest, resulting in a blurred image of poor contrast. The problem is therefore, how to capture only the light coming immediately from the object point in focus, while obstructing the light coming from out-of-focus areas of the specimen. • The light reflected, or the fluorescence light produced, at the focus of the high-NA objective is projected onto a variable aperture diaphragm by the same objective and a tube lens. The focus inside the specimen and the aperture are situated at optically conjugate points (confocal imaging). The decisive advantage of this arrangement is the fact that essentially no other light than that coming from the object plane of interest can pass the narrow aperture and be registered by a detector. Unwanted light coming from other specimen areas is focused outside the aperture, which passes only a small fraction of it. The smaller the aperture, the less stray light or fluorescence from out-of-focus areas will get on the detector. The image point thus generated is largely free from blur caused by unwanted light. • In order to obtain an image of the selected object plane as a whole, it is necessary to scan the object plane in a point-by-point, line-by-line Fig 3-1 Principle of confocal imaging raster by means of an XY light deflection system. The detectors - as a rule, photomultipliers - convert the optical information into electric signals. This allows the image of any object plane to be generated and stored within less than a second. By a defined focusing (Z axis) movement it is possible to look at any object plane of interest. By scanning a succession of object planes in a specimen, a stack of slice images can be produced. This way, the LSM technique in conjunction with ICS optics (Infinity Color-Corrected System) has brought decisive improvements over conventional microscopy in terms of resolving power and confocal depth contrast: Object features in the order of 0.2 μm can be resolved, and height differences of less than 0.1 μm made visible, without the use of interference methods. 3-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE Duo Scan 3.2 INTRODUCTION TO LASER SCANNING MICROSCOPY Three-Dimensional Presentation of LSM Image Stacks Carl Zeiss Three-Dimensional Presentations of LSM Image Stacks One of the advantages of the LSM technique is that it can present structures in three dimensions. This opens up many ways to process images. Outlined below are some of the possible methods to extract spatial information from stacks of slice images. • Gallery The simplest presentation of 3D information is a gallery showing the individual slice images (sections) of a stack arranged side by side, with each slice apart from the next by a defined, selectable interval on the Z axis. • Virtually infinite depth of focus The entire set of data can be imaged as a single projection. The computer establishes an image composed of all in-focus optical sections. The image produced by this so-called composite method has a virtually infinite depth of focus, since the result is made up of information from in-focus planes only. • Rotary animation A sequence of projections is computed, with the specimen being apparently rotated by a certain angle from image to image, for example by a full turn about an axis. If such a sequence is displayed on the monitor screen in rapid succession, the visual effect is that of a rotating three-dimensional object. • Stereo image pairs The computer establishes a pair of images corresponding to those we see with the right and the left eye, respectively. The two images forming the stereo pair can be shown on the monitor side by side. They can be seen as a 3D image with suitable optical aids. Another possibility is to present both images in registration, with one image in the red channel and the other in the green one (anaglyph). Viewed through red and green color filters in a spectacle frame, which only pass the image intended for the respective eye, the two images form a 3D image in the brain • Color-coded height slices Each level, i.e. each slice is assigned a different color. For direct evaluation, a color scale is shown, indicating the actual height above the bottom slice. • Orthogonal sections This computation produces a triplet of mutually perpendicular sectional images. • Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates, i.e. X, Y, Z, angle of rotation, and angle of tilt. • Topography (option) A computing program for surface topography presentations (as required in materials research) is available. • Physiology (option) With a special software, kinetic processes can be tracked, which is especially of interest to physiology. • Image VisArt (option) Three-dimensional display of floating transparent structures (cells) or opaque structures (material) • 3D Deconvolution (option) 03/06 B 45-0019 e 3-3 Carl Zeiss 3.3 INTRODUCTION TO LASER SCANNING MICROSCOPY Optical Diagram of the LSM 5 LIVE LSM 5 LIVE LSM 5 LIVE DuoScan Optical Diagram of the LSM 5 LIVE (Schematic) AOTF y (d, x, y) AOTF Acousto-Optical Tunable Filter Fig. 3-2 Optical path, schematic (2-channel configuration) The diagram above is a schematic representation of the LSM 5 LIVE system. Laser light is focused onto the specimen through an objective in a diffraction-limited mode. Light emitted at the focal plane and at planes below and above it is directed via an Y scanner onto the Achrogate beam splitter, which separates the emissions from the excitation light. The fluorescences are separated from each other by a Secondary Dichroic Beam Splitter and directed to individual photodetectors (CCD Line1 and CCD Line2). 3-4 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 5 LIVE 3.4 Performance Features of the LSM 5 LIVE 3.4.1 Optical and Mechanical Aspects Carl Zeiss The highly integrated system design makes for the shortest possible optical paths, top-grade optical precision and high stability. The compact scanning module can be fitted to an inverted (Axiovert 200 M SP) or upright (Axioskop 2 FS MOT, Axio Imager.Z1, Axio Imager.M1) microscope in less than three minutes. On the Axiovert 200 M, the scanning module may be mounted either to the base port directly below the microscope or to the side port. For the VIS (visible-light) Laser Module, the user can select from up to four lasers with wavelengths of 635, 532, 488, 440, and 405 nm. Coupling of the laser light is through polarization-preserving singlemode optical fibers. One beam collimator for the visible ranges provides optimum adaptation of the respective laser wavelength to the objective used and, thus, optimum correction for Z aberrations. The two simultaneous image acquisition channels, usable for fluorescence are ideal for the investigation of multiple fluorescence specimens. In the two channels, the diameters of the aperture and their XY positions can be optimized, and the desired emission filter placed into the beam path, by servo-motor control. In the simultaneous registration of multiple fluorescences, identical optical sections can be obtained in each confocal channel. This is of importance, e.g., with the FISH method (fluorescence in-situ hybridization) used for genome analysis in cytogenetic studies. It is possible to superimpose a multiple fluorescence image on a brightfield, differential interference or phase image. In addition to the emission filters for all standard and special applications, available in motor-controlled filter wheels, the user can easily install his own emission filters in two of the channels. The high-NA C-APOCHROMAT objectives specially developed for the LSM technique reach the physical limit in resolving power, and can be used throughout the 350...700 nm spectral range with the same high quality, producing brilliant images. A mirror scanner system, controlled by Realtime Electronics, offers several advantages. The large deflection angle of the scanning mirrors allows a wide area to be scanned. With a 1.25x objective, the object area scanned is 10 x 10 mm². The scanning field size can be freely selected between 4 x 1 and 512 x 2048 pixels. It is possible to carry out fastest XY scans without having to rotate the specimen itself under laser radiation load. Selection of the specimen detail of interest for zooming is fast and convenient, and the zoomed image is automatically centered. This saves the job of specimen centration with the microscope stage. Using a bi-directional scanning facility will double the scanning rate to 120 frames/sec (at 512 x 512 pixels); if two different lasing wavelengths are used for the scanning, two fluorochrome dyes can be viewed and documented in a quasi-simultaneous mode. This will absolutely prevent "bleeding". 03/06 B 45-0019 e 3-5 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 5 LIVE Carl Zeiss 3.4.2 LSM 5 LIVE LSM 5 LIVE DuoScan Microscope Equipment of the LSM 5 LIVE System The LSM 5 LIVE system is equipped either with the inverted Axiovert 200 M SP microscope or with the upright Axio Imager.Z1, Axioskop 2 FS MOT or Axio Imager.M1 microscopes. Referring to the delivered operating manual "Axiovert 200 M" only differences to this manual will be explained. (1) Stand a) The motorized objective nosepiece 5x H DIC is firmly fixed to the stand, where no operating elements can be found for the nosepiece. Operation will be done LSM 5 software controlled. The "Restriction of revolver height to protect the objectives when changing the objectives motorized" is inactivated. The nosepiece will be moved down automatically before each motorized objective change. b) The reflector mount is motorized and provided with the Axiovert 200 M reflector turret. The reflector turret has 5 positions: One transmitting light position, which is identical to the LSM position, and four further positions for fluorescence filter sets (reflector modules). If you want to use more than five conventional fluorescence filter sets, it is advisable to use a further reflector turret. When changing the reflector turret position you must make sure that the turret will click into position, since otherwise the image area will be cut. c) The stand has a motorized focusing drive (fine coarse). Sensitivity of the focusing drive is adjusted to the delivered objectives by the manufacturer. If you want to use other objectives, sensitivity and parfocality can be adjusted via the Axioset program. d) The stand features an integrated power supply for the internal motors and stand electronics. The power supply can be switched on at the right side of the stand. External power supply units will be used for the mercury vapor short arc lamp. e) The analyzer slider for conventional DIC methods will be operated from the right side and is located just below the nosepiece. When the rod is pushed in, the analyzer is located in the beam path. In LSM-mode the analyzer must not be located in the beam path, analyzer rod must be pulled out. f) The stands dispose of five additional ports: two side ports, front ports and base ports. The side port or the front is equipped with the LSM 5 special interface, one of the others with the TV interface. The LSM 5 LIVE scanning module can be mounted to the special interface port. Different camera systems can be adapted to the TV interface using the TV adapters 452982/83/92/94/95/97/980000-000. (2) Specimen stages and fine focus drives a) Mechanical stage The stage must be mounted with the coaxial drive on the right side of the stand. b) Scanning stage c) Piezo objective focus drive 3-6 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 5 LIVE Carl Zeiss (3) Transmitted-light illumination a) The illuminator support contains a security circuit, which activates a shutter preventing laser light from reaching the stand when the support is moved to back. A complementary shutter built-in the stand prevents laser light from reaching the eye pieces during scanning mode. b) The illuminator support is equipped with a rotatable polarizer. The Axiovert 200 M description contains the adjustment for DIC mode during conventional observation. For scanning transmitted light DIC mode the polarizer in the transmitted light support works like an analyzer and must be adjusted in such a manner, that direct laser light will be blocked. The conventional analyzer slider in the stand is not allowed be located in the beam path because of the laser light already is polarized. c) On the illuminator support as an option there is mounted a LSM 5 software controlled switching mirror fully motorized. Alternatively the light is directed to the LSM 5 T-light detector or enables conventional transmitted-light observation. d) The focusing screen for conventional transmitted-light is located in a support in front of the halogen lamp housing. e) Further information to halogen lamp and condensers you will find in the Axiovert 200 M operating manual. (4) Reflected light fluorescence All Axiovert 200 M fluorescence accessories exceptional the reflector slider can be used. Further information you will find in the Axiovert 200 M operation manual. (5) Imaging optics Optovar sliders are not usable. The analyzer for conventional DIC mode will be operated from the right side and is located just below the nosepiece. Use of sliders with auxiliary objects (473704/14-0000-000) is not possible. (6) Photo equipment The stand hasn’t have an integrated SLR-port, but microscope cameras, as described in the Axiovert 200 M operation manual, can be used. (7) TV adaptation The TV port aside and the tubes can be used as described in the Axiovert 200 M operation manual. The TV interface side port or base port can be used with TV adapters 44 or LSM adapters. 03/06 B 45-0019 e 3-7 Carl Zeiss 3.4.3 INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 5 LIVE LSM 5 LIVE LSM 5 LIVE DuoScan Computer Hardware and Software The LSM 5 LIVE is controlled through a standard high-end Pentium PC. Linking with the electronic control system is via an ultrafast interface. The PC comes with the WINDOWS XP operating system. The instrument is fully motorized, permitting fast change-over between methods as well as automatic operation. Parameters once set or complex examination sequences once established can be saved and reproduced; this way, complete application programs can be loaded and executed by pushbutton control. The software of the LSM 5 LIVE offers perfect facilities for individual settings of functions and parameters. Conversion of the light signals into a digital image is effected by means of four 12-bit A/D converters, each of which can generate 4096 brightness levels. The software provides a enormously wide range of image processing functions, including all standard 2D/3D (stereo, projection) functions same as sophisticated 3D reconstruction capabilities (surface and alpha rendering), digital processing of voxels and 3D measurement functions (surface areas, volumes). As all files and images are recorded in MS Access databases, elegant image database editing is just as easy as transferring the records to other programs. 3-8 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan IMPORTANT NOTES FOR CHAPTER 4 Carl Zeiss IMPORTANT NOTES FOR CHAPTER 4 Bleach series When doing bleach series on LSM DuoScan systems, the time stamps and bleach markers indicated in the images will depend on the start position of the scanners in the image frame. Since these are not identical for the imaging and the photomanipulation scanners, sometimes imaging seems to start too early after a bleach event. Ignore the last two digits in the time stamps and bleach markers in this case. Macro When using the modify images macro to convert time series data into Z stacks, an arbitrary section step of 1 µm is applied to the stack. This will also lead to an arbitrary time stamp when reconverting the Z stack into a time series. The order of the images however is preserved under all conditions. Fast Z-stack and When acquiring fast Z-stacks with line scan mode, the starting point of the stacks line scan acquisi- and the vertical dimensions can differ depending on uni- or bidirectional scanning mode. This is a mechanical effect which increases with rising line scanning speed. tion Avoid extremely fast speeds in the hundred(s) of frames per second range when acquiring Z-stacks. Piezo objective focus units 03/06 The piezo objective focus units 1325-210, 1325-212 and 1325-213 with an increased travel range of 250 µm are controlled by a 14 bit logic. Due to the increased travel range, this implies a smallest step of 15 nm, in contrast to former fine focus solutions with a smaller travel range but a smallest step size of <10 nm. B 45-0019 e 4-I IMPORTANT NOTES FOR CHAPTER 4 Carl Zeiss 4-II B 45-0019 e LSM 5 LIVE LSM 5 LIVE Duo Scan 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 4 OPERATION Purpose Carl Zeiss OPERATION CONTENTS Page 4 OPERATION......................................................................................................... 4-9 4.1 Purpose ........................................................................................................................... 4-9 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 Software ........................................................................................................................... 4-9 Windows and Window Elements ..................................................................................... 4-10 Convention for the Text in this Manual ........................................................................... 4-11 Backup............................................................................................................................ 4-11 Software Operation......................................................................................................... 4-11 4.2 Switching on the System............................................................................................. 4-12 4.2.1 4.2.2 Switching on the HBO 100.............................................................................................. 4-13 Starting the LSM 5 Program ............................................................................................ 4-13 4.3 Main Menu ................................................................................................................... 4-15 4.4 File Menu ...................................................................................................................... 4-17 4.4.1 4.4.2 4.4.3 4.4.3.1 4.4.3.2 4.4.3.3 4.4.3.4 4.4.3.5 4.4.3.6 4.4.3.7 4.4.3.8 4.4.3.9 4.4.3.10 4.4.4 4.4.5 4.4.6 4.4.7 4.4.7.1 4.4.7.2 4.4.8 Create New Image Database ........................................................................................... 4-18 Open Existing Image Database ........................................................................................ 4-19 Image Database Window ................................................................................................ 4-19 Form Display Mode ......................................................................................................... 4-21 Gallery Display Mode ...................................................................................................... 4-23 Table Display Mode......................................................................................................... 4-24 Load an Image into the Image Display Window ............................................................... 4-24 Load Image with Reduced Resolution (Subset) ................................................................. 4-25 Update Image Database Display (Refresh) ........................................................................ 4-25 Copy / Paste Image(s) to the CLipboard ........................................................................... 4-25 Display Images with Certain Features (Filter) .................................................................... 4-26 On Filter Function............................................................................................................ 4-28 Delete Function .......................................................................................................... 4-28 Save an Image to the Image Database ............................................................................. 4-29 Import of Images............................................................................................................. 4-32 Export of Images ............................................................................................................. 4-33 Multi Print....................................................................................................................... 4-34 Overlay Toolbar............................................................................................................... 4-34 Printing Images ............................................................................................................... 4-36 Exit ................................................................................................................................. 4-36 4.5 Acquire Menu ............................................................................................................... 4-37 03/06 B 45-0019 e 4-1 Carl Zeiss 4.5.1 4.5.1.1 4.5.1.2 4.5.1.3 4.5.1.4 4.5.2 4.5.2.1 4.5.2.2 4.5.2.3 4.5.2.4 4.5.2.5 4.5.3 4.5.3.1 4.5.3.2 4.5.3.3 4.5.3.4 4.5.3.5 4.5.3.6 4.5.3.7 4.5.3.8 4.5.3.9 4.5.4 4.5.4.1 4.5.4.2 4.5.4.3 4.5.4.4 4.5.5 4.5.5.1 4.5.5.2 4.5.6 4.5.6.1 4.5.6.2 4.5.6.3 4.5.6.4 4.5.6.5 4.5.6.6 4.5.6.7 4.5.6.8 4.5.7 4.5.8 4.5.8.1 4.5.8.2 4.5.8.3 4.5.8.4 4.5.8.5 4.5.9 4.5.9.1 4.5.9.2 4-2 OPERATION Purpose LSM 5 LIVE LSM 5 LIVE DuoScan Laser Control ...................................................................................................................4-38 Switching on the Enterprise UV Laser ...............................................................................4-38 Opening / Closing the Laser Control window ...................................................................4-38 Function Description ........................................................................................................4-39 Settings............................................................................................................................4-39 Microscope Control..........................................................................................................4-40 Open the Microscope Control Window ............................................................................4-40 Microscope Control Window for Axio Imager.Z1 ..............................................................4-41 Microscope Control Window for Axiovert 200 M .............................................................4-46 Working in LSM Mode .....................................................................................................4-50 Microscope Control for Axioskop 2 FS MOT.......................................................................4-50 Configuration Control......................................................................................................4-51 Open / Close the Configuration Control Window .............................................................4-52 Spectra Button .................................................................................................................4-52 Config Button ..................................................................................................................4-53 Settings for Single Track in the Channel Mode .................................................................4-55 Settings for Multi Track in the Channel Mode ..................................................................4-60 Transmitted light preset ...................................................................................................4-64 Ratio Settings Panel..........................................................................................................4-65 Camera Detection Panel...................................................................................................4-66 LSM 5 LIVE / DuoScan Panel .............................................................................................4-67 Scan Control ....................................................................................................................4-68 Open / Close the Scan Control Window ...........................................................................4-69 Frame ..............................................................................................................................4-71 Line..................................................................................................................................4-87 Camera Control ...............................................................................................................4-88 Edit ROI (Region Of Interest).............................................................................................4-90 Open / Close the Edit ROI Window...................................................................................4-90 Function Description ........................................................................................................4-90 Time Series.......................................................................................................................4-94 Open / Close the Time Series Control Window .................................................................4-94 Start Series Panel..............................................................................................................4-95 Stop Series Panel..............................................................................................................4-97 Cycle Delay / Time Interval Panels .....................................................................................4-98 Marker Panel..................................................................................................................4-100 Time Series of a Frame ...................................................................................................4-102 Time Series of a Frame over Z Stack (option)...................................................................4-104 Time Series with Mean ROI.............................................................................................4-106 Stripe bleach function (LSM 5 LIVE without LSM DuoScan) .............................................4-108 Edit Bleach (LSM 5 LIVE DuoScan) ..................................................................................4-110 Open / Close the Edit Bleach Window ............................................................................4-110 Settings Panel ................................................................................................................4-111 Bleach Parameter Panel ..................................................................................................4-112 Excitation of Bleach Track Panel .....................................................................................4-113 Start / End a Bleaching Procedure...................................................................................4-113 Stage .............................................................................................................................4-114 Open / Close the Stage and Focus Control Window .......................................................4-114 Focus Position Panel .......................................................................................................4-115 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Purpose Carl Zeiss 4.5.9.3 4.5.9.4 4.5.10 Stage Position Panel ...................................................................................................... 4-116 Tile Scan Panel .............................................................................................................. 4-118 VIS, TV and LSM Buttons............................................................................................... 4-120 4.6 Process Menu.............................................................................................................. 4-121 4.6.1 4.6.1.1 4.6.1.2 4.6.1.3 4.6.1.4 4.6.1.5 4.6.2 4.6.2.1 4.6.2.2 4.6.3 4.6.3.1 4.6.3.2 4.6.4 4.6.4.1 4.6.4.2 4.6.4.3 4.6.5 4.6.5.1 4.6.5.2 4.6.6 4.6.7 4.6.7.1 4.6.7.2 4.6.7.3 4.6.7.4 4.6.8 4.6.9 4.6.9.1 4.6.9.2 4.6.9.3 4.6.9.4 4.6.10 4.6.10.1 4.6.10.2 4.6.10.3 4.6.10.4 4.6.11 4.6.11.1 4.6.11.2 4.6.11.3 4.6.12 4.6.12.1 4.6.12.2 Add .............................................................................................................................. 4-121 Open / Close the Add Window...................................................................................... 4-121 Source Panel ................................................................................................................. 4-122 Destination Panel .......................................................................................................... 4-122 Add Panel ..................................................................................................................... 4-123 Preview Panel................................................................................................................ 4-123 Subtract ........................................................................................................................ 4-124 Open / Close the Subtract Window ............................................................................... 4-124 Performance of the Subtract Function ........................................................................... 4-124 Multiply ........................................................................................................................ 4-125 Open / Close the Multiply Window................................................................................ 4-125 Performance of the Multiply Function............................................................................ 4-125 Ratio ............................................................................................................................. 4-126 Open / Close the Ratio Window .................................................................................... 4-126 Performance of the Ratio Function ................................................................................ 4-126 Formula Panel ............................................................................................................... 4-127 Copy (Channel) ............................................................................................................. 4-127 Open / Close the Copy Window .................................................................................... 4-127 Performance of the Copy Function ................................................................................ 4-128 Duplication (Image) ....................................................................................................... 4-128 Filter ............................................................................................................................. 4-129 Open / Close the Filter Window..................................................................................... 4-129 Image Panel .................................................................................................................. 4-129 Filter List and Edit Panel................................................................................................. 4-129 Preview Panel................................................................................................................ 4-132 Contrast........................................................................................................................ 4-132 Interpolate .................................................................................................................... 4-133 Open / Close the Interpolate Brightness and Contrast Window...................................... 4-133 Image Panel .................................................................................................................. 4-133 Interpolation Panel ........................................................................................................ 4-134 Preview Panel................................................................................................................ 4-135 Channel Shift ................................................................................................................ 4-135 Open / Close the Channel Shift Window................................................................... 4-135 Image Panel.............................................................................................................. 4-136 Shift Panel ................................................................................................................ 4-136 Preview Panel ........................................................................................................... 4-137 Unmix ........................................................................................................................... 4-137 Open / Close the Unmix Window.............................................................................. 4-138 Source Panel............................................................................................................. 4-138 Definition of Channels Panel..................................................................................... 4-138 Ion Concentration ......................................................................................................... 4-141 Open / Close the Ion Concentration Window............................................................ 4-141 Function Description................................................................................................. 4-141 03/06 B 45-0019 e 4-3 Carl Zeiss OPERATION Purpose LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.12.3 4.6.12.4 4.6.12.5 4.6.12.6 4.6.12.7 4.6.12.8 4.6.13 4.6.14 4.6.15 4.6.16 4.6.17 4.6.17.1 4.6.17.2 4.6.17.3 Single Wavelength Dyes – Offline Calibration ............................................................4-142 Ratiometric Dyes .......................................................................................................4-143 Ratiometric Dyes - Online Ratio..................................................................................4-143 Ratiometric Dyes - Calibration....................................................................................4-144 Ratiometric Dyes - Equation Calibration (Grynkiewicz) ...............................................4-145 Options for Calibration Image Selection (Equation- or Titration Calibration) ...............4-145 Image Scaling.................................................................................................................4-145 Copy Window Contents.................................................................................................4-145 Copy Full Resolution.......................................................................................................4-146 Paste Bitmap ..................................................................................................................4-146 Kinetic ...........................................................................................................................4-146 Open / Close the Kinetic Analysis window .................................................................4-146 Function Description..................................................................................................4-147 Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins ....................4-148 4.7 3D View Menu.............................................................................................................4-152 4.7.1 4.7.1.1 4.7.1.2 4.7.1.3 4.7.1.4 4.7.2 4.7.2.1 4.7.2.2 4.7.2.3 4.7.2.4 4.7.3 4.7.3.1 4.7.3.2 4.7.3.3 4.7.3.4 4.7.4 4.7.4.1 4.7.4.2 4.7.4.3 3D DepthCod (Color Coded Depth Map).......................................................................4-152 Open / Close the Depth Coding Window .......................................................................4-152 Source Panel ..................................................................................................................4-153 Depth Coding Panel .......................................................................................................4-153 Preview Panel.................................................................................................................4-155 Projection.......................................................................................................................4-155 Open / Close the Projection Window..............................................................................4-155 Source Panel ..................................................................................................................4-155 Projection Panel .............................................................................................................4-156 Preview Panel.................................................................................................................4-157 Stereo ............................................................................................................................4-159 Open / Close the Stereo Images Window .......................................................................4-159 Source Panel ..................................................................................................................4-159 Stereo Images Panel .......................................................................................................4-160 Preview Panel.................................................................................................................4-162 3D DeConVolution (DCV)...............................................................................................4-163 Open / Close the 3D Deconvolution Window .................................................................4-164 Source Panel ..................................................................................................................4-165 Deconvolution Panel ......................................................................................................4-165 4.8 Macro Menu ................................................................................................................4-167 4.8.1 4.8.2 4.8.2.1 4.8.2.2 4.8.2.3 4.8.2.4 4.8.3 4.8.4 4.8.4.1 4.8.4.2 4.8.4.3 4.8.5 Macro Language ............................................................................................................4-167 Macro Control ...............................................................................................................4-168 Open / Close the Macro Control Window.......................................................................4-168 Edit Macro Function .......................................................................................................4-168 Assign Macro to Button Function ...................................................................................4-171 Editing and Debugging of Macros ..................................................................................4-172 Overview of Available Macros ........................................................................................4-173 Sample Macros ..............................................................................................................4-175 Distance Macro ..............................................................................................................4-175 Profile Macro .................................................................................................................4-175 Multiple Time Series Macro ............................................................................................4-176 Kollimatic.......................................................................................................................4-177 4-4 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Purpose Carl Zeiss 4.8.6 4.8.7 4.8.8 4.8.9 4.8.9.1 4.8.9.2 4.8.9.3 Optimize Macro ............................................................................................................ 4-186 ReInit Macro ................................................................................................................. 4-186 Image Match (Scanfield Transformation) Macro............................................................. 4-187 Visual Macro Editor ....................................................................................................... 4-189 Open / Close the Visual Macro Editor Window .............................................................. 4-189 Function Description ..................................................................................................... 4-190 Editing a Macro............................................................................................................. 4-195 4.9 Options Menu............................................................................................................. 4-198 4.9.1 4.9.2 4.9.2.1 4.9.2.2 4.9.2.3 4.9.2.4 4.9.2.5 4.9.2.6 4.9.2.7 4.9.2.8 4.9.2.9 4.9.2.10 4.9.2.11 4.9.2.12 4.9.2.13 4.9.2.14 4.9.2.15 4.9.2.16 4.9.2.17 Dye DB Function ........................................................................................................... 4-199 Settings Function .......................................................................................................... 4-200 Open / Close the Settings for user : ... Window ............................................................. 4-200 Autosave....................................................................................................................... 4-201 Database General.......................................................................................................... 4-202 Database Table Viewer.................................................................................................. 4-203 Database Gallery Viewer ............................................................................................... 4-203 Import / Export.............................................................................................................. 4-203 Scan Information........................................................................................................... 4-204 Image Status Display ..................................................................................................... 4-204 Print Status Display........................................................................................................ 4-204 Recording / Reuse..................................................................................................... 4-205 Time Series ............................................................................................................... 4-205 Mean of ROIs ........................................................................................................... 4-205 Temporary Files ........................................................................................................ 4-206 Program Start ........................................................................................................... 4-207 Hardware ................................................................................................................. 4-207 Image Display ........................................................................................................... 4-207 Save ......................................................................................................................... 4-208 4.10 Maintain Menu........................................................................................................... 4-210 4.10.1 4.10.1.1 4.10.1.2 4.10.2 4.10.2.1 4.10.2.2 4.10.2.3 4.10.3 4.10.3.1 4.10.3.2 4.10.3.3 4.10.3.4 4.10.4 4.10.5 4.10.6 4.10.7 Scanner......................................................................................................................... 4-210 Electrical Calibration ................................................................................................. 4-210 Important Notes and Hints........................................................................................ 4-211 Objective....................................................................................................................... 4-212 Objective Change ..................................................................................................... 4-212 Focus Speed Change ................................................................................................ 4-214 Parfocality Correction ............................................................................................... 4-214 Pinhole Adjustment....................................................................................................... 4-215 Open / Close the Pinhole and Collimator Window .................................................... 4-216 Pinhole Panel............................................................................................................ 4-216 Collimator panel....................................................................................................... 4-217 Aperture and collimator Adjustment......................................................................... 4-218 Camera......................................................................................................................... 4-220 Reboot.......................................................................................................................... 4-220 HW/Admin.................................................................................................................... 4-220 Test Grid ....................................................................................................................... 4-220 4.11 Window Menu............................................................................................................ 4-221 4.11.1 Full Screen .................................................................................................................... 4-221 03/06 B 45-0019 e 4-5 Carl Zeiss OPERATION Purpose LSM 5 LIVE LSM 5 LIVE DuoScan 4.11.2 4.11.3 Close All Image Display Windows...................................................................................4-221 Scan and System Information .........................................................................................4-222 4.12 Help Menu ...................................................................................................................4-223 4.12.1 4.12.2 4.12.3 Help...............................................................................................................................4-223 About ............................................................................................................................4-223 FRAP Guide....................................................................................................................4-223 4.13 Display and Analysis of Images .................................................................................4-224 4.13.1 Structure of the Image Display Window .........................................................................4-224 4.13.2 Display Modes................................................................................................................4-225 4.13.3 Select - Chan .................................................................................................................4-226 4.13.3.1 Assigning Another Color to a Channel.......................................................................4-227 4.13.3.2 Switching a Channel of a Multi Channel Image off or on...........................................4-227 4.13.3.3 Switching to Monochrome Image Display ..................................................................4-227 4.13.3.4 Defining a New Color ................................................................................................4-227 4.13.4 Select - Zoom.................................................................................................................4-228 4.13.5 Select - Slice...................................................................................................................4-229 4.13.6 Select - Overlay ..............................................................................................................4-230 4.13.6.1 Functional Description ...............................................................................................4-230 4.13.6.2 Buttons in the Overlay Toolbar...................................................................................4-231 4.13.7 Select - Contr.................................................................................................................4-234 4.13.7.1 Ramp ........................................................................................................................4-235 4.13.7.2 PolyLine.....................................................................................................................4-235 4.13.7.3 Spline........................................................................................................................4-236 4.13.7.4 Gamma .....................................................................................................................4-236 4.13.8 Select - Palette ...............................................................................................................4-237 4.13.8.1 Editing and Storing a Palette......................................................................................4-238 4.13.8.2 Delete a Palette .........................................................................................................4-239 4.13.8.3 Import a Palette.........................................................................................................4-239 4.13.9 Select - Anim .................................................................................................................4-239 4.13.10 Select - Reuse.................................................................................................................4-241 4.13.11 Select - Crop ..................................................................................................................4-242 4.13.12 Select - Copy..................................................................................................................4-243 4.13.13 Select - Save...................................................................................................................4-243 4.13.14 Select - Save As..............................................................................................................4-244 4.13.15 Display - xy.....................................................................................................................4-244 4.13.16 Display - Split xy .............................................................................................................4-245 4.13.17 Display - Ortho...............................................................................................................4-246 4.13.17.1 Ortho - Select Function..............................................................................................4-247 4.13.17.2 Ortho - Distance Function..........................................................................................4-248 4.13.17.3 Ortho - 2D DeConVolution Function..........................................................................4-249 4.13.18 Display - Cut ..................................................................................................................4-250 4.13.19 Display - Gallery .............................................................................................................4-251 4.13.20 Display - Histo ................................................................................................................4-253 4.13.20.1 Display - Histo - Overview ..........................................................................................4-253 4.13.20.2 Area Function............................................................................................................4-255 4-6 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Purpose Carl Zeiss 4.13.20.3 Colocalisation Function............................................................................................. 4-257 4.13.21 Display - Profile ............................................................................................................. 4-261 4.13.21.1 Function ................................................................................................................... 4-261 4.13.21.2 Function Elements .................................................................................................... 4-263 4.13.21.3 Display - Mean ......................................................................................................... 4-265 4.13.22 Display - 2.5 D .............................................................................................................. 4-266 4.13.22.1 Direct Setting in the Image ....................................................................................... 4-266 4.13.22.2 Setting via Scrollbars................................................................................................. 4-267 4.13.23 Display - 3D (Image VisArt)............................................................................................ 4-268 4.13.23.1 Shadow Projection.................................................................................................... 4-269 4.13.23.2 Transparency Projection............................................................................................ 4-272 4.13.23.3 Surface Rendering .................................................................................................... 4-273 4.13.23.4 Appearance (Settings)............................................................................................... 4-274 4.13.23.5 Series ....................................................................................................................... 4-275 4.13.24 Display - Topography for LSM ....................................................................................... 4-278 4.13.24.1 Channel Selection..................................................................................................... 4-279 4.13.24.2 Generate Topography............................................................................................... 4-279 4.13.24.3 Topography Thresholds ............................................................................................ 4-280 4.13.24.4 Processing by Filtering .............................................................................................. 4-281 4.13.24.5 Display Modes .......................................................................................................... 4-284 4.13.24.6 Context Menu of the 3D Display Mode..................................................................... 4-287 4.13.24.7 Measurement Functions ........................................................................................... 4-294 4.13.24.8 3D Measurement Functions ...................................................................................... 4-304 4.13.24.9 Export Data .............................................................................................................. 4-308 4.13.24.10 Topo ReUse .............................................................................................................. 4-308 4.13.25 Display - Prev. ............................................................................................................... 4-309 4.13.25.1 Context Menu for Scan Information Text .................................................................. 4-310 4.13.25.2 Context Menu for Topography Images ..................................................................... 4-310 4.13.25.3 Arranging and Printing the Print Preview .................................................................. 4-310 4.13.26 Display - Info................................................................................................................. 4-311 4.13.27 Additional Display Mode in Time Series ......................................................................... 4-312 4.13.27.1 Display - Mean ......................................................................................................... 4-312 4.14 Image Optimization ................................................................................................... 4-315 4.14.1 Single Channel.............................................................................................................. 4-315 4.14.1.1 Requirements ........................................................................................................... 4-315 4.14.1.2 Confocal Aperture / Detector Gain / Ampl. Offset ..................................................... 4-319 4.14.1.3 Exposure Time, Scan Average and Pixel Depth .......................................................... 4-321 4.14.2 Multiple-channel ........................................................................................................... 4-322 4.14.2.1 Requirements ........................................................................................................... 4-322 4.14.2.2 Image Optimization .................................................................................................. 4-323 4.15 Shut-Down Procedure................................................................................................ 4-325 4.15.1 4.15.2 4.15.3 4.15.4 Exiting the LSM Program ............................................................................................... 4-325 Shut Down the WINDOWS Operating System................................................................ 4-326 Turning Power Off ........................................................................................................ 4-326 Turning off the HBO 100............................................................................................... 4-326 4.16 Software and Hardware Options.............................................................................. 4-327 03/06 B 45-0019 e 4-7 Carl Zeiss OPERATION Purpose LSM 5 LIVE LSM 5 LIVE DuoScan 4.16.1 4.16.2 Software ........................................................................................................................4-327 Hardware.......................................................................................................................4-329 4.17 Courses on "How to Operate the System in an Optimized Way" ...........................4-330 4-8 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Purpose 4 OPERATION 4.1 Purpose Carl Zeiss This chapter describes the operation of the LSM 5 LIVE Laser Scanning Microscope exemplified by typical applications in conjunction with the LSM 5 software and its graphic user environment. When starting up and operating the microscope system, mind the operating instruction manuals for the Axio Imager.Z1, Axiovert 200 M and Axioskop 2 FS MOT microscopes: − B 46-0046 Axio Imager, Operating Manual − B 40-080 Axiovert 200 M, Operating Manual − B 40-076 Axioskop 2 FS MOT, Operating Manual 4.1.1 Software The LSM 5 software, Version 4.0: − controls the microscope, the scanning and laser modules, tools (filters, stand, Axioset) and the image acquisition process − displays and analyzes the images It is based on the network-capable graphic 32-bit Microsoft ® WINDOWS XP operating system. Portions © Copyright 1981 - 2001, Microsoft Corporation. All rights reserved. The installation of the software for the LSM 5 LIVE and the basic settings of the equipment components are carried out by Carl Zeiss service staff. This job includes the creation of a customized software configuration in line with the specific hardware components of the customer's microscope system. The LSM 5 software is menu-controlled and normally uses its own windows for the activation of the various functions; within these windows, further submenus (panels) can be displayed and removed. The screen layout used in this manual is based on the former Windows ® standard layout and may vary from yours. Images of the specimens to be examined, created by scanning, are displayed in separate Image Display windows. Theoretically, the number of simultaneously opened windows for software operation or image display is unlimited, but should not be too excessive so that an overview is still possible. Identical functions, e.g. Laser Control, can be performed in several software windows. Changes made by the software are recorded immediately and are automatically transferred to all the other windows concerned. 03/06 B 45-0019 e 4-9 OPERATION Purpose Carl Zeiss 4.1.2 LSM 5 LIVE LSM 5 LIVE DuoScan Windows and Window Elements Window element Description / Explanation Window (e.g.: Laser Control window) − Window displayed after activation of a function button (e.g.: Laser button in the toolbar of the Main menu). Panel (e.g.: Lasers panel) − Limited function range within a window List box or selection box − Selection of one of the displayed options at a click of the mouse. − Open the box by clicking on the arrow button. Input box − Input of text or numeric values via the keyboard. Scrollbar with slider − Setting of numbers in the relevant input box by moving the slider or clicking on the arrow buttons or clicking on the slider and moving via the arrow keys of the keyboard. Press the Shift or Ctrl key while clicking on the arrow button to change the numeric values in coarse or fine steps. Check box − Activates / deactivates setting options. Button − Selection / performance of a function via mouse click. 4-10 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.1.3 OPERATION Purpose Carl Zeiss Convention for the Text in this Manual All the originally used terms of the software interface, e.g. − names of windows, − panels, − input boxes, − list / selection boxes, − check boxes, − menu items, − names of buttons and − keyboard keys, are displayed in bold letters to allow easier identification. 4.1.4 Backup System backup − A complete backup is contained on the enclosed backup CD-ROM. User files backup The following user-generated files need to be included in a backup procedure (keep directory structure): − − − − − − − Image database files: *.mdb (but not system_configuration_*.mdb) LSM Image files: *.lsm Exported images: *.* (*.Tiff, *.LSM-Tiff, *.BMP, …) Palette files: AIM \ Palette \ *.lut Filter files: AIM \ Filter \ *.krn Pibhole (aperture setting files: AIM \ PH*.pos Log files: AIM \ *.log The following files generated during the system integration should also be included in a backup procedure: − − − − 4.1.5 Parameter file for pinhole (aperture) setting: AIM \ *.set Parameter file after pinhole (aperture) adjustment: AIM \ *.adj Scanner files: AIM \ bin \ *.bin Microscope stand database: AIM \ database \ system_configuration_*.mdb Software Operation The LSM 5 software can be operated using the mouse, the PC keyboard, or both. The operation of the mouse and the keyboard is identical to that of the Microsoft ® WINDOWS operating system and is therefore not dealt with in detail in this manual. If required, see the Microsoft manual or online help for relevant information. 03/06 B 45-0019 e 4-11 OPERATION Switching on the System Carl Zeiss 4.2 LSM 5 LIVE LSM 5 LIVE DuoScan Switching on the System The LSM 5 LIVE system is equipped with a main power switch located at the front of the System Electronic Rack and two further switches labeled System/PC and Components. The main switch has to be set to ON to be able to switch on and off the system using the System/PC and Components switches. The main switch can be used to switch off the complete system with one switch only. The electronics to run the computer and the microscope are switched on with the System/PC switch. The lasers and the scan head are switched on with the Components switch. These switches are also accessible via the remote power control switch (Fig. 4-1). Note that the full performance and correct adjustment of all internal elements is achieved after a warm-up time of 40 min.! • When set to ON the REMOTE CONTROL switch labeled System/PC provides power to the microscope and the computer. This allows to use the microscope and the computer without running the LSM Software. The drives for floppy discs and CD/DVD of the computer must not contain any data storage item. • To switch on the system completely put the Components switch also to ON. Now the complete system is ready to be initialized with the LSM Software. • After switching on the computer type in the user name and password to log on to the computer. Fig. 4-1 REMOTE CONTROL switch • After entries, confirm by clicking the OK button or Enter. − The WINDOWS XP operating system desktop appears on the screen, showing a number of icons. Change Filters icon Change Filters The Change Filters tool is used to update the filter data in the software after a change of filters in the reflector turret. See printed manual CHAPTER TOOLS. Stand Select icon Stand Select The Stand Select tool permits a new or updated database to be assigned to the LSM 5 software program. This function should preferably be performed by authorized service personnel. See printed manual CHAPTER TOOLS. LSM 5 LIVE icon LSM 5 Live 4-12 Start the LSM 5 software program for the LSM 5 LIVE laser scanning microscope. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.2.1 OPERATION Switching on the System Carl Zeiss Switching on the HBO 100 • If the HBO 100 is required, switch it on via the toggle switch of the HBO 100 power supply. 4.2.2 Starting the LSM 5 Program The LSM 5 software program can be operated in two different modes (with or without connected instrument system). − In the on-line mode, the entire program package (image recording and analysis) is available, while only a part of the software functions (image analysis only of already stored images) Fig. 4-2 Starting the LSM 5 software − In the off-line mode no hardware functions are available. Of course, the off-line mode can also be started when the instrument system is connected. In that case, it is not necessary that the lasers and the microscope are switched on. • Double-click on the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5 software program (Fig. 4-2). − The LSM 5 LIVE Switchboard window appears on the screen (Fig. 4-3). Fig. 4-3 03/06 LSM 5 LIVE Switchboard menu B 45-0019 e 4-13 OPERATION Switching on the System Carl Zeiss Online Mode LSM 5 LIVE LSM 5 LIVE DuoScan Clicking on this button activates the complete LSM hardware (on-line mode). Please note that the Online Mode button must be activated before clicking the Start button. Otherwise, the hardware can not be controlled by the LSM 5 software. Offline Mode This item allows you to process and analyze previously acquired images with the LSM 5 software. In this mode, control of the hardware (laser module ...) is not possible (off-line mode). Start Use of this button starts the software system. After the start, instrument initialization is performed and can be monitored in the Initialization window and interrupted with a click on the Cancel button, if required. Fig. 4-4 OFF LINE Initialization window Depending on the selected option (Online Mode Images or Offline Mode), initialization is performed in the offline or online mode. Some printers (for example KODAK Thermo Printer) will produce an error message "hard key not found" in case the printer is not switched on. Remedy: turn on the printer before starting the LSM 5 software. Don’t switch off the KODAK printer during the scanning process. 4-14 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.3 OPERATION Main Menu Carl Zeiss Main Menu The major functions can be selected in the Main menu either via the pull-down menus in the menu bar or via the Main menu toolbar which can be displayed or removed as required. Further subordinate toolbars are available below this toolbar, depending on which button has just been pressed (File, Acquire, etc.). In the standard setting of the LSM 5 software, the toolbars are automatically displayed after clicking Start. However, since the LSM 5 software is operated more conveniently with the help of the toolbars, only this method of function activation will be described in the following. • Click on the Start button in the LSM 5 LIVE Switchboard window. − The LSM 5 LIVE Main menu appears on the screen. The Acquire button is active automatically, and the submenus selectable in it are shown in the second (bottom) toolbar. Fig. 4-5 03/06 LSM 5 LIVE Main menu File button Open, save, import and export of image data. Printing individual or several images on one page. Ending (Exit) the Main menu. Acquire button Calling up and setting the necessary operating parameters. During the preparation for and execution of laser scan image acquisition, this menu item is used as the working dialog between the computer and the microscope. Process button Used for processing of acquired images. 3D View button Used for three-dimensional reconstruction. B 45-0019 e 4-15 OPERATION Main Menu Carl Zeiss Macro button Options button LSM 5 LIVE LSM 5 LIVE DuoScan Makes it possible for the user to store frequently used processes (Macro recorder) and to run them automatically (Macro play). It is possible to write new macros or to edit existing ones. For custom-configuration of software and hardware options. This menu item enables access to the coloring table. In the Settings for User window you can specify essential operating modes and informative help, organized by tabs, which have an effect on the user interface. Maintain button 4-16 Service mode for adjustment and setting of other parameters (e.g. objectives). B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4 OPERATION File Menu Carl Zeiss File Menu The functions of the File menu permit images and the relevant information to be managed and handled completely in a database system. You can also create your own databases. The databases allow images to be stored, loaded and deleted. The additional functions Import and Export permit images from other systems to be made available to the LSM 5 software, or the export of images to other software packages. The Print function allows individual or several images to be arranged on a print page for printout. The Main menu can be ended via the Exit function. In the pull-down Main menu File two additional functions not displayed in the Main Menu Toolbar are available. Clicking Recent Database opens up a choice of the last 5 databases opened by the program. Clicking the appropriate line will open up that database. A similar menu exists for Recent Image Files. • In the Main menu toolbar, click on File. − This opens another, subordinate toolbar in the Main menu. Fig. 4-6 File menu Fig. 4-7 Subordinate File menu toolbar 03/06 B 45-0019 e 4-17 OPERATION File Menu Carl Zeiss 4.4.1 LSM 5 LIVE LSM 5 LIVE DuoScan Create New Image Database The New function permits the creation of a new image database. • Click on the New button in the subordinate toolbar of the Main menu. File − This opens the Create New Database window for the selection of drives, directories and subdirectories. Fig. 4-8 • Enter the name of the image database you want to create in the File name text box, e. g. Convallaria. Create New Database window • If you want to create the image database in a certain folder (drive / directory), click on the arrow button next to the Create in box. − This opens a drop-down list box showing all folders available for selection. To move up one layer of folders, click on the • If you want to create a new folder, click on the button. Cancel allows you to stop the process. button. • Click on the Create button. − This creates the new image database in the selected drive and directory. − The database files of the LSM 5 software have the filename extension *.mdb. The Convallaria.mdb window appears, presenting the opened image database with 0 of 0 image entries (see Image Database window, page 4-19). The new image database can be used to store an acquired or processed image (see Saving an Image, page 4-29). The start settings and the extent of the parameters displayed in the image database window can be changed as required via the Settings function in the Options subordinate toolbar (see Settings Function, page 4-200). 4-18 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.2 OPERATION File Menu Carl Zeiss Open Existing Image Database The Open function allows one or several databases to be opened. The images stored in the database(s) are displayed in thumbnail form; they can be selected and loaded into the Image Display window (see page 4-224). • Click on the Open button in the File subordinate toolbar of the Main menu. − This opens the Open Database window for selection of image databases. • If you want to load an image database from another folder (drive / directory), click on the arrow button to the right of the Look in box. Fig. 4-9 Open Database window − This opens a drop-down list box in which you can select from all available folders. The window displays the various image databases with the file extension *.mdb. • Open the image database by a double click on the respective key icon (e.g. Convallaria.mdb), or click on the name of the image database for selection and open it by clicking on the Open button. − This opens the image database window, e.g. Convallaria.mdb - AIM, in which you can select from a variety of options. • Click on the selection. 4.4.3 button in the title bar of the Database window to close this window and make no Image Database Window The Image Database window allows you to choose one of three different display modes: − Form − Gallery − Table To choose the required mode, activate the relevant button on the right-hand side of the Image Database window. Loading of images into the Image Display window is possible in every display mode. The buttons on the right have the following functions: 03/06 Form button Show image database in form display mode. Gallery button Show image database in gallery display mode. Table button Show image database in table display mode. B 45-0019 e 4-19 OPERATION File Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Load button Load image and parameter from image database to Image Display window. Subset button Load image and parameter with size reduction from image database to Image Display window. Reuse button Reuse scan parameters of the selected image without loading the image. Refresh button Refresh Image Database window. Copy button Copy selected images to clipboard. Paste button Paste images from clipboard into image database. Filter button Select or edit search filter from image in the image database. On Filter button Switch search filter on or off. Delete button Delete selected images from the image database. The status line, which displays the following current parameters, is at the bottom of the Image Database window: Total: … Display of storage volume of the entire image database Selected: … Display of storage volume of the selected image(s) Current: … Display of storage volume of the current image Clipboard: … Display of storage volume of the image(s) contained in the clipboard 4-20 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.3.1 OPERATION File Menu Carl Zeiss Form Display Mode When a image database is opened, the Form display mode is used, if no other settings were made under Settings in the Options subordinate toolbar. Fig. 4-10 Image Database window (Form display mode) In the Options menu in the function Settings it is possible to define − the start mode of the image database (Form, Gallery, Table) − the Recordset displayed (first, last, middle) and − the parameters shown. The number of the image currently displayed from a set of images is indicated in the Recordset text box. • From the image database you can display images using the recording slider, or in one of the following ways: show the next image show the previous image show the last image of the image database show the first image of the image database 03/06 B 45-0019 e 4-21 Carl Zeiss OPERATION File Menu LSM 5 LIVE LSM 5 LIVE DuoScan The currently selected image is displayed as thumbnail in the Image Display window on the right. In the case of Z Stacks or time series, the Slice slider appears on the screen beside the Image Display window. • You can browse through the series by dragging the Slice slider using the mouse. • Click on the Load button to open the selected image. A double-click on the Image Display window of the database will also load the selected image. For a description of the toolbars Select and Display see Display and Analysis of Images, page 4-224. The name of the image is displayed in the Name input box of the Image Database window. • If you want to change the name, click on the input box and enter the new name directly via the keyboard. • The Description and Notes input boxes allow you to subsequently add descriptions or special notes on the recorded image via the keyboard. Fig. 4-11 Opened image displayed in the Image Display window The acquisition parameter settings of the image are displayed in the Acquisition panel. Changes to an original scan image are automatically recorded in the Processing panel under History. If, for example, the image was added to the database via the clipboard, the entry Imported file will be shown under History. Under Image Size, the size of the image in pixels for XY(Z) and the number of used channels are displayed. 4-22 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.3.2 OPERATION File Menu Carl Zeiss Gallery Display Mode • Click on the Gallery button. All images of the image database, e.g. Convallaria.mdb, (image series) are shown in a tiled arrangement of thumbnails on the screen. Fig. 4-12 Database window (Gallery display mode) In the Options menu in the function Settings it is possible to define − the start mode of the image database (Form, Gallery, Table) − the recordset displayed (first, last, middle) and − the parameters shown. • To select one of the images of the database for normal-size presentation, double-click on the desired image. The same can be achieved by clicking on the desired image in the gallery and then clicking on the Load button. • To select several single images press & hold down the Ctrl-key and select each desired image by a click of the mouse. If several images have been selected, they will all be opened and displayed one after the other. • To select a number of consecutive images press & hold down the Shift-key, click on the first and the last image to be selected. All the images between these two will also be included in the selection. If several images have been selected, they will all be opened and displayed one after the other. Selected images are highlighted in blue. Furthermore, the current image selected last receives a patterned frame. 03/06 B 45-0019 e 4-23 OPERATION File Menu Carl Zeiss 4.4.3.3 LSM 5 LIVE LSM 5 LIVE DuoScan Table Display Mode • Click on the Table button. − All images of the image database, e.g. Convallaria.mdb, (image series) are shown in Table display mode on the screen. Fig. 4-13 Database window (Table display mode) In the Options menu in the function Settings it is possible to define − the start mode of the image database (Form, Gallery, Table) − the recordset displayed (first, last, middle) and − the parameters shown. • To select one of the images of the database for normal-size presentation, double-click on the desired line. The same can be achieved by clicking on the desired image in the table and then clicking on the Load button. If several images have been selected, they will all be opened and displayed one after the other. The selection of several images is performed in the same way as in the Gallery mode, i.e. by pressing the Shift and Ctrl keys. 4.4.3.4 Load an Image into the Image Display Window • Click on the Load button to load the selected images into the Image Display window or double click on the appropriate image in the database window. 4-24 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.3.5 OPERATION File Menu Carl Zeiss Load Image with Reduced Resolution (Subset) The Subset function allows images to be loaded with reduced resolution. For this purpose, the image pixels in XY(Z) are reduced. It is also possible to reduce the number of slices (in stacks and time series). • Click on the Subset button to open the Load with reduction in size window. • Enter one value each for n under Pixel (x), Pixel (y), Pixel (z) and Stack (Time) in the Load every nth panel. • If required, turn on the Fig. 4-14 Load with reduction in size window Load 12 bit as 8 bit check box. • Click on the Load button to load the selected images with reduction in size, time slices and stack slices. • Use Cancel to exit the window without any selection. 4.4.3.6 Update Image Database Display (Refresh) • Click on the Refresh button to update the Image Database window. 4.4.3.7 Copy / Paste Image(s) to the CLipboard • Select the image(s) to be copied. You can use the Shift and Ctrl keys for multiple selection. • Click on the Copy button. − The image(s) is(are) copied to the clipboard and can be inserted in either the same or another database or in other software packages. • Click on the Paste button to insert the image in the current database. Identical to the WINDOWS function "Drag & Drop", one or several images can be copied or moved from one database to another. The Form mode allows only one image to be copied or moved. The Gallery and Table modes permit several images to be copied or moved simultaneously by multiple selection (keeping the Shift key pressed on clicking). • Open the relevant databases and position both windows side to side. • Select the required images (multiple selection by keeping the Shift key pressed) from one database. • Click on a selected image and keep the mouse button pressed, move the mouse button to the window of the other database (a small rectangular appears near the mouse button) and release the mouse button again (Drag & Drop). 03/06 B 45-0019 e 4-25 OPERATION File Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The images are now being moved to the other image database, i.e. they are deleted from the first image database and are then only available in the second image database. • If the images shall only be copied, also press the Ctrl key during the Drag & Drop procedure (in addition to the rectangular, a "+" sign will also appear near the mouse button). The images will then be available in both image databases. 4.4.3.8 Display Images with Certain Features (Filter) The Filter function permits the display of the database to be modified in such a way that only images with certain features are displayed. This requires the definition and following activation of a relevant filter. Defined filters can be stored, reloaded and also deleted. Fig. 4-15 Filter window Edit panel The following features can be used as filter functions under Field: Name Words or row of letters from the name of the image Description Words or row of letters from the description of the image Scan Mode Scan Mode in which the image was created: Stack or Plane Date / Time Date / Time of image acquisition # Planes (z) Pixel size of the image in the Z-direction (e.g.: 10) # Lines (y) Pixel size of the image in the Y-direction (e.g.: 512) Samples (x) Pixel size of the image in the X-direction (e.g.: 512) Z-Step Distance of Z Slices in a Z Stack in µm User Name of the user as entered in the WINDOWS NT login Time series Selection of time series • Open the Field list box and select the filter feature (e.g.: Scan Mode). 4-26 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION File Menu Carl Zeiss The following operator signs can be activated under Operator: = equals > larger < smaller >= larger, equals <= smaller, equals <> smaller, larger • Select the relevant operator sign (e.g.: =) from the Operator list box. The relevant value or a combination of characters for the filter function (Field) is entered under Value via the keyboard: • Enter the relevant text or value (e.g.: Stack). • Click on Add. The defined filter feature is displayed in the work box of the Edit panel and is therefore activated (e.g.: Scan Mode = stack). If a further filter feature is to be linked with the already defined one, proceed as follows: • Activate the relevant entries under Field and Operator and enter a value or text (e.g.: Name = Convallaria) under Value. If groups of letters shall be searched, the * sign can be entered for undefined letters (e.g.: if you search for the letter row Conv, enter Conv*). • Activate the required link type And, Or or Not with a click of the mouse (e.g.: And). • Click on the Add button. The created filter feature is added to the work box of the Edit panel (e.g.: AND Name = Convallaria). The Modify button enables you to edit an already defined filter feature: • Activate the required feature on the work box. • Make the necessary changes under Field, Operator and Value. Select the link type And, Or or Not. • Click on Modify. The filter feature will be changed accordingly. The Delete button enables you to delete a defined filter feature: • Activate the required feature in the work box. • Click on Delete. The filter feature will be deleted from the work box. • Clicking on OK will activate the filter (the entire set of filter features) displayed in the work box and close the Filter window. On Filter is activated right on in the Database window and the filter function will be performed. Only those images which fulfill with the defined filter features will then be displayed. The procedure is interrupted via Cancel. 03/06 B 45-0019 e 4-27 OPERATION File Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan If required, the filter features displayed in the work box can be stored. • Click on the Add to List button. The Add to List window will be opened. • Enter a name for the filter and click on OK. The filter will be included in the Filter List panel. Fig. 4-16 Add Filter to List window Filter List panel All the stored filters are displayed in the Filter List panel and can be activated any time at a click of the mouse. • Click on the name of the required filter. The linked filter features will be displayed in the work box. Fig. 4-17 Filter List panel Filters which are no longer needed can be deleted. • Click on the filter to be deleted in the Filter List panel. • Click on Remove from List. The filter will be removed. 4.4.3.9 On Filter Function The On Filter function is a toggle switch to activate or deactivate selected filter settings. 4.4.3.10 Delete Function • Select the images to be deleted from the image database. • Click on the Delete button. Confirm the safety inquiry then displayed by pressing OK. − The images and the acquisition parameters will be removed from the image database. 4-28 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.4 OPERATION File Menu Carl Zeiss Save an Image to the Image Database The Save function allows to store an image together with the acquisition parameters (and processing information) to be stored in an image database. In the Options menu in the function Settings it is possible to define an Autosave function. When Autosave is off, the Save dialogue is the Save As dialogue. Proceed as follows to save an acquired or an edited / processed image: • Click on the Save or Save As button in the File subordinate toolbar of the Main menu. − The Save Image and Parameter As window appears on the screen. Save Stores a newly created or changed image. Newly created images must be given a name and assigned to an existing or new database. Save As Stores a previously stored and called up image under a different name. If images are called up and stored again, the original data and time display will be retained. Clicking on either of these buttons opens the Save As window to create and open an image database. check box is activated, the images are stored in a compressed When the Compress Files form. • If necessary, enter a description of the image or comments on it in the appropriate text boxes. • The default display in the User text box is the name of the logged-on user. If you want, you can enter a different user name for the current image. • Click on the Open MDB button if you want to open an existing image database in which you want to save the current image. Click on the New MDB button if you want to create a new database to save the current image. Fig. 4-18 Save Image and Parameter As window • Enter the name of the image in the Name text box, e.g. Conv-7. 03/06 B 45-0019 e 4-29 OPERATION File Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click on the New MDB button. − This opens the Create New Database window in which you can create a new image database, e. g. Projections.mdb (see Create New Image Database, page 4-18). − After creating the new database Projections.mdb - AIM window appears. Fig. 4-19 4-30 Save Image and Parameter As window and Create New Database window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-20 OPERATION File Menu Carl Zeiss Database window • Click on the OK button in the Save Image and Parameter As window. − The Projections.mdb - AIM window now shows the saved image. − The Recordset box indicates the current number of the image in the image series contained in this database. • In the Description text box you can enter, for example, the configuration of the image. • In the Notes text box you can enter further information about the image content. 03/06 B 45-0019 e 4-31 OPERATION File Menu Carl Zeiss 4.4.5 LSM 5 LIVE LSM 5 LIVE DuoScan Import of Images The Import function enables the import of externally created images into the Image Display window and the image database of the LSM 5 software. • Click on the Import button in the File subordinate toolbar of the Main menu. − This opens the Import Images window. Fig. 4-21 Import Images window • Select the data medium and the directory where the relevant image is contained in the Look in selection box. • Select the image file by clicking on it. − The selected image will be shown for checking in the Image Display window (on the left) together with the relevant details (size, channels, storage volume). • Select the image type (Single Image or Image Series) in the Image type selection box. • Click on Open. − The image is displayed in a new Image Display window. All the usual image and movie formats (e.g. .tif, .jpg, .bmp, .pcx, .avi, .mov etc.) are supported. When importing series, please make sure to select the first image for the representation of the entire series and to select the Image Series option under Image type. • Finally, save the image in the desired image database via the Save As function. • In Processing History this file is marked as imported file. 4-32 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.4.6 OPERATION File Menu Carl Zeiss Export of Images The Export function allows the export of acquired images and images loaded from the image database. • Select the image to be exported. • Click on the Export button in the File subordinate toolbar of the Main menu. − This opens the Export Images and Data window. • Under Save in, select the data medium and the directory to which the image is to be exported. • Enter a name for the image under File name. • Select the image format into which the image is to be exported under Image type (Single Image with raw data, Contents of the Image Display window, Full resolution). • Chose a compression level. For some file formats lossless compression or various other compression levels are available. The degree of losses for the image quality are listed according to the type of compression. • Click on the Save button. − The image is stored on the relevant data medium / directory. All the usual image and movie formats (e.g. .tif, .jpg, .bmp, .pcx, .avi, .mov etc.) are supported. When stacks or time series are exported, each frame is stored as an individual image. Fig. 4-22 03/06 Export Images and Data window B 45-0019 e 4-33 OPERATION File Menu Carl Zeiss 4.4.7 LSM 5 LIVE LSM 5 LIVE DuoScan Multi Print This function permits you to arrange several images on one print page and to print them out together. • Click on the Multi Print button in the File subordinate toolbar of the Main menu. − This opens the Print - AIM window. The main area of the Print – AIM window is used for the display of the print page in the selected paper orientation and for the arrangement of the images to be printed. The Print toolbar with the following buttons is displayed on the right-hand side of the window: Fig. 4-23 4.4.7.1 Print - AIM window Paste Paste from clipboard to sheet. Delete Delete marked image. Print Start printing. Setup Printer setup. Landsc. Landscape paper orientation. Portrait Portrait paper orientation. Zoom slider Zoom function for page preview. Overlay Toolbar The following functions can be performed on activation of the buttons in the Overlay toolbar (left-hand side): Arrow (selection) button: Activation of the mouse button for selection, resizing or movement of an overlay element in the Image Display window. Resizing: Click on the handle and hold down the mouse button, drag the handle, release the mouse button. Movement: Click on the line and hold down the mouse button, move the entire element, release the mouse button. Line button: Creation of a straight line in the Image Display window. Click and hold down the mouse button, draw a line in any required direction, release the mouse button to end the procedure. Rectangle button: Creation of a rectangle in the Image Display window. Click and hold down the mouse button, draw a rectangle in any required direction, release the mouse button to end the procedure. 4-34 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION File Menu Carl Zeiss Closed polyline button: Creation of a closed polyline figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Open polyline button: Creation of an open polyline figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button ends the procedure. Ellipse button: Creation of an ellipse in the Image Display window. The first click sets the center point, the displayed line permits the determination of the first dimension, the second click sets the first dimension, the second dimension and rotation direction can then be determined, the third click sets the second dimension and direction and ends the procedure. Closed free-shape curve button: Creation of a closed Bezier figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Open free-shape curve button: Creation of an open Bezier figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Circle button: Creation of a circle in the Image Display window. Clicking and holding down the mouse button sets the center point, drag the diameter and release the mouse button to end the procedure. Line with arrow button: Creation of a line with arrow in the Image Display window. Click and hold down the mouse button, drag the line in any required direction, release the mouse button to end the procedure. A (Text) button: Creation of a text box in the Image Display window. After clicking on A, the Text window will be displayed, and text can be entered via the keyboard. The Font ... button enables you to select the font style and size in the Font window. The entered text will be displayed in the left upper corner of the Image Display window after clicking on OK and can be moved to the required position using the mouse. The Text window can also be activated with a double-click on a created text box, and the entered text can be edited subsequently. Insert opens up a further window which allows you to annotate coordinates, time and Z-position with either automatic or user definable units and precision. This annotation is updated during image acquisition and can be exported with the image. The annotation can be stamped into already existing images. 03/06 B 45-0019 e 4-35 OPERATION File Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Recycle bin button: All the overlay elements and dimensions dragged to the scanned image are deleted. If one overlay element was marked before, this element is now deleted from the scanned image. Line button: This button allows you to determine the line thickness of the area outline. Color button: After clicking the Color button, the Color selection box will be opened. The colors displayed in the Color selection box can be assigned to the overlay elements with a click of the mouse. The currently selected color is displayed in the Color button. A selected color is automatically assigned to the currently selected overlay element and then to all the elements created afterwards. 4.4.7.2 Printing Images To print several images on one page, proceed as follows: • Use the Overlay functions to additionally illustrate the graphics and images to be printed. • Select the paper orientation by clicking on the Landsc. or Portrait button. • Open the image to be printed or select it from the relevant image database. • Click on the Copy button. The image is copied to the clipboard. • In the Print - AIM window, click on the Paste button. The copied image appears in the work area of the Print - AIM window. You can click on it with the mouse and move it to any position on the print page or you can adapt the image size. • Proceed in the same way with all other images you want to print. • Finally, arrange all images on the print page as required. • Click on the Print button to start the printout. • Close the Print - AIM window by clicking on the 4.4.8 button. Exit • Make sure to save all required images in the image database or export them. • Close all open windows of the LSM program by clicking on the closing icon of each window. in the top right corner • Click on the Exit button in the File subordinate toolbar of the Main menu. − The LSM 5 LIVE Main menu will be closed and the LSM 5 LIVE Switchboard menu appears on the screen. 4-36 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5 OPERATION Acquire Menu Carl Zeiss Acquire Menu • In the Main menu toolbar, click on Acquire. − This opens another, subordinate toolbar in the Main menu. Fig. 4-24 Acquire menu For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the subordinate toolbar in the following order: • Conventional microscope setting. • Laser setting. • Configuring the optical system for the Scanning Mode. • Setting of scan parameters. • EditROI permits up to 99 regions within a frame to be defined and analyzed (option). • TimeSeries permits user-specific time series to be selected for the scan procedure. • Upon selecting Stage you can set the focus (Z coordinate) and the Z step size between successive slices. If the optional, motorized X/Y-stage is connected, the X and Y-positions of the sample can also be selected. • The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in the binocular tube of the microscope (VIS for viewing, TV for camera observation, LSM for laser operation with monitor observation). For the scanning process, the LSM button in the toolbar subordinate to the Acquire item must be activated. 03/06 B 45-0019 e 4-37 OPERATION Acquire Menu Carl Zeiss 4.5.1 LSM 5 LIVE LSM 5 LIVE DuoScan Laser Control The Laser Control panel shows the types, excitation wavelengths and operating status of all lasers available. The subordinate laser settings panel shows the relevant and currently set Maximum Power, Wavelength, Status and Tube Current (only Sapphire 488) of the current laser. The buttons On, Off and Standby permit the current laser to be set in the required status. Fig. 4-25 The name of the selected laser (Toptica ..., etc.) is displayed in the headline of this setting panel for checking. Laser Control window 4.5.1.1 Switching on the Enterprise UV Laser • If the UV laser is required, switch it on via the toggle switch (4-26/1) of the power supply. − It will be ready for operation after a few seconds. Fig. 4-26 Power supply of UV-laser 4.5.1.2 Opening / Closing the Laser Control window • Click on the Laser button in the Acquire subordinate toolbar. − This opens the Laser Control window, which shows all lasers connected to the system. When the setting of the required lasers has been finished, the Laser Control window can be closed again. • Click on the Close button to close the Laser Control window. − The Laser Control window will be closed. 4-38 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss 4.5.1.3 Function Description Lasers panel (upper) List of available lasers, including the display of relevant wavelengths and switching status. Selection of the laser to be switched on / off and setting of the laser output is performed in the subordinate setting panel. Laser settings panel (lower) Switch on / off the required laser or set Standby operation. Display Maximum Power, Wavelength and Status of the relevant laser. 4.5.1.4 Settings • Click on the desired laser on the (upper) Lasers panel. − This highlights the selected laser. On the lower panel of the Laser Control window, activate the laser as follows: This applies to the 405 Diode laser (405 nm): • Click on the On button directly (not on Standby) This applies to the Toptica and the Sapphire lasers (635 / 488 nm): • Click on the Standby button. − Wait for the laser to heat up, until the Status ready - Standby message appears (approx. two minutes). • Click on the On button. − Status ready - On appears. This applies to Compass 315M laser (532 nm): • After selecting the laser, click on the On button. − The required laser for image acquisition is now available. After switching on the lasers in the laser control window and their status ready the system can be used for imaging. However, for quantitative imaging it is recommended to let the system warm up for 40 minutes. This applies to the Coherent UV-laser 653 II (Enterprise) and the Ar-multiline laser • Click on the Standby button. − Wait for the laser to heat up, until the Status ready - Standby message appears (approx. two minutes). • Click on the On button. − Status ready - On appears. 03/06 B 45-0019 e 4-39 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Use the Output [%] slider to set the laser power which is ideal for the measurement job. Thus, the laser needed for image acquisition is available. Argon: Set output between 25 and 100 % of the maximum tube current. Optimum operation is at 8 A (lowest laser noise). However, the laser life is reduced if the laser is constantly operated at 8 A. Therefore, 8 A should be used only if this is absolutely necessary. Enterprise: Set output between 50 and 100 % of the maximum tube current. Optimum operation is at 20 A (Tube Current; lowest noise). However, the laser life is reduced if the laser is constantly operated at 20 A. Therefore, 20 A should be used only if this is absolutely necessary. To switch on the Enterprise laser, proceed as follows: • The internal water cooling LP 5 is running. • Start the PC, wait until NT system is booted. • Switch on the power supply of the Enterprise laser, power potentiometer turned to maximum. • Start the LSM 5 software. Please bear in mind that a cooling phase of at least 5 minutes is required between switching off of the laser via the software and switching off of the entire system via the REMOTE CONTROL main switch or the Power Supply switch of the Enterprise UV laser. If the LSM 5 software is already running and you want to use the UV laser, do the following: • Close the LSM 5 software. • Switch on the power supply of the Enterprise, power potentiometer turned to maximum. • Start the LSM 5 software again. 4.5.2 Microscope Control The Microscope Control (Micro button) window permits motorized functions (objective and reflector change, condensor, filter and diaphragm settings) and the illumination mode (transmitted or reflected light) of the connected microscope to be controlled via the software. Without any difference to software control, these microscope functions can also be operated directly on the stand via the relevant controls. In that case, any changes are recorded by the software and displayed in the relevant windows / panels. 4.5.2.1 Open the Microscope Control Window • Click on the Micro button. − This opens the Microscope Control window on the screen. 4-40 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss After conclusion of the conventional setting of the connected microscope, the Microscope Control window can be closed again. • Click on the Close button in the Microscope Control window. − The Microscope Control window will be closed. 4.5.2.2 Microscope Control Window for Axio Imager.Z1 • Click on the Micro button in the main frame. • The microscope window opens in the last saved configuration. • By clicking on the More / Less button the microscope window is displayed with or without detailed microscope beampath panel. Reflected Light button The shutter is switched on and off. When using the X-Cite 120 additional controls are available. See below for description. Reflector button Push and click reflector cube can be selected via graphical pop-up menu. Objective button Objective can be selected via graphical pop-up menu. Condensor button Numerical aperture of the condensor is set via input box or slider. Turret position selected from graphical pop-up menu (only for motorized condensors). By clicking on the Close button the value is stored and the Condensor frame is closed. Field Stop button Opening of luminous-field diaphragm (transmitted and reflected light) can be set via input box or slider. By clicking on the Close button the value is stored and the Field Stop frame is closed. Filter button Transmission values for attenuation filter (transmitted and reflected light) is set via input box or slider for the front or rear filter wheel in accordance with the available filter steps. By clicking on the Close button the value is stored and the Filter frame is closed. Aperture Diaphragm button Diaphragm opening is set via input box or slider. By clicking on the Close button the value is stored and the Aperture Diaphragm frame is closed. Transmitted Light button Transmitted light is switched on / off via ON button in the Transmitted Light frame, setting of light intensity can be varied via input box or slider. 3200 K color temperature for photo documentation can be switched on via 3200 K button in the Transmitted Light frame. By clicking on the Close button the Transmitted Light frame is closed. 03/06 B 45-0019 e 4-41 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Recording of microscope settings The upper part of the Axio Imager Control window shows the recording functionality of microscope configurations. Complete microscope created and applied. configurations can be The Store button permits existing microscope configurations to be stored under any name. The Apply button permits existing microscope configurations to be loaded. stored The Delete button permits existing microscope configurations to be deleted. The Assign button permits the assignment of a microscope configuration to a button. Load a microscope configuration An existing microscope configuration can be loaded as follows: • Click on the arrow button. Fig. 4-27 − This opens a list box of all stored microscope configurations. Axio Imager.Z1 Control window • Browse through the microscope configurations by clicking, or use the scroll bar at the side of the list box. • Click on the desired microscope configuration. − The selected microscope configuration is shown in the first line of the Microscope Configurations list box. • Click on the Apply button. • Click on the Close button to close the microscope window. Only those microscope settings which are encoded and motorized can be reloaded. Store a Microscope Configuration A newly created or changed microscope configuration can be stored under a new name as follows: • Enter the desired name in the first line of the microscope setting list box. • Click on the Store button. • The actual configuration with the chosen name is added to the microscope settings list. • Click on the Close button to close the microscope window. 4-42 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Delete a Microscope Configuration A no longer required microscope configuration can be deleted as follows: • Select the microscope configuration to be deleted from the microscope configuration list box. • Click on the Delete button. • Click on the Close button to close the microscope window. Assignment of a microscope configuration to a button A microscope configuration can be assigned to a button as follows: • Click on the Assign button. • This opens the Assign-Microscope-SettingsTo-Button window. Fig. 4-28 • Click on the arrow in the Button list box and select a button out of the list. Assign-Microscope-Settings-To-Button window With increasing numbers the buttons are arranged from the upper to the lower row from lefthand side to right-hand side. • Click on the arrow in the Settings list box and select a microscope configuration. • Click on the Apply button. A new button with the name of the selected microscope configuration has been created. • Click on the Close button to close the Assign-Microscope-Settings-To-Button window. • Click on the Close button to close the microscope window. Conventional setting of the microscope Axio Imager • Click on the VIS button in the Acquire subordinate toolbar. • Place specimen on microscope stage. − The cover slip must be facing up. • Click on the Micro button to open the Microscope Control window. • Via the Objective button, select the required objective as follows: − Open the graphical pop-up menu by clicking on the Objective button. − Click on the objective you want to select. − The selected objective will automatically move into the beam path. • Use the focusing drive (4-29/5) to focus the required object plane. • Select specimen detail by moving the stage in X and Y using the XY stage fine motion control (4-29/6 and 7). 03/06 B 45-0019 e 4-43 OPERATION Acquire Menu Carl Zeiss Fig. 4-29 LSM 5 LIVE LSM 5 LIVE DuoScan LSM 5 LIVE with Axio Imager.Z1 Microscope settings on Axio Imager for transmitted-light observation • Set the reflector turret position to None and click on the On button for transmitted light. • Control the brightness of the halogen lamp with the potentiometer (4-29/4) or the Intensity % slider in the Transmitted Light panel. • Set the required transmission value of the gray filters in the Filter frame. • Set the condensor and the luminous-field diaphragm for KÖHLER illumination. With Transmitted Light activated (On), the halogen lamp is automatically occluded in the laser scanning mode. 4-44 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Microscope settings on Axio Imager for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 W power supply with switch (4-29/1). • Click on the RL reflected light button. The shutter opens. To avoid excessive bleaching of biological samples, expose the specimen to the minimum possible irradiation, i.e. keep the irradiation time as short as possible. For this, close the field stop to the necessary field of illumination and use additional filters if available. • By clicking on the reflector turret button, select the reflector module (filter sets) to suit the type of fluorescence excitation. Proceed as follows: • Click on the reflector turret button. • Click on the desired reflector module. − The reflector turret moves the selected reflector module into the beam path. The FITC filter set consists of an excitation filter for the 450 - 490 nm spectral range, an FT color splitter for 510 nm and an LP long pass filter, which passes emission light wavelengths greater than 510 nm (FSET 09 = FITC, FSET 15 = Rhodamine, FSET 01 = DAPI). Other filter sets: DAPI: BP 365 FSET01 FT 395 LP 397 Rhodamin: BP 546 FSET15 FT 580 LP 590 The filter sets described in this section are included in the standard configuration, but other sets are available on request. If the X-Cite 120 fiber coupled lamp for reflected-light illumination is used a further dialog is opened when clicking the Reflected light button. The lamp internal shutter and the attenuation function can be controlled via the LSM Software. The lamp has to be switched on first using the main switch on the lamp housing. Fig. 4-30 03/06 B 45-0019 e X-Cite 120 control panel 4-45 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Remote Enables the attenuation control via LSM Software. This is also enabled when clicking the ON button. ON Opens the shutter in the reflected light path and the internal shutter of the lamp. Both shutters are closed automatically when switching to LSM mode. Shutter Opens and closes the shutter in the reflected light path independent of the status of the internal lamp shutter and the attenuation. Intensity Sets the light via input box or slider. Lamp Hours Indicates the total time the lamp has been in use. The aperture setting on the condensor of the Axio Imager.Z1 is performed in fixed steps. 4.5.2.3 Microscope Control Window for Axiovert 200 M Transmitted Light button Transmitted light is switched on / off via ON button in the Transmitted Light frame, setting of light intensity can be varied via input box or slider. 3200 K color temperature for photo documentation can be switched on via 3200 K button in the Transmitted Light frame. The transmission light control potentiometer on the stand is disabled via the Remote button. By clicking on the Close button the Transmitted Light frame is closed. Condensor button Numerical aperture of the condensor is set via input box or slider. Turret position selected from graphical pop-up menu (only for motorized condensors). By clicking on the Close button the Condensor frame is closed. Objective button Objective can be selected via graphical pop-up menu. Reflector button Push and click, reflector cube can be selected via graphical pop-up menu. Tube Lens button Push and click, tube lens can be selected via graphical pop-up menu. Reflected Light button The shutter is switched on and off. When using the X-Cite 120 additional controls are available. See below for description. 4-46 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Recording of microscope settings The upper part of the Axiovert Control window shows the recording functionality of microscope configurations. Complete microscope created and applied. configurations can be The Store button permits existing microscope configurations to be stored under any name. The Apply button permits existing microscope configurations to be loaded. stored The Delete button permits existing microscope configurations to be deleted. The Assign button permits the assignment of a microscope configuration to a button. Load a microscope configuration An existing microscope configuration can be loaded as follows: • Click on the arrow button. − This opens a list box of all stored microscope configurations. Fig. 4-31 Axiovert 200 Control window • Browse through the microscope configurations by clicking, or use the scroll bar at the side of the list box. • Click on the desired microscope configuration. − The selected microscope configuration is shown in the first line of the Microscope Configurations list box. • Click on the Apply button. • Click on the Close button to close the microscope window. Only those microscope settings which are encoded and motorized can be reloaded. Store a microscope configuration A newly created or changed microscope configuration can be stored under a new name as follows: • Enter the desired name in the first line of the microscope setting list box. • Click on the Store button. • The actual configuration with the chosen name is added to the microscope settings list. • Click on the Close button to close the microscope window. 03/06 B 45-0019 e 4-47 Carl Zeiss OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan Delete a microscope configuration A no longer required microscope configuration can be deleted as follows: • Select the microscope configuration to be deleted from the microscope configuration list box. • Click on the Delete button. • Click on the Close button to close the microscope window. Assignment of a microscope configuration to a button A microscope configuration can be assigned to a button as follows: • Click on the Assign button. • This opens the Assign-Microscope-Settings-To-Button window. • Click on the arrow in the Button list box and select a button out of the list. With increasing numbers the buttons are arranged from the upper to the lower row from lefthand side to right-hand side. • Click on the arrow in the Settings list box and select a microscope configuration. • Click on the Apply button. A new button with the name of the selected microscope configuration has been created. • Click on the Close button to close the Assign-Microscope-Settings-To-Button window. • Click on the Close button to close the microscope window. For the conventional setting of the Axiovert 200 M, proceed as follows: • Click on the VIS button in the Acquire subordinate toolbar. • Place specimen on microscope stage. − The cover slip must be facing down. • In the Objective list box, select the required objective. • Use the focusing drive (4-32/4) to focus the required specimen plane. • Select specimen detail by moving the stage in X and Y via the XY stage fine motion control (4-32/3 and 2). 4-48 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss 1:1 ZE OB RO JEC TIV RE FO Fig. 4-32 CU E FLE CT OR S LSM 5 LIVE with Axiovert 200 M BP Microscope settings on Axiovert for transmitted-light observation • Click on the Reflected Light button and set the shutter to the Closed position. • Click on the Transmitted light button. Click on the On button in the Transmitted Light panel and set the transmitted light intensity via the slider or click on 3200 K. Click on Close to close the Transmitted Light panel. • Click on the Condensor button and set the aperture via the slider in the Condensor panel. Set the filter in the Filter selection box. Click on Close. • Click on the Objective button and select the objective by clicking on it. • Click on the Reflector button and select the None. 03/06 B 45-0019 e 4-49 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Microscope settings on Axiovert for reflected-light observation (Epi-fluorescence) • Turn on the HBO 100 power supply switch (4-32/1). • Click on the Reflected Light button and set the shutter in the Open position. • Click on the Reflector button and select the desired filter set by clicking on it. • The filter is automatically moved into the beam path to enable observation in epi-fluorescence. • Click on the Tube Lens button and select the tube lens. • Click on the Objective button and select the objective. Fig. 4-33 If the X-Cite 120 fiber coupled lamp for reflected-light illumination is used a further dialog is opened when clicking the Reflected light button. The lamp internal shutter and the attenuation function can be controlled via the LSM Software. The lamp has to be switched on first using the main switch on the lamp housing. X-Cite 120 control panel Remote Enables the attenuation control via LSM Software. This is also enabled when clicking the ON button. ON Opens the shutter in the reflected light path and the internal shutter of the lamp. Both shutters are closed automatically when switching to LSM mode. Shutter Opens and closes the shutter in the reflected light path independent of the status of the internal lamp shutter and the attenuation. Intensity Sets the light via input box or slider. Lamp Hours Indicates the total time the lamp has been in use. 4.5.2.4 Working in LSM Mode Switchover to the scanning mode is then required. • Click on the LSM button in the Acquire subordinate toolbar. 4.5.2.5 Microscope Control for Axioskop 2 FS MOT For setting the Axioskop 2 FS MOT, proceed in the same way as with Axio Imager.Z1 and Axiovert 200 M. Since the Axioskop 2 FS MOT is not motorized (except the external Z drive and the tube), all microscope settings have to be made manually. Especially, the change of objectives is made manually. The used objective must be set in the Scan Control window. 4-50 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.3 OPERATION Acquire Menu Carl Zeiss Configuration Control The setting of the beam path for the scanning procedure, i.e. the definition of channels (PMT photomultiplier) and tracks and the setting of the attenuators of the various laser lines is performed in the Configuration Control window. A track is: - a set of parameters for the detection channels and for illumination (wavelength and intensity) - scanned simultaneously and identified and handled by the system with one name The Configuration Control window has a different appearance, depending on which selection button has been activated (Single Track and Multi Track). Use the Single Track and Multi Track buttons to toggle between the two image acquisition modes single tracking and multitracking. Performed settings can be stored as Track Configurations for single tracking. In case the number of available channels is not sufficient for the scanning procedure, further tracks can be added and configured. The combination of these tracks can also be stored as Recording Configurations for multitracking (option). A recording configuration may contain the maximum of 4 tracks. Regardless of the number of included tracks, the maximum of 8 channels (incl. transmission) can be used in a recording configuration in multitracking. If several tracks have been activated, they are processed one after the other during the scan procedure. If the maximum number of channels to be used in a Single Track or a Multi Track has already been achieved, it is no longer possible to add further channels or tracks. If a second track or further tracks are used, the scan parameters can be changed as required. This avoids "cross-talk" from one channel to another when different tracks are used. 03/06 B 45-0019 e 4-51 OPERATION Acquire Menu Carl Zeiss 4.5.3.1 LSM 5 LIVE LSM 5 LIVE DuoScan Open / Close the Configuration Control Window • Click on the Config button in the Acquire subordinate toolbar. − The Configuration Control window is opened with the display last selected. The Beam Path and Channel Assignment panel differs according to the hardware configuration supplied. • Click on the Close button to quit the Configuration Control window. Fig. 4-34 Configuration Control window, Single Track activated 4.5.3.2 Spectra Button The Spectra button opens the Detection Spectra & Laser Lines[Help74] window. This window displays the laser wavelengths activated for excitation (as colored vertical lines) and the activated channels (as colored horizontal bars). The color of the bar corresponds to the one assigned to the relevant channel. Non-active channels receive a gray bar over the entire spectral range. The length and position of the bar corresponds to the emitted spectral range which is overlaid with the filter and beam splitters selected in the Configuration Control window or number of selected channels of the META detector. Fig. 4-35 4-52 Detection Spectra & Laser Lines window • Click on the Spectra button to open the Detection Spectra & Laser Lines[Help75] window and to check the settings you made. The window is closed via Close. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss All amendments made in the Configuration Control or Laser Control window are updated on-line in the Detection Spectra & Laser Lines window. • A click on the Laser button enables you to open the Laser Control window, switch lasers on and off, if required, and control the laser output. 4.5.3.3 Config Button The Config button permits existing track configurations to be loaded, stored under any name, or deleted. (1) Fig. 4-36 Track panel Fig. 4-37 Track Configurations window Load a track configuration A configuration stored in the system, whether factory-supplied or user-created, can be accepted or selected for active operation as follows: • Click on the Config button, the Track Configurations window appears on the screen. • On the Store / Apply Configuration panel, click on the arrow button . − This opens a list box of all stored track configurations. • Browse through the configurations by clicking, or use the scroll bar at the side of the list box. • Click on the desired configuration. − The selected configuration is shown in the first line of the Configurations list box (e.g.: FITC/Rhod). • Click on the Apply button. − This results in the stored instrument parameters being taken over for active use. The track configuration selected before is overwritten. The optical diagram of the configuration selected appears on the Beam Path and Channel Assignment panel. The newly loaded track has been automatically activated for the scanning procedure. The Track Configurations window is closed automatically. In the Options menu in the function Settings it is possible to define the parameters to be used when applying a track configuration. (2) Store a track configuration A newly created or changed track configuration can be stored under a new name as follows: • Click on the Config button, the Track Configurations window appears on the screen. • Enter the desired name in the first line of the Configurations list box. • Click on the Store button. • Close the window by clicking on Close. 03/06 B 45-0019 e 4-53 Carl Zeiss OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan During storage via the Store/Apply function, all the data of the Beam Path and Channel Assignment and the Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of the current track (single tracking) will be stored. (3) Delete a track configuration A no longer required track configuration can be deleted as follows: • Click on the Config button, the Track Configurations window appears on the screen. • Select the configuration to be deleted from the Configurations list box. • Click on the Delete button. • Close the window by clicking on Close. 4-54 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.3.4 OPERATION Acquire Menu Carl Zeiss Settings for Single Track in the Channel Mode The settings of the beam path for the scanning procedure with regard to the main beam splitter (Achrogate), secondary dichroic beam splitter (NFT), emission filters (EM) to be used and the assignment of channels, excitation wavelengths and laser intensities are performed in the Beam Path and Channel Assignment panel. The setting can be performed manually or by using existing track configurations. • Click on the Single Track button, unless it has already been activated. − The Configuration Control window for single tracking is displayed. (1) Beam Path and Channel Assignment panel The Beam Path and Channel Assignment panel displays the selected track configuration which is used for the scan procedure. You can change the settings of this panel using the following function elements. Activation / deactivation of the excitation wavelengths (check box) and setting of excitation intensities (slider). Open the Laser Control window via the Laser button. Selection of the main beam splitter (Achrogate) or secondary dichroic beam spliter (NFT) position through selection from the relevant list box. Selection of an emission filter through selection from the relevant list box. Activation / deactivation of the selected channel for the scanning procedure by assigning an existing color icon or defining a new one. Deactivation of the channel via deactivation of the check box. For the configuration of the beam path, please refer to the application-specific configurations depending on the used dyes and markers and the existing instrument configuration (e.g.: module LSM 5 LIVE 1 channel biomedical configurations – VarioOne GB, 488, 532 (635) nm, 1 channel FL) listed in the ANNEX of the printed manual. The assignment of the numbered emission filters (1-2), NFT secondary dichroic beam splitters and Achrogate main beam splitters in the Beam Path and Channel Assignment panel is shown in the Configuration Control window (Fig. 4-38). The numbers of the emission filters correspond to those of the channels lying behind (PMT photomultipliers). 03/06 B 45-0019 e 4-55 OPERATION Acquire Menu Carl Zeiss Fig. 4-38 (2) LSM 5 LIVE LSM 5 LIVE DuoScan Configuration Control window Beam path - Achrogate main beam splitters and NFT secondary dichroic beam splitters • On the Beam Path and Channel Assignment panel, click on the Achrogate main beam splitters (see Fig. 4-38). − This opens a graphical pop-up window of all beam splitters available. • To select a beam splitter, click on the respective line of the list. − The selected beam splitter moves into the beam path. • Proceed accordingly to configure the NFT secondary dichroic beam splitters 4-56 B 45-0019 e . 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (3) OPERATION Acquire Menu Carl Zeiss Beam path - Emission filter • On the Beam Path and Channel Assignment panel, click on the emission filter symbol. − This opens a graphical pop-up window of all available emission filters (e.g. BP for band pass, or LP for long pass) with their wavelengths. • To select an emission filter, click on the respective filter in the pop-up window. − The emission filter selected moves into the beam path in front of the PMT photomultiplier. • Depending on the application, it may be necessary to insert additional mirrors, secondary dichroic beam splitters or neutral glass filters between the Achrogate main beam splitter and the PMT photomultiplier. To select these symbols. components, click on the respective For channels 1 and 2, it is possible to change the filters directly on the LSM 5 LIVE scan module (see CHAPTER ANNEX: Filter Change in the Detection Beam Path of Channels 1 and 2 of the printed manual). (4) Fig. 4-39 Configuration Control window Fig. 4-40 Channel Color Selection window Beam path - Activation / Deactivation of Channels and Channel Color Assignment • On the Beam Path and Channel Assignment . panel, click on the channel symbols, e.g. − This opens the Channel Color Selection window on the Beam Path and Channel Assignment panel. • Click on the desired color bar. This changes the color of the channel symbol. • To close the Channel Color Selection box, click on the Close button. 03/06 B 45-0019 e 4-57 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Further colors for the corresponding channel can be produced as follows: • Clicking on the Define button will open a further Channel Colors window. All the available colors are shown as buttons in the Current Set of Channel Colors panel. • Via a reticule in the Define Color panel, any desired color can be produced. • Clicking on the Add button allows the color to be used for further channel coloring. • Choose the desired color with the reticule (the reticule is in the left corner at the bottom of the color range). • Define the brightness by use of the scroll bar. • Use the Add button to add the color to the color range. • To delete a defined color, click on the relevant color button and then on the Remove button. Fig. 4-41 Channel Colors window Standard colors (black for OFF, red, green, blue and white) cannot be removed. • Click on the Close button to close the Channel Colors window. − Newly defined colors are accepted and displayed in the Channel Color Selection window. They can then be used in the same way as standard colors. The PMT1 photomultiplier is activated / deactivated by the check box. • Proceed in the same way for the other PMT photomultiplier. 4-58 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (5) OPERATION Acquire Menu Carl Zeiss Beam path - Laser attenuation • On the Beam Path and Channel Assignment panel, click on the Excitation button. − This opens a dialog box of all available lasers with their wavelengths and their usable attenuation. • To select the desired laser line, activate the check box for Line Active. • Use the Transmission [%] slider to set the utilizable laser intensity (recommendation: start at 50 %). − The transmittance of the attenuator changes accordingly. Fig. 4-42 Configuration Control window • This allows you to adapt the laser intensity very sensitively to the job. Activate the check box for Line Active. By clicking on the Excitation button you can check at any time which lasers are available for active operation. If you deactivate Line Active, the laser wavelengths for the lasers are deselected by means of the attenuator, i.e. these lasers change into standby status. Excitation filters, emission filters, Achrogate main beam splitters and NFT secondary dichroic beam splitters can be switched online, channels (PMT photomultipliers) only off-line. 03/06 B 45-0019 e 4-59 OPERATION Acquire Menu Carl Zeiss 4.5.3.5 LSM 5 LIVE LSM 5 LIVE DuoScan Settings for Multi Track in the Channel Mode The Multi Track function permit several tracks to be defined as one configuration (Recording Configuration) for the scan procedure, to be stored under any name, reloaded or deleted. The maximum of four tracks with up to 8 channels can be defined simultaneously and then scanned one after the other. Each track is a separate unit and can be configured independently of the other tracks with regard to channels, emission filters and dichroic beam splitters. • Click on the Multi Track button. − The Configuration Control window for multitracking appears, which means that the List of Tracks panel is additionally displayed. The tracks required for multitracking can either be configured manually one after the other (identical to single tracking) and then stored as recording configuration, or already existing recording configurations can be used and changed as required. It is also possible to load already stored track configurations (single tracking) in a recording configuration. Fig. 4-43 (1) Configuration Control window, Multi Track activated Beam Path and Channel Assignment panel The Beam Path and Channel Assignment panel displays the track configuration of the track currently selected in the List of Tracks panel (highlighted in blue or gray). The settings for this panel are performed separately for each track, in the same way as for single tracking. To do this, select the track to be configured from the List of Tracks panel (see the following description of the List of Tracks panel). 4-60 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION Acquire Menu Carl Zeiss List of Tracks panel In the List of Tracks panel, the available tracks are displayed with names, activated channels and laser lines. The sequence of tracks to be processed can be changed for the scan procedure. The Add Track, Store/Apply Single Track and Remove buttons permit individual tracks to be added, saved or deleted. Fig. 4-44 List of Tracks panel In addition, this panel is used to activate / deactivate the tracks for the scan procedure. • To activate or deactivate one or several tracks for the scan procedure, activate / deactivate the check box of the relevant tracks. The configuration of the selected track is displayed in the Beam Path and Channel Assignment - ... panel. • To select a track for the display of the beam path configuration, click on its name. − The selected track is highlighted in gray or blue. When you switch from multitracking to single tracking, the track selected in the multitracking mode (highlighted in blue or gray) is always transferred and automatically activated for the scan procedure. All other tracks are deactivated, and they remain deactivated when you switch back to the multitracking mode afterwards. The following functions are available in the List of Tracks panel. Add Track button An additional track is added to the configuration list. The maximum of four tracks can be added. One track each with basic configuration is added, i.e.: one ChL 1 channel is activated, all laser lines are switched off, emission filters and dichroic beam splitters are set in accordance with the configuration last used. Remove button The single track previously marked in the List of Tracks panel in the Name column is deleted. Store/Apply button Opens the Track Configurations window. A selected track defined in a Recording Configuration can also be stored as a single track for single tracking applications. Also, it's possible to load a single track in a multitracking configuration. A click on this arrow button will move the selected track (highlighted in blue) one position upwards in the list box. A click on this arrow button will move the selected track (highlighted in blue) one position downwards in the list box. 03/06 B 45-0019 e 4-61 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan When adding new tracks, the following sequence should be followed: • Add a track by clicking on the Add Track button. • Determine the configuration of the track in the Beam Path and Channel Assignment panel or select an existing one via the Store/Apply Single Track button of the List of Tracks panel. • Store the name of a track configuration defined via the Store/Apply button of the List of Tracks panel. The new track name will then be displayed in the List of Tracks panel. If this way of storing is performed, the created track will also be available as a single track and can therefore also be activated individually. • Add the next track via the Add Track button and then configure and store it again. The name of a track can also be changed directly in the List of Tracks panel. In that case, however, the edited track is not available as a single track configuration, but only within the recording configuration. To edit a track name within Recording Configurations, proceed as follows: • To select the track, click on the relevant track name in the List of Tracks panel. Then click on the name again to open the text editing field. • Change the track name via the keyboard. Use Esc to undo the procedure. • Click once in the area outside the text editing box to close this box. The channels of the individual tracks with the relevant scan parameters can be displayed in the Scan Control window after activation of the Channels button. The description of channel 1 in Track 1, for example, is ChL1-T1. (3) Config button in Multi Track mode The Config button in the Multi Track mode permits all tracks to be loaded, stored under any name, or deleted. (a) Load a Channel Mode Configuration An existing recording configuration can be loaded as follows: • Click on the Config button, the Channel Mode Configurations window appears on the screen. • On the Store / Apply Configuration panel, click on the arrow button . Fig. 4-45 Channel Mode Configurations window − This opens a list box of all stored recording configurations. • Browse through the configurations by clicking, or use the scroll bar at the side of the list box. 4-62 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss • Click on the desired configuration. − The selected configuration is shown in the first line of the Configurations list box (e.g.: DAPI). • Click on the Apply button. − The program loads those parameters of the selected Channel Mode Configuration which have been activated in the Options menu under Settings / Recording Configuration (see Settings Function, page 4-200). The Channel Mode Configurations window is automatically closed. The optical diagram of the configuration selected appears on the Beam Path and Channel Assignment panel. The entire recording configuration has been activated for the scanning procedure. (b) Store a Channel Mode Configuration A newly created or changed recording configuration can be stored under a new name as follows: • Click on the Config button, the Channel Mode Configurations window appears on the screen. • Enter the desired name in the first line of the Configurations list box. • Click on the Store button. • Close the window by clicking on Close. During storage via the Config button, all the data of Beam Path and Channel Assignment and the Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of all the defined tracks (multitracking) are stored. Furthermore, the used objective, the Frame Size, Zoom, Rotation & Offset and Scan Direction parameters are stored. (c) Delete a Channel Mode configuration A no longer required recording configuration can be deleted as follows: • Click on the Config button, the Recording Configurations window appears on the screen. • Select the configuration to be deleted from the Configurations list box. • Click on the Delete button. • Close the window by clicking on Close. 03/06 B 45-0019 e 4-63 OPERATION Acquire Menu Carl Zeiss 4.5.3.6 LSM 5 LIVE LSM 5 LIVE DuoScan Transmitted light preset To add a transmitted light image, the light level of the Halogen lamp can be preselected in the configuration control window. The illumination is activated when the actual scan is started. To separate the transmitted light signal from the emitted fluorescence, choose e.g. the BP 700-750 in Ch1. Fig. 4-46 4-64 Transmitted light preset B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.3.7 OPERATION Acquire Menu Carl Zeiss Ratio Settings Panel The Ratio Settings panel permits you to activate two additional Ratio channels. • Click on the Ratio button. − The Ratio Settings panel is displayed at the bottom of the Configuration Control window. The settings of the selected tracking mode (Single Track / Multi Track) remain unchanged. • The Ratio Settings panel is only available in the Single Track and Multi Track mode. The following function elements are provided in the Ratio Settings panel: Activation of the Ratio channel (R1, R2) through assignment of an existing color or definition of a new one. Activation / deactivation of the Ratio channel via the check box. Selection of the channels of which the ratio is to be formed from the relevant list box. Source 2 in ratio settings includes the option to select "1st Image" for R1 and/or R2 (e.g. to calculate F/F0 for single wavelength dyes). A suitable color can be assigned to each of the two Ratio Channels R1 and R2, in the same way as for the photomultiplier channels. The channels of which a ratio will be formed are selected via the Source 1 and Source 2 list boxes. • Click on the opened. arrow button to select the required channel for Source 1 and 2 from the list box now The ratio to be formed between the selected channels can be defined more precisely using one of the four preset formulas in the Scan Control window after activation of the Channels button and a click on the relevant ratio button (e.g.: R1) for online display of radiometric or single wavelength dyes. The Set by min/max function (in Scan Control window - Channel mode) allows the definition of the display scaling according to the expected minimal and maximal values 03/06 B 45-0019 e 4-65 OPERATION Acquire Menu Carl Zeiss 4.5.3.8 LSM 5 LIVE LSM 5 LIVE DuoScan Camera Detection Panel The use of this function permits the use of a Zeiss AxioCam (HRm, HRc MRm) camera as an alternative external detector. • Click on the Config button in the Acquire subordinate toolbar of the main menu. − The Configuration Control window opens. • Activate one of the Single Track or Multi Track buttons and click on the Camera button. − The Beam Path and Channel Assignment panel for camera detection is opened. Control buttons Fig. 4-47 Configuration Control window; camera detecting activated TV Menu for selecting a display color for the camera image. Reflector Selects a beam splitter for the excitation/emission. Add Track Adds a second track to the acquisition in Multi Track mode, e.g. a different fluorescence filter cube or transmitted light. If TV and LSM tracks are mixed, the active detection port of the microscope has to be set according to the first track. 4-66 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss 4.5.3.9 LSM 5 LIVE / DuoScan Panel The use of this function permits the use of a LSM 5 LIVE or a LSM DuoScan in combination with a T-PMT as an alternative transmitted light detector. • Click on the Config button in the Acquire subordinate toolbar of the main menu. − The Configuration Control[Help92] window opens. • Activate one of the Single Track or Multi Track buttons and click on the LSM 5 LIVE or LSM DuoScan button. − The Beam Path and Channel Assignment panel for camera detection is opened. If LSM 5 LIVE and LSM DuoScan tracks are mixed, the active detection port of the microscope has to be set according to the first track. Fig. 4-48 03/06 B 45-0019 e Configuration Control window; LSM 5 LIVE / Duo Scan activated 4-67 OPERATION Acquire Menu Carl Zeiss 4.5.4 LSM 5 LIVE LSM 5 LIVE DuoScan Scan Control The scan parameters for image acquisition are set in the Scan Control window. The microscope must be in the LSM mode, i.e. the LSM button on the tube of the microscope stand must be pushed or the LSM button in the Acquire subordinate toolbar must be activated to set the LSM mode. The scanning actions are started via the buttons on the right-hand side of the Scan Control window, and the scan parameters are set in the main part of the window. An acquired image is displayed in a separate Image Display window. If an Image Display window is not yet available, a new Image Display window is automatically opened during the acquisition. The following scanning modes can be performed: Line Fig. 4-49 Scan Control window − scanning of a line in the XY-plane (Line, Line + Time Series) − scanning of a line with different Z-values (Line + Z Stack, Line + Z Stack + Time Series) Frame − scanning of an XY frame (Frame, Frame + Time Series) − scanning of XY frames with different Z-values (Frame + Z Stack, Frame + Z Stack + Time Series) 4-68 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.4.1 OPERATION Acquire Menu Carl Zeiss Open / Close the Scan Control Window • Click on the Scan button in the Acquire subordinate toolbar of the Main menu. − This opens the Scan Control window, which shows all lasers connected to the system. • Click on the Close button to quit the Scan Control window. The following main function buttons are available in the Scan Control window: Generally available buttons When the button is activated, the following panels are available for the Mode button setting of the scanning parameters for the line and frame modes: Objective Lens, Image Size & Line Step Factor, Speed, Pixel Depth, Scan Direction & Scan Average and Zoom, Rotation & Offset. Channels button When the button is activated, the Channel Settings and Excitation of Track ... panels are available for the setting of the channels and the laser excitation. Line button Activates the Line scan mode. Frame button Activates the Frame scan mode. Z Stack button Activates the Z Stack scan mode, display of additional buttons on the right-hand side of the Scan Control window. Z Settings button When the button is activated, the Z Settings panel is available for the Zscan parameter definition. The Z Stack scan mode must be active. Close button Closes the Scan Control window. New button Opens a new Image Display window. Find button Automatic optimization of image brightness and contrast. The settings for the Find function can be varied as required using the Maintain menu. Fast XY button Continuous scan with high speed. This function should be used to a limited extent and only for a short period of time. Fast XY switches temporarily to 512 x 512 frame size. Single button Single scan (named Start in the Z Stack mode). Stop button Stops the current scan procedure, no matter in which window the button is pressed (also see the Time Series Control window). Cont. button / Finish button Continuous scan (not available in Spot Scan mode and Z Stack mode). If you select the option Frame for Mode and the option Continuous for Number in the Pixel Depth, Scan Direction & Scan Average panel, the Finish button is displayed instead of the Cont. button. In this case, continuous averaging is performed when you have started the scan. If you click on the Finish button, the scan/averaging process is stopped after the scan of the current image has been completed. 03/06 B 45-0019 e 4-69 Carl Zeiss OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan Additional button in the Line mode Automatically defines a line in the center of the Image Display window Line Sel button (Frame) for creation of the intensity profile; using the mouse, the line for the intensity profile can then be positioned anywhere in the Image Display window. Additional buttons in the Z Stack mode Triggers the scan of a stack. Start button XYscan button Triggers a single XY-scan XYcont button Triggers continuous XY-scan. Line Sel button To prepare the Range function, a cutline is created in the scanned XYframe to determine the position at which the XZ-scan through the specimen is to be produced. Using the mouse, the line for the XZ-scan can be positioned anywhere in the scan frame. The cutline can be defined either as a straight line or free shape curve. Range button Produces an XZ-scan through the specimen within the limits determined in Num Slices and Interval; the cutline is determined via the Line Sel function. 4-70 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.4.2 OPERATION Acquire Menu Carl Zeiss Frame When the Frame button is activated, a frame of variable size is scanned pixel by pixel and line by line. The laser beam is moved over the specimen line by line. The scan parameters and the channels (single detector) are set via the Mode and Channels buttons, and the laser settings can be checked again or changed. (1) Mode When the Mode button is activated, the Objective Lens, Image Size & Line Step Factor, Pixel Depth, Scan Direction & Scan Average and Zoom, Rotation & Offset panels are displayed in the Scan Control window. Objective Lens, Image Size & Line Step Factor panel • Open the Objective list box and select the objective to be used via a click of the mouse (identical to Microscope Control). When using immersion oil objectives, make sure to perform immersion as required. • Select the Frame Size from the default sizes via the buttons 128, 256, 512, 768, 1024 or enter the required values via the keyboard. Recommended setting to start with: 512 x 512 pixels. Fig. 4-50 Scan Control window - Mode/Frame Fig. 4-51 Objective Lens, Image Size & Line Step Factor panel − It is also possible to enter different values for X and Y. The value for Y is freely selectable between 1 and 1024 pixels (integers). The value for X depends on the chosen image format. − The most common image formats can be set directly with the Frame size buttons. Note that more formats are available in the format pull down list. • Use the Scan Speed slider to adjust the exposure time. • Previous the slider is shown the number Frames per Second. Start with 2 to 4 frames per second. 03/06 B 45-0019 e 4-71 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The time slider displays the following information: − Frames per second (it is possible to type in the required FPS directly, or to move the slider accordingly), − Pixel time and − Frame (scan) time. Pixel Depth, Scan Direction & Scan Average panel • Select 8 Bit or 12 Bit Data Depth, i.e. 256 or 4096 gray values. • Select the Unidirectional or Bi-directional Scan Direction. Fig. 4-52 Pixel Depth, Scan Direction & Scan Average panel − Unidirectional: The laser scans in one direction only, then moves back with beam blanked and scans the next line. − Bi-directional: The laser also scans when moving backwards, i.e. the Scan Time is halved. Unidirectional scan is always optimal regarding image quality. For possible artefacts caused by bidirectional scan (partially caused by fluorochromes behaviour), (jitter or flicker) the Scan Corr Y correction slider is available. Move slider to either side in continuous scan mode till images are stable. The Auto button sets the right correction automatically. Fig. 4-53 4-72 Bidirectional Scan & Scan Correction B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Example Fig. 4-54 Example for Scan Correction Intensity fluctuations at the end of the frame (left image) are fully compensated by the Auto button (right image). Average • Select the Line or Frame mode for averaging. • Select the desired scan average method Mean or Sum in the Method selection box. • Select the desired scan average from the available values 2, 4, 8 and 16 in the Number selection box or Continues (only for Frame average mode). The greater the number of averages selected for Mean average Method, the better the image quality will be; the scanning time will be prolonged accordingly. Averaging can be performed in different ways, depending on whether the Mean or Sum method has been activated. If you are using the Mean method, the image information is generated by adding up all scans pixel by pixel and then calculating the mean value. In the Sum method, the pixel values of all scans are only added up, without a mean value being calculated. To create the image information using the Line average mode, each line (depending on the setting) is scanned 2, 4, 8 or 16 times during Scan Average, and then the average value per pixel is calculated. This minimizes noise interference during the scanning procedure. If the Frame average mode is used to create the image information, the complete frame is scanned 2, 4, 8 or 16 times, depending on the setting. The average value is recalculated after each frame scan. The Frame average mode also permits continuous averaging. 03/06 B 45-0019 e 4-73 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan For this, select the Continuous option in the Number selection box. If you have selected the Continuous option, the Finish button for ending continuous averaging is displayed instead of the Cont. button. Use the Single button in this case to start continuous scanning. When you click on the Finish button, the scan currently in progress will be completed before the process is stopped. Zoom, Rotation & Offset panel In this panel, the scan range is set for zoom and offset in relation to the field of view of the microscope. The diagonals of the outer square on the right-hand side correspond to the field of view of the microscope. Fig. 4-55 The inner square contained in it (rectangle in the case of differently set frame size) represents the scan range and immediately shows the changes made to zoom and offset. Zoom, Rotation & Offset panel The blue line at the top of the scan range is helpful for orientation when the scan range is rotated in the direction of the field of view. • Set the desired zoom factor via the slider (Zoom) or by clicking on the arrow buttons. − The zoom factor can be set continuously in the range from 0.5 to the maximum of 2.0, and is displayed in the relevant input box. The value 0.5 corresponds to factor 1, and value 2.0 to factor 4, related to the field of view of 18 mm in the intermediate plane. Clicking on button 1 enables immediate resetting to the zoom factor 1. − Recommended setting to start with: Zoom 1. • Move the scan area by clicking on the 4 arrow buttons (Offset). − The offset of the scan area from the center of the field of view is displayed online in µm for X and Y. − A click on the center button will recenter the scan area to the field of view. − Clicking, holding and drawing the rectangle with the mouse permits the scan area to be moved directly within the field of view. − Recommended setting to start with: Offset X = 0, Y = 0 During the scan procedure, the functions Objective change, Speed, Scan Corr, Zoom, Rotation and Offset can be influenced online. By clicking on the Reset button the scan zoom is set to 1 and the XY offsets are set to the zero position. 4-74 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION Acquire Menu Carl Zeiss Channels If the Channels button is activated, the Channel Settings and Excitation of Track ... panels are displayed in the Scan Control window. Channel Settings panel In the Channel Settings panel, the channels (incl. ratio channels) defined in the Configuration Control window are listed track by track as selectable buttons. Depending on the selected Channels button (e.g. ChL1-T1), the currently used settings of Pinhole (confocal aperture), Detector Gain, Amplifier Offset and Amplifier Gain are displayed. • The slider near Pinhole (Confocal Aperture) enables you to change the aperture size of the relevant channel. − The aperture size is indicated in µm, Optical Slice and Airy Units. The Airy value depends on the aperture of the objective, excitations and the emission wavelength. − A small aperture size will increase the depth of focus, but reduce the light intensity received by the PMT photomultiplier. − When you vary the aperture size, an Optical Slice value is displayed. For optimum depth resolution, Airy values should be small, but in fluorescence applications not below 1.0 to keep the intensity loss within a reasonable limit. Fig. 4-56 Scan Control window – Channels − A click on the 1 button sets the aperture size to 1 Airy unit. A click on the Max button sets the aperture size to the maximum. • The sliders (and the relevant arrow buttons) near Detector Gain, Ampl. Offset and Exposure Time enable you to set the photomultiplier of the selected channel during continuous scanning. − Detector Gain: Setting of the sensitivity of the detector - setting of image contrast and brightness (values available between 0 and 50) − Amplifier Offset: Setting of the electronic offset - background of the image can be set (values available between -2 and 0.1) − Exposure Time: Setting of exposure time with display field for frame rate (Frames per Second) 03/06 B 45-0019 e 4-75 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Gain and Find The Find button always sets an ideal gain for a clear background, best sensitivity and full grey resolution. This is maximally a gain value of 25. One can manually increase this gain by moving the gain slider beyond 25 and use the digital post amplification by doing this. Be aware that from value of 37,5 on, background artefacts can be visible, since those are amplified too. When using 12 bit grey resolution, gaps can occur in the histogram of the acquired image, in case the Gain slider is moved beyond a gain level of 25. Use the Amplifier Offset slider to remove underexposed pixels in the image background (no blue pixels visible with “range” palette). Fig. 4-57 Gain and Find The parameters Detector Gain, Ampl. Offset and Ampl. Gain are described in section Confocal Aperture / Detector Gain / Ampl. Offset (see page 4-319) in the context of image optimization. 4-76 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss The parameters of a ratio channel are set in a separate dialog box. • Click on the button of a ratio channel (e.g. R1). The dialog box for the setting of the ratio parameters is displayed. Clicking on the required tabs enables you to choose from four formulas (Type 1 to 4) for ratio calculation. The relevant decimal values can be entered in the input boxes via the keyboard. The entered values remain unchanged even after switchover to another formula and can be reactivated any time. The formula type activated last is always used for ratio formation during the scan procedure. If the input box does not contain any value at all or no suitable value, the useful value last used will be activated. The ratio channels are displayed in the Image Display window (see Fig. 4-61). • Select the required formula and enter the relevant values. Letters can be entered into the formula fields which will be valued as 1; it is also possible to make no entry, which will also be valued as 1, but will not be displayed. Set by min/max (in Scan Control window Channels mode) allows the definition of the display scaling according to the expected minimal and maximal values. Fig. 4-58 Channel Settings panel of a Ratio Channel Fig. 4-59 Excitation of Track ... panel Excitation panel • In the Excitation panel you can select other lasers and vary laser intensities (in the same way as in the Laser Control or Configuration Control window). All parameters under Channels can be varied online. 03/06 B 45-0019 e 4-77 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Acquisition of a frame Once you have set up your parameter as defined in the above section, you can acquire a frame image of your specimen. • Click on the Single button in the Scan Control window. The system will automatically start the acquisition of a frame. The individual channels and the overlay image can be viewed by changing to the Split xy mode. This button is located on the right-hand side of the Image Display window. The following scan image shows the result with two defined tracks and the overlay (see Fig. 4-61). See the appropriate Channel Settings panel in the Scan Control window (Fig. 4-60). Fig. 4-60 Channel Settings panel for two defined tracks plus Ratio channel Fig. 4-61 4-78 Image Display window with two tracks plus ratio track (Split xy mode) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (3) OPERATION Acquire Menu Carl Zeiss Z Stack This function permits a series of XY-images to be produced in different focus positions (Z slices). When the Z Stack button is pressed, the Z Settings button is automatically activated and the Z Settings panel is displayed in the Scan Control window. However, it is possible at all times to switch over to setting / changing the scan parameters or the PMT photomultipliers and lasers via the Channels and Mode buttons. The additional XYscan, XYcont, Line Sel and Range buttons are available on the right-hand side of the Scan Control window, and the labeling of the Single button changes to Start. The Z Stack function is deactivated by clicking again on the Z Stack button. Z Settings panel - overview The parameters of the Z Stack to be created are defined and displayed online in the Z Settings panel. Fig. 4-62 Scan Control window – Z Settings Stack Z Size: The dimension of the Z Stack in µm. The stage (nosepiece) is moved in such a way that the stack size, dependent on the refractive index, is achieved optically. Focus Position: The current Z position. If the refractive index (Refr. Corr.) changes, the value of the focus position in relation to the "0" also changes (online). Z Slice: Opens the Optical Slice window (Fig. 4-63). The Optical Slice window contains three buttons (Optimal Interval: ... µm, Optimal Pinhole Diameter (confocal aperture) and Undo) to allow the setting of the optimal interval and the optimal aperture size of fluorescence stacks. Both values influence each other and depend on the objective used. In the case of a fixed aperture size, half the value of the smallest aperture size used is taken to determine the optimum interval. Accordingly, the aperture size to be used in the case of a preset interval is determined by doubling the value of the selected interval. Undo resets the just before altered values. The Optical Slice window displays the following information: Black: Stack Z Size (µm) = intervals x (number of slices - 1) Optimal Interval = depending on the objective used and the aperture size setting Red and other colors: Presentation of the actual data set by the operator helps to optimize stack creation. 03/06 B 45-0019 e 4-79 OPERATION Acquire Menu Carl Zeiss Fig. 4-63 LSM 5 LIVE LSM 5 LIVE DuoScan Optical Slice window Tabs Z Sectioning: Tab for setting of Number of Slices, Interval and Current Slice via slider / arrow button. Mark First/Last: Tab for determination of the Z-value for the first and last XY-image of the stack, combined with manual focusing or Stage control. Hyperfine Z Sectioning: Tab for production of a Z Stack using the optional Piezo focus for objectives. First: Scanning / Display of the beginning (first XY-image) of the stack. Mid: Scanning / Display of the center (XY-image in the center) of the stack. Last: Scanning / Display of the end (last XY-image) of the stack. Refr. Corr.: Considers the different refractive index between the immersion medium of the objective (n') and the embedding medium of the specimen (n), which can be set between 0.5 and 3 via the slider / arrow buttons Ratio = n n' X:Y:Z=1:1:1 Clicking on this button will set the Z-interval in such a way that the voxel has identical dimensions in the X-,Y- and Z-directions (cube). Auto Z Corr. This function permits the set values of the scan parameters Detector Gain, Ampl. Offset and Ampl. Gain (as measure for the brightness level) to be varied between two freely selectable slices of a stack to be recorded. During the scan procedure, the interim values of these three parameters are automatically linearly interpolated between the initial and end values (see page 4-84). 4-80 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss The parameters of a Z Stack can be defined using the Z Sectioning tab, the Mark First/Last tab or - if the optional Piezo focus for objectives is connected - the Hyperfine Z Sectioning tab. Z Sectioning tab Num Slices: Entry of the number of sections (single XY-images) to be recorded with the stack via the slider / arrow buttons. The entry does not influence the interval. Interval: Entry of the step width (Z-distance between the single XY-images) via slider / arrow buttons. The entry has no influence on Num Slices. Current Slice: Display of the current position of the slice within the stack. Change of position via slider / arrow keys. Reset of the current slice position in the center of the stack by clicking on the C button. Of course, the borders of the stack are also changed if the current slice position is changed. Keep Interval: The interval remains constant when the stack limits or number of slices are changed. Keep Slices: The number of slices remains constant when the stack limits or interval are changed. The Stack Z Size is slightly adjusted to hold the number of slices with no interval changes or the interval size if the number of slices is varied. Fig. 4-64 03/06 Scan Control window - Z Sectioning tab activated B 45-0019 e 4-81 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click on the Range button. − The XZ-scan will be performed and displayed in the Image Display window. At the same time, the position of the current slice is shown with a green line and the positions of the first and last slice with two red lines. Fig. 4-65 Scan Control window and Image Display window • Moving the green line (current slice) enables you to change the current focus position (moving the stage or nosepiece in the process). The stack limits are also changed, while interval and Num Slice remain unchanged. • Shifting one of the red lines enables you to change the stack size; in that case, the interval size is matched, and the Num Slice remains constant. − Changing the values of Num Slice, Interval and Current Slice in the Z Sectioning tab will, of course, also change the positions of the red and green lines in the Image Display window. • A click on the Start button will start the recording of the Z Stack. − The settings of the entire Scan Control window (Mode, Channels, Z settings) will be used when the stack is produced. In Frame mode the stack is acquired as a stack of images in xyz. In Line mode the stack is displayed by a xz image. Continuous imaging of the xz stack is also possible. 4-82 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Mark First/Last tab The determination of the optimum stack size is performed here via focusing during a continuous scan. • Click on the XYcont button. − A continuous XY-scan of the set focus position will be performed. − If you have reduced the scan speed or have set image averaging, you should use the fast scanning mode to find the lowest and highest points of focus. These settings are made under Mode in the Scan Control menu, or directly via the FAST XY button. Fig. 4-66 Mark First/Last tab • Use the manual focusing drive or the Stage and Focus Control window (see Stage, page 4-114) to focus on the upper position of the specimen area where the Z Stack is to start. • Click on the Mark First button to set the upper position of the Z Stack. • Then focus on the lower specimen area where the recording of the Z Stack is to end. • Click on the Mark Last button to set this lower position. • Keep Slice enables the Num Slices slider which allows to vary the number of slices. The limits of the Z Stack remain constant, the interval is matched accordingly. • Keep Interval enables the Interval [µm] slider which allows to vary the interval between the slices. The number of slices is matched accordingly, the limit of the Z Stack is adjusted. • Click on the Start button to start the recording of the Z Stack. In case the upper and lower limits of the stack have been switched round, automatic matching will be performed by the software, since the stage of the Axio Imager.Z1 always moves from bottom to top and the nosepiece of the Axiovert 200 M always moves from top to bottom. Setting via Range is not possible via the Mark First/Last function, i.e. the lines cannot be shifted. The Fast Z Line functions is not available in frame mode. When you change from Mark First/Last to Z Sectioning or vice versa, the values are updated in the Z Sectioning tab. 03/06 B 45-0019 e 4-83 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Hyperfine Z Sectioning tab Activation of this tab is only possible if the piezo objective focusing device has been connected. The piezo objective focusing device can be controlled via software (see Stage, page 4-114). Fig. 4-67 Hyperfine Z Sectioning tab The accuracy of the piezo objective focusing device regarding the step width in the Z-direction lies in the range of 60 nm. The piezo objective focusing device allows stacks to be produced considerably quicker than via the focus of the microscope stand. The focus position remains unchanged. • Clicking on the Leveling button moves the piezo objective focus to the zero position, while the motor focus moves into the opposite direction at the same time, i.e. the position of the object in relation to the objective remains unchanged. This function is used to set defined initial conditions. • Use the slider or the arrow keys to set the number of slices for the Z Stack. • Use the slider or the arrow keys to set the size of the interval. Num Slices and Interval can be varied independently of each other within the piezo objective focus work range of ±250 µm. When change is made to Z Sectioning, or vice versa, values are also taken over, provided they are within the piezo objective focus work range. If a larger range is set for the Z Stack under Z Sectioning or Mark First/Last, the Interval is matched accordingly when changing to Hyperfine Z Sectioning, while Num Slice remains constant. • Use XYcont, Line Sel and Range to determine the parameters of the Z Stack (identical to Z Sectioning). If the green line (Current Slice) is shifted after the creation of Range, the focus position will change (the piezo objective focusing device remains in the center position). The red lines (stack limits) can only be changed symmetrically to the Current-Slice position within the piezo objective focusing device work range. Since the piezo objective focusing device moves from bottom to top during the creation of the Z Stack, top and bottom of the Axiovert 200 M have been switched round. Bidirectional Z-Scan in time series (4D) Similar effects can occur when using the fast piezo Z-drive in Bidirectional mode with multiple stacks. Like for X/Y-scanning, there is a uni- and a bidirectional mode here too. Fig. 4-68 Corr Z Slider • To compensate possible image deviations in the Z-direction in bidirectional focus mode, use the Corr Z slider by typing in the deviation occurring in the adjacent image stacks of a time series. If only one time point (stack) is acquired, the Corr Z slider has no effect. 4-84 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Auto Z Corr. The function Auto Z Correction allows a linear variation of Detector Gain, Ampl. Offset, and Ampl. Gain values between the different slices of a stack. • Click on the Auto Z button, the Auto Z Brightness Correction panel opens. The buttons Set A and Set B permit definition of two distinct gain / offset settings at two different Z positions A and B. Pressing the Move A and Move B buttons permits the defined Z-position to be directly approached. The Enable test check box permits simulation of the value changes for Detector Gain, Ampl. Offset, Ampl. Gain and Attenuation in the Scan Control window without the scanners being in operation. Fig. 4-69 Z Brightness level control panel If a Z Stack is performed and the Auto Z Brightness Correction window is opened this correction is automatically performed equal whether the Enable test box is enabled or disabled. • Use the focusing drive to set the Z-position where the brightness level correction is to be started. • In the Scan Control window, set the initial values for Detector Gain, Ampl. Offset and Ampl. Gain. If required, start the continuous scan procedure for this purpose. Click on the Set A button. • Use the focusing drive to set the Z-position where the brightness level correction is to be ended. • Set the end value for Detector Gain, Ampl. Offset and Ampl. Gain in the Scan Control window. Click on the Set B button. • If required, check the change of the set values by activating Enable test. After the start of the scan procedure, the brightness level values are linearly interpolated between the defined references A and B. Note that the total Z range where the interpolation takes place can exceed the Z reference A and B. 03/06 B 45-0019 e 4-85 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Acquisition of a Z Stack Once you have set up your image as defined in the above section, you can collect a series of confocal images through the different focal planes of your specimen. • Click on the Start button on the Scan Control window. The system will automatically start the creation of a Z Stack. Be careful not to bump the air table or the microscope until Z sectioning is completed. Each successive Z Slice can be viewed by changing to the Gallery Mode. This button is located on the right-hand side of the image. A black bar will be shown under the image and will move from left to right, showing that the LSM 5 LIVE is in the process. The laser will automatically stop scanning when the Z Stack is completed. The entire stack of images can be saved using the Save or Save As buttons on the right-hand side of the image. Fig. 4-70 4-86 Image Display window of a Z Stack B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.4.3 OPERATION Acquire Menu Carl Zeiss Line In the Line mode, fluorescent or reflected light along a freely definable line is displayed in the form of an intensity profile. All the possibilities of creating an image (Frame, Z Stack) are also available in the Line mode. The Line and Frame buttons are activated alternately and exclude each other. • Set all the parameters for the Scan procedure (Mode and Channels or Z Settings) in the same way as for the scanning of a frame or a Z Stack. • Then click on the Line Sel button. Fig. 4-71 03/06 B 45-0019 e Scan Control window - Mode/Line 4-87 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.4.4 Camera Control The use of this function permits the control of the external CCD-camera settings. (1) Open / Close the Scan control window for camera control • In the Configuration Control window, activate the Camera button. • Click on the Scan button in the Acquire subordinate toolbar of the main menu. • Click on the Close button. Fig. 4-72 Scan Control window - Mode, settings for camera control (2) Function description Mode button Displays the selected objective, frame size and pixel depth. Frame Size Selects between square formats or free defined frame sizes. Format Selects between a range of default camera resolutions. The 5x5 binning mode can be used for focusing without delay of the image display. Data Depth Sets the pixel depth. Zoom/Offset Shifts a subregion in the frame. Reset Resets the frame/subregion to default value selected in Format. 4-88 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Fig. 4-73 Carl Zeiss Scan Control window - Channels, settings for camera control Channels button Displays the activated channels and possible settings. Exposure time Sets the exposure time of the camera. Find Starts a prescan and sets the exposure time automatically. In case of a camera multitracking, only one channel should be selected in Configuration Control in order to speed up the find function. Fast X/Y Starts a fast online scan mode, e.g. for focusing. Also, the 5x5 binning mode can be used (to be set in Mode / Format). Single Starts a single image acquisition (The Image Display window appears.). Cont. Starts acquisition of a series of images (The Image Display window appears.). Crop Defines a ROI for camera acquisition in the Image Display window. Note that this is just a Crop function, while the whole sample is illuminated. Rotation of the ROI is not possible. Info button Shows the acquisition parameters in the Image Display window. Close Close the Scan Control window. 03/06 B 45-0019 e 4-89 OPERATION Acquire Menu Carl Zeiss 4.5.5 LSM 5 LIVE LSM 5 LIVE DuoScan Edit ROI (Region Of Interest) A scan image allows certain areas (ROIs) to be defined. Definition and activation of the ROIs for the analysis is performed in the Edit ROI window. 4.5.5.1 Open / Close the Edit ROI Window • Click on the Edit ROI button in the Acquire subordinate toolbar of the Main menu. The Edit ROI window appears on the screen and the ROIs defined last are visible in the Image Display window. • Click on the Close button in the Edit ROI window. The Edit ROI window is closed and the ROIs disappear from the Image Display window. Fig. 4-74 Edit ROI window and Image Display window with ROIs When Edit ROI is activated and the first ROI is drawn in the Image Display window, the Use ROI is activated automatically. 4.5.5.2 Function Description The following functions are available on the right-hand side of the Edit ROI window: Close button The Edit ROI window is closed. Remove button An entry marked in ROI Lists (stored ROI configuration) is deleted. Add to Lists button The Add ROI List window is opened. 4-90 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (1) OPERATION Acquire Menu Carl Zeiss ROI Lists panel In the ROI Lists panel, all the currently defined and stored ROI configurations are shown. • Click on the ROI configuration which you want to use for the scan procedure. − The selected ROI configuration is highlighted in blue and displayed in the opened Image Display window. Fig. 4-75 ROI Lists panel Fig. 4-76 Interactive ROI Definition panel • To produce a new ROI configuration, an already stored configuration can be activated, changed and stored under a new name using the Add to List button. • To delete a stored ROI configuration from the list, click on its name first (highlighted in blue) and then on the Remove button. (2) Interactive ROI Definition panel In the Interactive ROI Definition panel, the parameters of the ROI configuration just selected from the ROI Lists panel are displayed. Furthermore, it contains all the functions required for the creation of ROIs. The X and Y values for Center Position and Dimension can be edited. • Activate the relevant text box with a mouse click and enter the new value via the keyboard. • If you click outside the edited text box, the new value will be taken over and the ROI figure be shifted to the new position. The upper part of the panel gives an overview of all the individual figures stored under the selected name according to type, position within the Image Display window (in pixels) and greatest dimension in X and Y (in pixels). The origin of the position indication lies in the left top corner of the Image Display window. 03/06 B 45-0019 e 4-91 Carl Zeiss OPERATION Acquire Menu LSM 5 LIVE LSM 5 LIVE DuoScan Check box (e.g.: 1 - 4): Clicking on this check box allows a ROI to be deactivated. The tick disappears from the check box, as does the relevant marked area from the scanning image. Clicking on the check box again will reactivate the ROI. Arrow button: Activation of the mouse button to change the size or move the ROIs in the Image Display window. Rectangle button: Draw of a rectangle in the Image Display window; click and keep mouse button pressed, drag the rectangle in any direction, let go off the mouse button to end the procedure. Bezier button: Draw of a bezier figure in the Image Display window; first click sets the starting point, each additional click adds a line, double-click on the starting point closes the figure and ends the procedure. Ellipse button: Draw of an ellipse in the Image Display window; first click sets the center point, displayed line permits determination of the extension, second click sets the first dimension, then the second dimension and the rotation direction can be determined, third click sets the second dimension and direction and ends the procedure. Circle button: Draw of a circle in the Image Display window; click and keep the mouse button pressed to set the center point, drag the diameter, let go off mouse button again to end the procedure. Polyline button: Draw of a polyline figure in the Image Display window; first click sets the starting point, each further click adds a line, double-click on the starting point closes the figure and ends the procedure. Recycle bin button: All the ROIs dragged to the scanning image are deleted. If an area outline was marked before, this area is now deleted in the scanning image. Auto / Color button: A defined color from the list of colors can be assigned to the ROIs. In that case, the same color is assigned to all the individual figures. In the Auto position, the outlines of the dragged ROIs are automatically colored differently. Line button: This button allows you to determine the line thickness of the area outline. This is for display purposes only. The scanned line is not effected. Fit Frame Size to bounding Rectangle of all ROIs check box: If this check box is ticked, the scan procedure is displayed only within a rectangle which is defined by the greatest extension in X and Y of all the individual figures together, i.e. the pixel number and the data quantity of the Image Display window are reduced. 4-92 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss • In the toolbar of the Interactive ROI Definition panel, click on the symbol of the area you want to use to mark the region of interest in the scanning image. Five different area symbols are available in the form of buttons. • Click on the marking area and keep the mouse button pressed to drag the area into the region of interest in the scanning image. The marking area will be numbered automatically and entered in the Interactive ROI Definition panel with its position and dimension parameters and the appropriate number. • The dragged marking area is marked by clicking on its outline; its size can be changed by clicking on the marking points. Clicking on the area edge beside the marking points allows repositioning of the area on the scanning image. The digits of the ROIs can be shifted independently of the contours of the figure. • If you have framed all the required ROIs in accordance with steps 2 to 4, you can store these ROIs under any required name via the Add to Lists button. • The Add ROI List window will appear. Enter any required name to store the ROIs and click on the OK button. • This stored ROI configuration appears in the ROI Lists panel of the Edit ROI window. 03/06 Fig. 4-77 B 45-0019 e Add ROI List window 4-93 OPERATION Acquire Menu Carl Zeiss 4.5.6 LSM 5 LIVE LSM 5 LIVE DuoScan Time Series The Time Series Control window allows the definition of parameters for time series. The Time Series function offers the following options for the creation of image series: • Definition of break times between 0.1 ms and 10 hours. • Determination of the number of steps from 1 to 10,000 for one scanning procedure. • Setting of markers. • Interruption of time control via pause function, and resume of the time series function. • Triggering of time series via: − numeric input − external trigger pulses − time (of the PC) 4.5.6.1 Open / Close the Time Series Control Window • Click on the Time Series button in the Acquire subordinate toolbar of the Main menu. Fig. 4-78 Time Series Control window The Time Series Control window appears on the screen. • Click on the Close button to close the Time Series Control window. The following functions are available on the right-hand side of the Time Series Control window: Close button Closes the Time Series Control window. New button Opens a new Image Display window. Start T button Starts the Time Series. Stop button Stops the entire Time Series. A current scan is interrupted. Pause button Interrupts the Time Series. Button labeling is changed to Resume. A current scan is performed until the end. When the button is pressed again, the Time Series is immediately continued with the next scan procedure. 4-94 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Mean ROI button OPERATION Acquire Menu Carl Zeiss Creates a Time Series with the intensity values of the Frame or the default ROIs. An average value is formed of the intensity values of the Frame or the ROIs determined. These average values are displayed in an extended Image Display window as a function of the time which has passed. The status line, in which the phases of the current Time Series or notes for the user are displayed, is in the lower part of the Time Series Control window. 4.5.6.2 Start Series Panel In this panel, the parameters for the start of the time series are set. Fig. 4-79 Start Series panel The following functions are available: Manual button The time series is started manually with a click on the Start T or Start B button. Trigger button The time series is started via a trigger signal from Trigger Control. Time button The time series is started when the set time is reached. The internal computer time applies. Time input box Input of the time for the start of the time series (Time button activated). Trigger in list box Selection of the trigger key (1-4) with which the start is to be triggered (Trigger button activated). Trigger out list box Selection of the trigger keys (1-4) for the out signal. Pre-Scan If this box is checked before starting the Time Series a continuous scan is started , which then appears on the but no images are acquired until the button Go lower right hand side of the control window, is clicked. 03/06 B 45-0019 e 4-95 OPERATION Acquire Menu Carl Zeiss (1) LSM 5 LIVE LSM 5 LIVE DuoScan Start via Trigger For the start via trigger control (Trigger button activated), first determine the trigger key which is to trigger the start of the Time Series. Fig. 4-80 Start Series panel • Open the Trigger in list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger1). It is also possible to trigger an out signal via trigger control. • Open the Trigger out list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger1). In this example, the scan procedure is triggered on pressing key 1 of the trigger control, and an out signal is given at the same time. When starting a Time Series via Trigger, the Start T or Start B button must be pressed first. Waiting for Trigger will then be displayed in the status line. Then the relevant trigger key on the Trigger Control must be pressed to start the first scan procedure of the Time Series. (2) Start via Time For the start via the time set on the PC (Time button activated), the start time must be entered first in the Time input box. Fig. 4-81 Start Series panel • Click in the Time input box to open it. • Enter a start time via the keyboard. Then click outside the input box once to close it again. When starting a Time Series via the time, the Start T or Start B button must also be pressed in this case. Waiting for Start Time will be displayed in the status line. The Time Series is started when the starting time has been reached. The starting time for the Time Series can be changed online. (3) Pre-Scan The Pre-Scan allows to image the sample continuously before actually starting the image acquisition. This function should only be used for starting the time series manually. After clicking the Start or Start B button, the button Go appears on the lower right hand side of the control window. By clicking this button the actual image acquisition is started. 4-96 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.6.3 OPERATION Acquire Menu Carl Zeiss Stop Series Panel In this panel, the parameters for the end of the time series are set and the number of cycles is determined. Fig. 4-82 Stop Series panel The following functions are available: Manual button The time series is finished manually with a click on the Stop button. Trigger button The time series is finished via a trigger signal. Time button The time series is finished when the set time has been reached. The internal computer time applies as the set time. Number input box / Determination of the number of images acquired or image stacks for the time series. arrow keys / slider Time input box Input of the time for the end of the time series (Time button activated). Trigger in list box Selection of the trigger keys (1-4) with which the end is to be triggered (Trigger button activated). Trigger out list box Selection of the trigger keys (1-4) for the out signal. • Use the slider near Number to select the images or image stacks for the time series. (1) Stop via Trigger To end the Time Series via Trigger Control (Trigger button activated), first determine the trigger key which is to end the Time Series. • Open the Trigger in list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger2). Fig. 4-83 Stop Series panel It is also possible to trigger an out signal via Trigger Control. • Open the Trigger out list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger2). In this example, the Time Series is ended on pressing key 2 of the Trigger Control, and an out signal is given at the same time. 03/06 B 45-0019 e 4-97 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan If the entered number of cycles has been processed without a trigger impulse having been given to end the procedure, the Time Series is finished. If a trigger signal is given before the cycles have been processed, the Time Series will only be interrupted. Waiting for Trigger will be displayed in the status line. The Time Series can now be continued via a new trigger signal or ended via Stop. (2) Stop via Time To end the Time Series via the time set on the PC (Time button activated), the end time must first be entered in the Time input box. • Click on the Time input box to open it. Fig. 4-84 Stop Series panel • Enter the end time via the keyboard. Then click outside the input box once to close the box. The Time Series is interrupted when the end time has been reached. If the entered Number of cycles has been processed, the Time Series is finished. If the number of cycles has not yet been processed, the Time Series is only interrupted. Waiting for Start Time is displayed in the status line. The Time Series can now be continued by entering a new start time, or finished via Stop. The end time for the Time Series can be changed online. 4.5.6.4 Cycle Delay / Time Interval Panels Depending on the settings in the Time Series tab (see Options menu, Settings), the time series interval is defined either as a Cycle Delay or Time Interval. Accordingly, either the Cycle Delay panel or the Time Interval panel is displayed in the Time Series Control window. Cycle Delay is the interval between the end of one scan process and the beginning of the next. Fig. 4-85 Cycle Delay panel Time Interval is the interval between the beginning of one scan process and the beginning of the next. The Cycle Delay (or Time Interval) panel permits the intervals to be activated and changed. Fig. 4-86 4-98 Time Interval panel B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Function description Cycle delay or Time List of the stored sets of Cycle Delays or Time Intervals for time series. Interval list box Apply button Application of the sets of delays for time series selected in the list box. Store button Storage of sets of delays for time series. Delete button Deletion of sets of delays for time series from the list box. Time buttons Activation of the time for the time series set for the relevant button. Time input box / Determination of the cycle time for the currently activated Time button. arrow buttons / slider Unit buttons Selection of time units: min, sec or ms. Trigger in list box Selection of the trigger key (1-4) to be used to activate the Time button for the delay time. Trigger out list box Selection of the trigger key (1-4) for the out signal. Setting delay time or time interval • The delay time or time interval to be used during the Time Series is set to a default value by activating a Time button. For this purpose, the relevant time must be assigned to the Time button first. • Activate a Time button with a click of the mouse. • Set the required delay time or time interval via the slider (arrow keys or input box) near Time. The set time is displayed online on the button. Select the time unit by clicking on the relevant button near Unit. You can assign different times to all the six Time buttons and store this assignment either as a set of delays or of time intervals. • Enter a name in the Cycle Delay list box or Time Interval list box and click on Store to store the set of delays. Activating delay time or time interval If required, a set of delays or time intervals can be activated again quickly. • Open the list box with a click on the arrow button and select the required set with a click of the mouse. • Then click on the Apply button to activate the set. The stored delays are assigned to the Time buttons. Sets of delays or Sets of time intervals which are no longer required can be deleted. • Open the list box and select the required set. 03/06 B 45-0019 e 4-99 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click on the Delete button. The set will be removed. The Time buttons can also be activated via keys 1 to 4 of the Trigger Control. • Click on the required Time button. • Open the Trigger in list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger3). It is also possible to trigger an out signal via Trigger Control. • Click on the required Time button. • Open the Trigger out list box with a click on the arrow button. • Choose one of the trigger keys 1 to 4 (e.g. Trigger3). In this example, the relevant Time button is activated on pressing key 3 of the Trigger Control, and an out signal is given at the same time. The delays or time intervals can be changed online with a click on another Time button. The new delay will be applied immediately. A change of the delay during a Time Series is displayed in the Image Display window if the Gallery button (Display toolbar) is activated. 4.5.6.5 Marker Panel The setting of a marker permits information about the moment in the current time series and any required comment to be assigned to the current scan. The time indication is set automatically, while comments must be defined before. The markers (red squares) are visible in the Image Display window if the Gallery button (Display toolbar) is activated. Fig. 4-87 Marker panel On storage of the image, all the markers, including the time indication and the comments, are stored along with the image contents. Function description Marker list box List of the stored combinations of markers. Apply button Application of the marker combinations selected from the list box. Store button Storage of a combination of markers. Delete button Deletion of a combination of markers from the Marker list box. 4-100 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Set 1-7 button Setting of a marker during the scan procedure. Description input box (1-7) Entry of the comments for the marker. Trigger in list box (1-7) Selection of the trigger key (1-4) with which the marker is to be set. Trigger out list box (1-7) Selection of the trigger key (1-4) for the out signal. Carl Zeiss Setting marker for the current scan • A marker for the current scan is set by clicking on one of the Set 1 to 7 marker buttons. The assignment of any required comment for the marker must be performed as follows: • Click in the Edit Text box of the required marker key (e.g.: Set 1) to open the editing box. • Enter the comments via the keyboard. Then click outside the editing box to close this box again. You can assign comments of any required length to all the seven Set buttons and store this assignment as a combination of marker keys. • Enter a name in the Marker list box and click on Store to store the combination. If required, a combination of markers can be activated again quickly. • Open the Marker list box with a click on the arrow button and select the required combination with a click of the mouse. • Then click on the Apply button to activate the combination. The relevant comments are displayed in the Edit Text boxes of the Set buttons. Combinations which are no longer required can be deleted. • Open the Marker list box and select the required combination. • Click on the Delete button. The combination will be removed. The marker buttons can also be activated via keys 1 to 4 of the Trigger Control. • Click on the required Set button. • Open the Trigger in list box with a click on the arrow button. • Select one of the trigger keys 1 to 4 (e.g. Trigger4). It is also possible to trigger an out signal via Trigger Control. • Click on the required Set button. • Open the Trigger out list box with a click on the arrow button. • Select one of the trigger keys 1 to 4 (e.g. Trigger4). In this example, the relevant Set button is activated on pressing key 4 of the Trigger Control, a marker is set in the Scan and an out signal given at the same time. 03/06 B 45-0019 e 4-101 OPERATION Acquire Menu Carl Zeiss 4.5.6.6 LSM 5 LIVE LSM 5 LIVE DuoScan Time Series of a Frame • Set the relevant parameters for time control in the Start Series, End Series and Cycle Delay panels. • Start the Time Series with a click on the Start T or Start B button. • If you use Trigger Control, confirm the relevant Trigger key to start the Time Series with the first scan procedure. • Use the Set 1 to Set 7 buttons to set markers during the scanning procedure which will allow you to evaluate interesting scanning images later. Time end will finish time series even if you have created a program which would exceed the time end. If a time series is interrupted before its programmed end, the programmed number of images will be taken over in the database. However, only those images are stored which were created before interruption of the time series. This is due to the fact that the original image parameters are to be taken over via the Reuse function. If a stop time for time series is entered via the Trigger button or the Time button, the recording of the series will not be definitely finished. It is possible to either continue the series via new settings of Trigger and Time or to definitely finish the time series via the Stop key. The following example of a scanning image was taken using the Time Series function. Both the time and the markers set during the scanning procedure are projected in the image series in different colors. If the cursor is moved to a marker position in the scanning image, the relevant information on the image detail is automatically provided in an additional window. 4-102 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-88 OPERATION Acquire Menu Carl Zeiss Image Display window of a Time Series Scan The image markers have different colors with the following meaning: • red: manually set marker with time indication and comments • blue: automatically set marker with change of delay 03/06 B 45-0019 e 4-103 OPERATION Acquire Menu Carl Zeiss 4.5.6.7 LSM 5 LIVE LSM 5 LIVE DuoScan Time Series of a Frame over Z Stack (option) • First, set all parameters required for recording a Z Stack in the Scan Control window. • Then set the parameters required for recording the time series in the Time Series Control window (identical procedure as for the time series of a frame). • Start the time series by clicking on Start T. − Complete stacks are now recorded at the defined time intervals. The result is displayed in the form of the combined Image Display window of the stack and time series (4D). Fig. 4-89 Image Display window of a Z Stack and a Time Series Scan The additional Z, Time and Z + Time buttons are available in the Gallery toolbar of the Image Display window. When you click on the Z button, the individual frames of the Z Stack are displayed for the selected Time Slice. When you click on the Time button, the individual frames of the time series are displayed for the selected Z Slice. 4-104 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss For Z Stacks over the time (4D) following offline functions will be enlarged: − Slice (Z slider and Time slider) − Gallery (Z, Time and Z + Time buttons) − 3D (slider for single time index) To select the Z or Time Slices, use the appropriate sliders which are displayed if the Slice button in the Image Display window has been activated. When you click on the Z + Time button, all individual frames will be displayed. 03/06 B 45-0019 e 4-105 OPERATION Acquire Menu Carl Zeiss 4.5.6.8 LSM 5 LIVE LSM 5 LIVE DuoScan Time Series with Mean ROI • Set all the parameters in the same way as for Time Series of a frame. • Then click on the Mean ROI button in the time series frame. A mean intensity profile of the defined ROIs (To be defined using the Edit ROI, see page 4-90) is created as a function of time. Fig. 4-90 Image Display window of a Time Series with Mean ROI The Image Display window of the Mean ROI function is structured differently than that of a frame. On the left-hand side of the Image Display window, the intensity time profiles per ROI are displayed graphically. The Select and Display toolbars, which are also available in the standard Image Display window, are positioned in the center. The Scan Mean of ROIs toolbar with further function elements is additionally displayed on the righthand side. The major purpose of these function elements is to vary the display of the recorded Mean ROI. By selecting the appropriate options (see Options menu, Settings – Scan Mean of ROIs) you can activate the following additional functions: − Display of the live image in the Image Display window of the Mean ROI function (used ROIs only) − Scan of the complete image (if Live Image has been activated) − Saving of the complete time series (if Live Image has been activated) 4-106 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss The following functions are available: Display of the data of the ROIs used for the creation of the MeanROI (identical to the Edit ROI window). If the check box of a ROI is deactivated, the ROI's intensity values are no longer displayed in the Intensity-Time diagram. 1 button: Intensity values for ROI and Channels are displayed in a diagram. Chan button: Intensity values are displayed separately for each channel used. ROI button: Intensity values are displayed separately for each ROI used. Mono button: Switches between color and monochromic display of intensity profiles. Automatic button: Automatic scaling of the display of Intensity-Time diagrams. Time Range button: Display of Intensity-Time diagrams is scaled depending on the Time Range set in the input box shown on the left. Number Cycles button: Display of Intensity-Time diagrams is scaled depending on the Number Cycle set in the input box shown on the left. Show Image button: Shows the scan image in the Image Display window to the side of the intensity diagram. This button is active only if the Live Image option is activated. Copy Table button: The table of intensity values is copied to the clipboard. Show Table button: The table of intensity values is displayed at the bottom left of the Image Display window. Save Table button: The table of intensity values can be stored as a text file. 03/06 B 45-0019 e 4-107 OPERATION Acquire Menu Carl Zeiss 4.5.7 LSM 5 LIVE LSM 5 LIVE DuoScan Stripe bleach function (LSM 5 LIVE without LSM DuoScan) Bleaching of selectable rectangular image regions with the LSM 5 LIVE Stripe Bleach is activated by the EditBleach button in the main menu. The Bleach Control window opens and can be used in the same way as in any other Zeiss LSM 5, e.g. the LSM 510. The only difference is that the bleach region has to be rectangular. Fig. 4-91 EditBleach Button Hence the Define Region button only allows to select the upper and lower border of a bleach region. It is possible to select more than one bleach region. Bleach Regions are drawn directly in the image window. The bleach wavelength is choosen in the Excitation (laser) wavelength menu on the bottom of the bleach control dialogue. Check the required line and set the power with the sliders. A single bleach event can be started interactively by pressing the Bleach button. Fig. 4-92 4-108 Stripe Bleach Function B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-93 OPERATION Acquire Menu Carl Zeiss Image Window Bleach series To set up a time series with a bleach event, the bleach region is selected as described above. In the Time Series window, a time series is set up. Fig. 4-94 03/06 Time Series Button B 45-0019 e 4-109 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan In the upper part of the Bleach Control window, the time parameters can be set. Check the required boxes and type in the number of the images where the bleach event is required. The number has to be lower than the overall number of images choosen in the time series dialogue. Start the bleach series with StartB (!) in the Time Series window. Fig. 4-95 Settings for Bleach Series 4.5.8 Edit Bleach (LSM 5 LIVE DuoScan) The use of this function permits the setting of bleaching parameters for spot, line or frame bleaching. 4.5.8.1 Open / Close the Edit Bleach Window • Click on the Edit Bleach button in the Acquire subordinate toolbar of the Main menu. The Bleach Control[Help190] window appears on the screen. • Click on the Close button to close the Bleach Control window. The following functions are available on the righthand side of the Bleach Control window: Fig. 4-96 4-110 Close button The Bleach Control window is closed. Bleach button Starts the bleaching procedure. Stop button Ends the bleaching procedure. Bleach Control Window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.8.2 OPERATION Acquire Menu Carl Zeiss Settings Panel The Settings[Help191] panel allows you to determine when, where and how the bleaching process shall be done (only works in connection with time series). Furthermore, all the settings of the Bleach Control window can be stored, reactivated or deleted in this panel. a Fig. 4-97 Settings panel Bleach after number scans: If this check box is ticked , the bleaching procedure is automatically performed in combination with a time series. Under Scan Number, you must enter after how many scanning procedures bleaching is to be performed. Scan Number: Number of Scans in a time series, after performance of which the bleaching procedure shall be started. Bleach repeat after number scans: If this check box is ticked , the bleaching procedure is automatically performed in combination with a time series. Under Scan Number, you must enter after how many scanning procedures bleaching is to be repeated. Scan Number: Number of Scans in a time series, after performance of which the bleaching procedure shall be repeated. Different Z Position: If this check box is ticked , you can set the current stage position as the one in which the bleaching will be done by clicking the Mark Position Z button. This function is only available using the Line or Frame scanning mode. Different Scan Speed If this check box is ticked , the speed for scanning during the bleach process can be defined independently of the scan speed during image acquisition. A lower speed results in a longer pixel dwell time which increases the efficiency of bleaching. Different XY Spot Bleach Position: If this check box is ticked , you can set a different XY position for spot bleaching. This function is only available using the Spot scanning mode. Click on the Spot Select button in the Scan Control window. A new image is produced and two crosshairs appear in the image. The red crosshair marks the spot that will be imaged. The green crosshair marks the spot that will be bleached. Move the center of the crosshairs to the desired positions and perform bleaching. Store settings of the Bleach Control Proceed as follows to store the entire settings of the Bleach Control window: • Enter a name in the Settings list box and click on Store to store the settings. If required, stored settings for the bleaching procedure can be reactivated quickly. • Open the Settings list box with a click on the arrow button and select the required name with a click of the mouse. 03/06 B 45-0019 e 4-111 OPERATION Acquire Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Then click on the Apply button to activate these settings. The Bleach Control window will be updated accordingly. Delete settings of the Bleach Control Settings which are no longer required can be deleted. • Open the Settings list box and select the required name. • Click on the Delete button. This stored setting will be removed. The bleaching procedure can also be activated via keys 1 to 4 of the Trigger Control. • Open the Trigger in list box with a click on the arrow button. • Select one of the trigger keys 1 to 4 (e.g. Trigger4). It is also possible to trigger an out signal via trigger control. 4.5.8.3 Fig. 4-98 Bleach Parameter panel Bleach Parameter Panel The Bleach Parameter[Help193] panel allows you to determine how often the bleaching process shall be performed, and to select the area for bleaching in the scan image via the Define Region button. • Enter the number of iterations of the bleaching procedure in the Iterations input box. • Click on the Define Region button. − The Bleach appears. Regions[Help194] window The definition of bleach regions corresponds to the Edit ROI function and is performed in the same way (see Edit ROI, page 4-90). ROIs already defined with Edit ROI are also available in the Bleach Regions window. They can be activated directly, modified - if required - and stored under a new name. ROIs newly defined in the Bleach Regions window will then also be available in the Edit ROI window. Fig. 4-99 4-112 Bleach Regions window • Define the required bleach regions in the scan image or use an existing ROI. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.8.4 OPERATION Acquire Menu Carl Zeiss Excitation of Bleach Track Panel In the Excitation of Bleach Track panel you can select the lasers and laser intensities for bleaching. The setting of the lasers for the bleaching procedure corresponds to that for the scanning procedure and must be performed accordingly (see Laser Control, Configuration Control and Scan Control). • Select the required laser wavelength and its intensity under Excitation. • If required, switch the relevant laser to On (Laser button). • If more than one ROI has been defined under Bleach Parameters, a different laser intensity and laser line can be defined for each region. Toggle between the buttons indicating the number of the ROI and check the laser line and intensity for each ROI. 4.5.8.5 Fig. 4-100 Excitation of Bleach Track panel Start / End a Bleaching Procedure • The bleaching process will be started via the Bleach button. However, it is also possible to start the bleaching process via the Bleach button in the Time Series Control[Help201] window or to combine it with a time series. When a trigger key is activated to start the bleaching procedure, the Waiting for Trigger message first appears in the status line of the Bleach Control window. In that case, the bleaching procedure is started after activation of the relevant trigger key. • The bleaching process can be finished via Stop[Help202] in the Bleach Control window. Stop does not only stop the bleaching process, but the entire scanning process. 03/06 B 45-0019 e 4-113 OPERATION Acquire Menu Carl Zeiss 4.5.9 LSM 5 LIVE LSM 5 LIVE DuoScan Stage The following software description applies to systems which are equipped with a motorized stage. This window enables you to activate both the motor focus and the scanning stage. The Focus Position and Stage Position panels include the function keys for the performance of defined moves and the display of the current Z and X, Y positions. By use of an LSM 5 LIVE scanhead on an Axiovert 200 M sideport system care should be taken when moving the motorized XY scanning stage to the maximum positions, so that fingers are not bruised between scan head and stage. 4.5.9.1 Open / Close the Stage and Focus Control Window • Click on the Stage button in the Acquire subordinate toolbar of the Main menu. Fig. 4-101 Stage and Focus Control window − The Stage and Focus Control window appears on the screen. • Click on the Close button in the Stage and Focus Control window to close this window. The following functions are available on the right-hand side of the Stage and Focus Control window: Close button The Stage and Focus Control window is closed. Start button Starts the tile scanning procedure. Stop button Ends the scanning procedure. 4-114 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.5.9.2 OPERATION Acquire Menu Carl Zeiss Focus Position Panel Focus buttons (Z Moves) Clicking on the ▲ button moves the specimen stage / nosepiece upwards. Clicking on the Z button sets the current Z-position to zero. Clicking on the ▼ button moves the specimen stage / nosepiece downwards. Fig. 4-102 Focus Position panel Focus Step slider 0.1 µm is the smallest value which can be set, and 100 µm the highest. • Clicking on the arrow keys changes the step size by 1 µm. • Pressing the CTRL key and clicking changes the step size by 0.05 µm. • Pressing the Shift key and clicking changes the step size by 10 µm. Work button Pressing the Work button moves the specimen stage / nosepiece back to the Work position. This is the position last set before the Load button was pressed. Load button Clicking on the Load button lowers the specimen stage / nosepiece to make it easier for you to change the specimen (or objective). Focus Wheel check box Clicking on this check box activates / deactivates the focus wheel of the microscope. Use of the optional piezo objective focusing device The HRZ Step slider is used to set the step width of the fine focusing device. • Use the arrows ▲ and ▼ of HRZ to move the fine focusing device upwards or downwards in steps. As soon as the focus position is changed (via handwheel or software), the piezo objective focus is automatically leveled. • A click on the L button moves the piezo objective focus in the center position of its travel range and the focus position is reset accordingly. Therefore, the same Z-level remains visible (the current position is not set to zero). The motor focus of the stand is operated in the same way via the relevant buttons. Moving into the Work or Load position is always performed via the motor focus and not via the piezo objective focus. Please see CHAPTER ANNEX of the printed manual for further information on the piezo objective focus. 03/06 B 45-0019 e 4-115 OPERATION Acquire Menu Carl Zeiss 4.5.9.3 LSM 5 LIVE LSM 5 LIVE DuoScan Stage Position Panel The Stage Position panel shows a symbolic specimen carrier in the left upper. The buttons for moving to a position and mark it are below or on its right. The Current Position display for X and Y is below. Below that, you will find the Marks selection box of marked positions and the possibility to activate and delete them. Moving the scanning stage Fig. 4-103 Stage Position panel The scanning stage can be moved using the joystick, or software-controlled using the Stage XY buttons, or manually. Stage XY buttons • Clicking on the arrow buttons moves the stage in X or Y direction. • Clicking on the Center button moves the stage in the XY = 0 position. XY Step slider 1 μm is the smallest value which can be set for XY movement, and 100 μm the highest. Manual check box This check box activates / deactivates the motor control of the stage and the joystick, if available. If Manual is active, the scanning stage can be moved manually via the knurled screws. The Move To and Center function buttons in Stage Position are without a function. The Current Position is updated. You can zero the display via ZERO and mark manually set positions (Mark pos.). The scanning stage cannot be moved via the software or the joystick. If Manual is deactivated, the scanning stage can be moved via the software or the joystick. All the functions of the Stage Position window are available. Current Pos(ition) field Current Pos displays the currently set stage position in relation to the zero position. Marks selection box Clicking on the arrow button displays the table of the session-related marked specimen areas. The table includes the ordinal number, the X-position and the Y-position. Click on the appropriate mark to select it for operation. 4-116 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Acquire Menu Carl Zeiss Move To button Clicking on the Move To button moves the stage to the position selected before from the Marks selection box. Remove The Remove command enables a selected position to be deleted from the table. The position then also disappears from the specimen carrier display. The selected position is deleted, the position with the next number in sequence moves up one number. Remove All The Remove All command deletes all the entries marked in the current session. Speed selection box Clicking on the arrow key displays the table of the available speeds for stage movement. Click on the appropriate speed to select it for operation. Zero button Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and Y. The already marked object areas thus receive new X and Y-coordinates. Mark Pos. button Mark Pos. allows the Current Position to be marked. This marked position is then stored in the Marks selection box in sequence. The marked position is shown on the specimen carrier with a cross and its ordinal number. HRZ Zero button Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and Y. The already marked object areas thus receive new X and Y-coordinates. 03/06 B 45-0019 e 4-117 OPERATION Acquire Menu Carl Zeiss 4.5.9.4 Fig. 4-104 LSM 5 LIVE LSM 5 LIVE DuoScan Tile Scan Panel This function permits a frame to be created as an overview image of the specimen with a maximum size of 4096 x 4096 pixels. According to settings, such a frame is divided in XY-tiles of 1 x 1 to the maximum of 15 x 15. A tile of special interest (target) can then be selected for scanning. Tile Scan window The application of the Tile Scan function requires an objective with a minimum magnification factor of 2.5x. Tiles Numbers X / Y input box Input of the number of tiles for X or Y from which the frame is to be composed. Tile Size X / Y display Display of the size of a single tile in µm (corresponds to the value selected in the Scan Control window). Frame Size X / Y display Display of the frame size of the tile scan for X or Y. Specification in pixels and µm. Move To button If the Move To button is activated, a rectangle with a target allowing the selection of the region of interest is positioned in the center of the scanned frame. Click and hold down the left mouse button to drag the rectangle to the required specimen area. When you release the mouse button, the stage moves to the selected position. Mark button If the Mark button is activated, marks previously set in the Tile Scan image are displayed, and further marks can be added at spots of special interest by a mouse click in the Tile Scan image. By activating the Move To button, the stage can be moved to the individual marks set in Tile Scan in the same way as it is moved to the marks set in the Stage Position panel. Creating an overview image • Set the number of tiles for the frame in the Tiles Numbers X / Y input boxes of the Tile Scan window. − The resulting frame size is displayed on-line. • Click on Start. − The overview frame is scanned and displayed on the screen in a new Image Display window. 4-118 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-105 OPERATION Acquire Menu Carl Zeiss Image Display window of a Tile Scan • Activate the Move To button. • In the tile scan image, move the target to the required spot of the frame (dragging with the mouse). − The microscope stage then travels to the selected position. Or: • Activate the Mark button. • Set a mark at the spot of interest by clicking with the mouse in the Tile scan image. A cross with the consecutive number of the mark is displayed in the Tile Scan image. The new mark is also displayed in the specimen carrier (Stage Position panel) and included in the Marks selection box. • Select the mark in the Marks selection box and click on the Move To button in the Stage Position panel. The stage moves to the selected position. • Then click on the Single button in the Scan Control window to scan the selected area as a single image. − The single image is scanned and displayed in a new Image Display window. Overlay functions cannot be activated in the Tile Scan Image Display window. The created overview frame can then be stored like any other scan image. If a stored overview frame is opened again, the rectangle with target will appear again. However, it can be deleted using the Overlay function. 03/06 B 45-0019 e 4-119 OPERATION Acquire Menu Carl Zeiss 4.5.10 LSM 5 LIVE LSM 5 LIVE DuoScan VIS, TV and LSM Buttons The VIS, TV and LSM buttons are included in the Acquire subordinate toolbar of the Main menu. They switch the beam path and indicate which beam path has been set in the binocular tube of the microscope: − VIS: observation via the eyepieces of the binocular tube, lasers are off − TV: camera observation (if connected) via camera adapter of the binocular tube − LSM: screen observation via laser excitation using the LSM 5 LIVE and software evaluation If the beam path of the microscope is changed manually via buttons on the tube this is recorded by the software and the relevant button is activated automatically. Vice versa, the beam path can be "switched" via activation of the appropriate button in the software. 4-120 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6 OPERATION Process Menu Carl Zeiss Process Menu • In the Main menu toolbar, click on Process. − This opens another, subordinate toolbar in the Main menu. Fig. 4-106 Process menu The functions of the Process menu permit already stored scan images to be subsequently linked and processed using mathematical functions and algorithms. 4.6.1 Add The Add function links two channels each of one or two images into a new channel through addition. The channel created in this way can be stored via the Save As function. 4.6.1.1 Open / Close the Add Window • Click on the Add button in the Process subordinate toolbar of the Main menu. − This opens the Add window. • Click on the Close button to quit the Add window. Fig. 4-107 03/06 B 45-0019 e Add window 4-121 OPERATION Process Menu Carl Zeiss 4.6.1.2 LSM 5 LIVE LSM 5 LIVE DuoScan Source Panel In the Source 1 panel, the first image source for the addition process is determined. The current image is displayed in the display box of the image selection box. Fig. 4-108 Source 1 panel Proceed as follows to select an image via the image selection box: • Click on the arrow button. The image selection box is opened and all the currently loaded images are displayed in a minimized form. • Click on the required image. This image will then appear in the display box of the image selection box and has been selected as Source 1. Use the Click into window button to directly select the opened image: • Click on the Click into window button first and then double-click on the relevant Image Display window. The selected image will then be displayed in the display box of the image selection box and has been activated as Source 1. The channel which is to be used for the Add operation is selected via the Channel selection box: • Click on the arrow button. The Channel selection box is opened and shows all the recorded channels of the relevant image. • Click on the required channel to activate it. In the Source 2 panel, the second image source for the addition process is determined. The procedure is identical to that for Source 1. • Select the image for Source 2 and the relevant channel. 4.6.1.3 Destination Panel In the Destination panel, it is determined in which Image Display window the Add operation is performed, and the data format which the newly created image shall have. Fig. 4-109 Destination panel The Add operation can be performed in an already opened window or in a new Image Display window. • Click on the arrow button of the image selection box to open this box. • Click on the relevant image if the Add operation shall be performed in an existing Image Display window. or • Click on New Image 8 bit or New Image 12 bit to use a new Image Display window. You can also use the Click into window button for image selection. Clicking on the New 8 bit or New 12 Bit button enables you to determine directly and quickly whether the new image is to be created in the 8-bit or 12-bit format. 4-122 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Process Menu Carl Zeiss If an existing Image Display window is used to perform the Add function, you must determine whether an existing channel shall be overwritten with the Add operation or whether a new channel shall be added. • In the Channel selection box, click on the channel which shall be overwritten, or click on New for a new channel. 4.6.1.4 Add Panel In the Add panel, the currently set formula for the Add operation is displayed. The editable input boxes permit the formula to be changed with any numeric values. Fig. 4-110 Add panel • Click in the required input box and enter the relevant value. • Click on the Apply button to perform the operation in the activated window or a new Image Display window. • The new image can then be stored via the Save As function. 4.6.1.5 Preview Panel The Preview function enables you to preview the result of the defined Add operation in a preview window. Fig. 4-111 Preview panel Preview check box with a click • Activate the of the mouse. The Add - Preview Image Display window is displayed with the operation result. • Deactivate the Preview check box to close the Add - Preview Image Display window. After a change of the formula in the Add panel, click in the Add - Preview Image Display window for an update. 03/06 B 45-0019 e 4-123 OPERATION Process Menu Carl Zeiss 4.6.2 LSM 5 LIVE LSM 5 LIVE DuoScan Subtract The Subtract function links two channels each of one or two images into a new channel by subtraction. The channel created in this way can be stored via the Save As function. 4.6.2.1 Open / Close the Subtract Window • Click on the Subtract button in the Process subordinate toolbar of the Main menu. − This opens the Subtract window. • Click on the Close button to quit the Subtract window. 4.6.2.2 Fig. 4-112 4-124 Subtract window Performance of the Subtract Function This function is performed in the same way as the Add function (see Add, page 4-121). The only difference is that the mathematical formula is based on subtraction. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.3 OPERATION Process Menu Carl Zeiss Multiply The Multiply function permits two channels each to be linked into a new channel by multiplication. The channel created in this way can be stored via the Save As function. 4.6.3.1 Open / Close the Multiply Window • Click on the Multiply button in the Process subordinate toolbar of the Main menu. − This opens the Multiply window. • Click on the Close button to quit the Multiply window. 4.6.3.2 Performance of the Multiply Function This function is performed in the same way as the Add function (see Add, page 4-121). The only difference is that the mathematical formula is based on multiplication. 03/06 Fig. 4-113 B 45-0019 e Multiply window 4-125 OPERATION Process Menu Carl Zeiss 4.6.4 LSM 5 LIVE LSM 5 LIVE DuoScan Ratio The Ratio function permits to create a new image or image series by offsetting two images or image series against each other. The channel created in this way can be stored via the Save As function. 4.6.4.1 Open / Close the Ratio Window • Click on the Ratio button in the Process subordinate toolbar of the Main menu. − This opens the Ratio window. • Click on the Close button to quit the Ratio window. 4.6.4.2 Performance of the Ratio Function This function is performed in the same way as the Add function (see Add, page 4-121). However, self edited formulas can be used for image calculation. Fig. 4-114 4-126 Image Calculation window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.4.3 OPERATION Process Menu Carl Zeiss Formula Panel The formulas can be typed in using the keyboard. • Click the Operators button to open the permissible operators list. • Select the operator(s) from this list (Fig. 4-115) and click the Insert button. The operators will be inserted into the formula at the cursor position. By highlighting the selected operator a description of the function of this operator is displayed in the lower part of the operators list. The calculation works pixel by pixel and starts with the upper left pixel irregardless of the image size of the two images or image series. When choosing images of different data depth the check box next to Intensity range 1…0 should be marked. The image intensity for all images will then be normalized to values between 1 and 0. 4.6.5 Fig. 4-115 Operators window Fig. 4-116 Copy window Copy (Channel) The Copy function permits one channel each of an existing image to be copied and stored as a new image. The selection of Source, Channel and Destination is made in the same way as in the Add function (see Add, page 4-121). 4.6.5.1 Open / Close the Copy Window • Click on the Copy button in the Process subordinate toolbar of the Main menu. − This opens the Copy window. • Click on the Close button to quit the Copy window. 03/06 B 45-0019 e 4-127 OPERATION Process Menu Carl Zeiss 4.6.5.2 LSM 5 LIVE LSM 5 LIVE DuoScan Performance of the Copy Function • Select Source, Channel and Destination and then click on the Apply button. − The image of the copied channel is then displayed in a new window or in the Image Display window activated for it. • The new image can be stored via the Save As function. For Z Stacks or Time Series, the entire series of the selected channel is copied. 4.6.6 Duplication (Image) This function permits images (including Z Stacks and Time Series) to be duplicated completely. • If several images have been opened, select the image to be duplicated. • Click on the Dup button in the Process subordinate toolbar of the Main menu. − The selected image is duplicated and displayed in a new Image Display window. • Use the Save As function to store the image under a new name. 4-128 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.7 OPERATION Process Menu Carl Zeiss Filter The filter function permits the subsequent processing of scanned images via the integrated Lowpass, Sharpness and Median filters. Furthermore, User-defined filters can be installed by the user. User-defined filters can be stored, reloaded and removed. 4.6.7.1 Open / Close the Filter Window • Click on the Filter button in the Process subordinate toolbar of the Main menu. − This opens the Filter window. • Click on the Close button to quit the Filter window. 4.6.7.2 Image Panel In the Image panel, the image or channel to be processed is selected. Fig. 4-117 Filter window The currently selected image is displayed in the image selection box. Proceed as follows to select an image via the image selection box: • Click on the arrow button. The image selection box is opened and all the currently loaded images are displayed in a minimized form. • Click on the required image, which will then appear in the display box of the image selection box and will be available for filtering. You can also use the Click into window button to select the image. • Open the Channel selection box with a click on the arrow button and select the channel to be processed. 4.6.7.3 Filter List and Edit Panel In the Filter List panel, the filters and the matrix size (Kernel Size) are selected. The matrix of the selected filter and the set filter parameters Factor, Divisor and Offset are displayed in the Edit panel. 03/06 B 45-0019 e 4-129 OPERATION Process Menu Carl Zeiss (1) LSM 5 LIVE LSM 5 LIVE DuoScan Kernel size The size of the filter matrix can be modified here. The effect of a filter increases along with the matrix size. However, this also increases the time required for filtering. • Select the required matrix size by clicking on one of the selection buttons 3 x 3, 5 x 5 or 7 x 7. (2) Lowpass filter With the lowpass filter, the gray value of each center pixel is replaced with the average value of the surrounding neighbor pixels. The viewed neighbor pixels are defined by a square. The modified pixel now is the center pixel of the filter matrix. Fig. 4-118 Filter List and Edit panel (Lowpass) Image noise will be reduced by the application of the lowpass filter. The cutoff of regions will blur. Local maxima will be flattened. The dynamic range will be reduced considerably. This filter permits the matrix size to be modified only in the 3 preset steps. (3) Sharpness filter With the sharpness filter, the original image is filtered with a lowpass filter first. The result of this filtering is then subtracted from the original image. This will improve image sharpness. The matrix size can be modified in the 3 preset steps. Furthermore, divisor values ranging from 1 to 78 can be entered. The higher the divisor value, the lower the image sharpness. Fig. 4-119 4-130 Filter List and Edit panel (Sharpness) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (4) OPERATION Process Menu Carl Zeiss Median filter With the median filter, the gray value of each center pixel is replaced with the median value of the surrounding neighbor pixels. The viewed neighbor pixels are defined by a square. The modified pixel now is the center pixel of the filter matrix. The median value is defined as the middle value (not average) of all the gray values sorted in ascending order within a matrix. Image noise will be reduced by the application of the median filter. The cutoff of regions will slightly blur. Local maxima will be flattened. The dynamic range will be reduced considerably. The settings of this filter can not be modified. (5) Fig. 4-120 Filter List and Edit panel (Median) Fig. 4-121 Filter window (User-defined filter) User-defined filter The User-defined function permits you to create your own filters. In addition to the Kernel Size, the parameters Factor, Divisor and Offset can be modified here. The filter result can be subtracted from the original image via the Subtract from Source check box. Proceed as follows to store User-defined filters: • Click on the Add To List button and enter a name in the Add Filter To List window. The name will be included in the Filter List. Proceed as follows to activate stored, Userdefined filters: • Click on the name of the filter in the Filter List. The filter will then be activated immediately. Proceed as follows to delete User-defined filters: 03/06 B 45-0019 e 4-131 OPERATION Process Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click on the name of the filter in the Filter List and then on the Remove button. The filter will be deleted. • After selection of the required filter, click on the Apply button to start the filter procedure. Fig. 4-122 Add Filter to List panel − Filtering will be performed and displayed in the current Image Display window. • In the case of images with several channels, activate the xy button in the Display image toolbar to display all the channels. Each channel must be filtered separately. • Use the Save As function to store the newly created image. 4.6.7.4 Fig. 4-123 Preview panel Preview Panel The Preview function allows you to have the result of the Filter operation displayed as a preview image. Preview check box with a click of the mouse. The Filter - Preview Image Display • Activate the window with the filter result will be displayed. • Deactivate the Preview check box to close the Filter - Preview Image Display window. After a change of the filter settings, click in the Filter - Preview Image Display window once to update it. 4.6.8 Contrast The Contrast function permits the subsequent modification of contrast and brightness of the stored image. • Open the image to be processed and click on the Contrast button. − The function is performed with firmly set parameters and the result is displayed in a new Image Display window. The procedure can be repeated as often as required. • The newly created image can be stored using the Save As function. 4-132 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.9 OPERATION Process Menu Carl Zeiss Interpolate This function permits the continuous contrast and brightness change in a Z- stack or Z- stacks over time through interpolation between the starting and end values. This permits the subsequent compensation of signal loss when imaging further into deeper tissue regions where the excitation efficiency decreases but also the detection efficiency. Interpolation can be defined for the entire image or only for individual channels. The Modify Series macro can be used to convert time series data for use of the Interpolate function. 4.6.9.1 Open / Close the Interpolate Brightness and Contrast Window • Click on the Interpolate button in the Process subordinate toolbar of the Main menu. − This opens the Interpolate Brightness and Contrast window. Fig. 4-124 Interpolate Brightness and Contrast window Fig. 4-125 Image panel • Click on the Close button to quit the window. 4.6.9.2 Image Panel The image to be processed is selected in the Image panel. The currently selected image is shown in the display box of the image selection box. Proceed as follows to select a series via the image selection box: • Click on the arrow button. The image selection box will be opened and all the currently loaded images will be displayed in a minimized form. • Click on the required image, which will then appear in the display box of the image selection box and has been selected for the interpolation procedure. You can also use the Click into window button for image selection. 03/06 B 45-0019 e 4-133 OPERATION Process Menu Carl Zeiss 4.6.9.3 LSM 5 LIVE LSM 5 LIVE DuoScan Interpolation Panel In the Interpolation panel, the parameters for the interpolation procedure are set. • Use the Start Image slider to select the slice at which the interpolation procedure shall start. Clicking on the First button permits the fast selection of the first slice in the series. • Use the Brightness and Contrast sliders to set the image brightness and contrast for the first slice (Start Image). Fig. 4-126 Interpolation panel • Use the End Image slider to select the slice at which the interpolation procedure shall end. Clicking on the Last button permits the fast selection of the last slice in a series. • Use the Brightness and Contrast sliders to set the image brightness and contrast for the last slice (End Image). • Use the available Channel buttons (e.g.: Ch1) to select the channel for interpolation or click on the All button if the entire image is to be interpolated. • Having set the parameters, click on the Apply button. Interpolation will be performed in a new Image Display window. • The newly created image (series) can be stored using the Save As function. If you activate the Overwrite Source Images check box, interpolation will be performed in the current Image Display window. If you activate the Ignore Images less than "Start Image" and greater than "End Image" check box, only the slices lying between Start Image and End Image will be taken into consideration for interpolation. Otherwise, brightness and contrast will also be changed for the other slices. 4-134 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.9.4 OPERATION Process Menu Carl Zeiss Preview Panel The Preview function enables you to see the result of interpolation for one slice each in a preview window. Fig. 4-127 • Activate the Preview panel Preview check box with a click of the mouse. − The Interpolate C&B - Preview Image Display window will be displayed. At the same time, the Slice slider with the relevant input box and arrow keys and the two buttons Start and End are displayed in the Preview panel. • Use the slider or input box / arrow keys to set the slice which shall be displayed in the preview window. • Clicking on the Start or End button permits the fast activation of the Start Image or End Image for previewing. • Deactivate the Preview check box to close the Interpolate C&B - Preview Image Display window. 4.6.10 Channel Shift The Channel Shift function is used to produce a congruent image with relation to the pixels of the various channels. This pixel correction function important in UV applications. 4.6.10.1 is particularly Open / Close the Channel Shift Window • Click on the Shift button in the Process subordinate toolbar of the Main menu. Fig. 4-128 − This opens the Channel Shift window. Channel Shift window • Click on the Close button to quit the window. 03/06 B 45-0019 e 4-135 OPERATION Process Menu Carl Zeiss 4.6.10.2 LSM 5 LIVE LSM 5 LIVE DuoScan Image Panel • Click on the arrow button. The image selection box will be opened and all the currently loaded images are displayed in a minimized form. Fig. 4-129 Image panel • Click on the required image, which will then appear in the display box of the image selection box and has been selected for the Shift function. You can also use the Click into window button for image selection. 4.6.10.3 Shift Panel • Select the channels required for processing in the Shift panel by clicking on the Ch1 or Ch3 will appear in the button buttons. A tick when the channels are activated. Fig. 4-130 Shift panel and buttons to • Use the scrollbar or the select the pixel shift in the horizontal and vertical direction. • Click on the Apply button to activate the setting. 4-136 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.10.4 OPERATION Process Menu Carl Zeiss Preview Panel • If Preview is activated, a preview of the shift is shown in a separate Image Display window. Fig. 4-131 Preview panel The following image shows the result of a pixel shift via the Shift function. This image change can be stored in the image database via the Save or Save As buttons. For applications requiring 3- or 4-channel scanning, proceed in the same way as described for the 1- or 2channel mode. Fig. 4-132 4.6.11 Image Display window with channel shift Unmix The Unmix functionality permits to extract the emission of single fluorescence dyes (e.g. GFP only, YFP only etc.) from the overall emission band of strongly overlapping multifluorescence signal intensities by a pixelwise linear unmixing procedure. Mathematically, experimental fluorescence spectra of monolabelled samples are taken as an external reference. Up to 8 different reference signals can be varied in this least-square-fit based algorithm to produce an 8 channel multifluorescence stack without any partial overlap between the channels. 03/06 B 45-0019 e 4-137 OPERATION Process Menu Carl Zeiss 4.6.11.1 LSM 5 LIVE LSM 5 LIVE DuoScan Open / Close the Unmix Window • Click on the Unmix button in the Process subordinate toolbar of the Main menu. − This opens the Linear Unmixing window. • Click on the Close button to quit the Unmix window. 4.6.11.2 Source Panel In the Source panel the image source for the linear unmixing process has to be defined. This has to be a Stack, a Stack Z series or a Stack T series. Proceed as follows to select an image via the image selection box: • Click on the arrow button. The image selection box is opened and all the currently loaded images, stacks, time series with a Lambda dimension are displayed in a minimized form. • Click on the required image. This image will then appear in the display box of the image selection box and has been selected. 4.6.11.3 Definition of Channels Panel In the Channels panel the number of reference spectra (number of fluorescence channels) can be selected from the channel selection boxes. • Select the references fluorescence dye spectra which are present in the sample with the check boxes. • If necessary change the colors of the relevant fluorescence channel. • A new window with the resulting channels of the unmixing procedure opens immediately. There are several settings that can be chosen for linear unmixing. Autoscale balances the intensity of the unmixed images. Generate channels with Residuals shows the difference between the offered spectrum and the resulting image in intensity values. The higher the intensity in this additional channel the worse the fit of the spectra to the dataset. This occurs for example when pixels are saturated and indicates to repeat the image acquisition with different settings to avoid overexposure or in extreme cases to chose different reference spectra. Ask for Background ROI, when this is checked a window appears before the calculation starts which asks for the spectrum of the background which is then subtracted from the images prior to unmixing. Advanced Linear Unmixing restarts the unmixing process again if negative values are generated. The negative values are set to zero and ignored for the next unmixing calculation. Ignore Overexposed Pixels and Ignore Underexposed Pixels will exclude this pixels for calculation of the unmixed images. 4-138 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Process Menu Carl Zeiss Multichannel Unmixing allows to apply the unmixing algorithm to a multichannel image (up to 8 channels) without the use of reference spectra. The calculation of residuals and the subtraction of background based on a background spectrum are not available when this procedure is chosen. It will not work for heavily overlapping signals. Try to avoid saturation of fluorescence signals in the stack to be unmixed. This will generate a high signal in the residuals channel. To get the highest quality unmixing results, please define an extra background channel, if possible. Fig. 4-133 03/06 B 45-0019 e Multichannel unmixing 4-139 OPERATION Process Menu Carl Zeiss Fig. 4-134 4-140 LSM 5 LIVE LSM 5 LIVE DuoScan Image Display window after unmixing B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.12 OPERATION Process Menu Carl Zeiss Ion Concentration The use of this function (option) permits the calibration of ion concentrations in physiological experiments. 4.6.12.1 Open / Close the Ion Concentration Window • Click on the Ion Conc button in the Process subordinate toolbar of the main menu. − This opens the Ion Concentration window. • Click on the Close button. Fig. 4-135 4.6.12.2 Ion Concentration window Function Description Ion Conc button Activates the Ion Concentration window. Source window Selects input of images to be processed. Destination window Select output and pixel depth of processed image. Calibration window Sets the six different calibration options, according to the dyes used (single wavelength, ratiometric) and required method. Show Curve button Shows resulting calibration curve. Image scaling window Sets min. and max. concentration. Preview window Activates Preview function. 03/06 B 45-0019 e 4-141 OPERATION Process Menu Carl Zeiss 4.6.12.3 LSM 5 LIVE LSM 5 LIVE DuoScan Single Wavelength Dyes – Offline Calibration • Subtract background/autofluorescence image from raw images to obtain. • Perform equation- or titration calibration (compare F with a calibration curve → titration calibration or put F values in calibration formula). Fig. 4-136 4-142 Ion Concentration window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.12.4 OPERATION Process Menu Carl Zeiss Ratiometric Dyes • Fura-2, Indo-, SNARF, Cameleon, Ratiometric Pericam, Phluorin, ... • Display fluorescence ratio R over time • Display fluorescence ratio R corrected for background/autofluorescence over time • Calculate absolute ion concentrations (pixel by pixel) via titration calibration (known ion concentrations applied to the cells – in situ – or in solutions – in vitro or equation calibration where possible [Fura-2, Indo-, SNARF] • Calculation of R eliminates artifacts and uncertainties caused by − inhomogenous dye distribution − photobleaching − may be applied with moving cells 4.6.12.5 Ratiometric Dyes - Online Ratio R(t1) = F1(t1) / F2(t1), R(t2) = F1(t2) / F2(t2) ... Fig. 4-137 03/06 Ratiometric Dyes – Online ratio B 45-0019 e 4-143 OPERATION Process Menu Carl Zeiss 4.6.12.6 LSM 5 LIVE LSM 5 LIVE DuoScan Ratiometric Dyes - Calibration • Subtract background/autofluorescence images from raw images to obtain Rkorr [=F1-F1Background)/(F2-F2Background)] when calibration reference is not obtained with the experimental sample (in situ) • Calculate ratio R • Perform equation- or titration calibration (compare R with a calibration curve -> titration calibration or put R values in calibration formula) Fig. 4-138 4-144 Ratiometric Dyes - Calibration B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.12.7 OPERATION Process Menu Carl Zeiss Ratiometric Dyes - Equation Calibration (Grynkiewicz) Fura-2, Indo-1,.. KD (dissociation constant) taken from literature Rmin: derived from ion-free state of the dye (e.g. 0 Ca2+) Rmax: derived from ion-bound state of the dye (e.g. saturated with Ca2+) Fmin2 and Fmax2 are the minimum and maximum fluorescence intensities at wavelength 2 Fig. 4-139 Ion Concentration button Rmin, Rmax, Fmin2 and Fmax2 may be determined in the cells under investigation (in situ) or in solutions (in vitro) Calibration parameters may be saved and reloaded (*.cal) 4.6.12.8 Options for Calibration Image Selection (Equation- or Titration Calibration) • Click into image window. • Select source channel(s). • Optional background subtraction • Optional calculation of parameters from overlay region(s) 4.6.13 Image Scaling The use of this function permits the scaling of a imported image in voxel dimensions. Setting the values will provide the voxel dimensions in the X-,Y- and Z-directions ( Z-direction not for 2D images). 4.6.14 Copy Window Contents The use of this function permits to duplicate the displayed image. 03/06 B 45-0019 e 4-145 OPERATION Process Menu Carl Zeiss 4.6.15 LSM 5 LIVE LSM 5 LIVE DuoScan Copy Full Resolution The use of this function permits to display the current image with full resolution in a new Image Display window. 4.6.16 Paste Bitmap This function allows to paste bitmap images from the clipboard into a Image Display window. Images can be incorporated from other programs such as MS Excel, MS Powerpoint or MS Word. 4.6.17 Kinetic The use of this function (option) permits − a correction of FRAP data for bleaching and background − the fitting of the FRAP data to a mono exponential or double exponential model for intensity recovery. 4.6.17.1 Open / Close the Kinetic Analysis window • Click on the Kinetic button in the Process subordinate toolbar of the main menu. − The Kinetic Analysis window opens. • Click on the Close button. 4-146 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.6.17.2 OPERATION Process Menu Carl Zeiss Function Description Click into window Allows to select the image series of the FRAP experiment to be analyzed by clicking into the appropriate image window. Channel pull down menu Allows to select single channels or all channels for analysis. Kinetic Model pull down menu Allows to select the mathematical model (mono or double exponential model) for fitting the data. Background Region Displays a button Select (Click) that allows to chose the region of interest which checkbox represents the mean background intensity that should be used to correct the data. First Click Select (Click), than click into the ROI that has been drawn using the drawing tools in the Mean ROI image display. If a region has been selected a cross appears next to the button. Reference Region checkbox Displays a button Select (Click) that allows to chose the region of interest which represents the fluorescence intensity of a reference cell which has not been bleached. The mean intensity within that region is used to correct the data at each time point for any bleaching artifact that occurred during the imaging process. First Click Select (Click), than click into the ROI that has been drawn using the drawing tools in the Mean ROI image display. If a region has been selected a cross appears next to the button. Combine Regions checkbox Opens a further dialogue with the following options: Add Region, New Group, Remove Group and Remove All. It provides the possibility to chose more than one ROI for analysis and also to group them together according to the experimental set up. First click Add Region, than click into the ROI that has been drawn using the drawing tools in the Mean ROI image display. For each added ROI a cross is displayed in the line. Click Add Region for each ROI to be added. Clicking New Group puts the cursor in the next line and further ROIs or Group of ROIs can be added for analysis. Apply Performs the calculations and the fitting of the data. 03/06 B 45-0019 e 4-147 OPERATION Process Menu Carl Zeiss 4.6.17.3 LSM 5 LIVE LSM 5 LIVE DuoScan Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins Display of the image series in the Mean ROI display mode: The drawing tools are used to define the ROI to be analyzed (ROI 1), the background ROI (ROI 2), and the reference ROI (ROI 3). The reference ROI must be a neighboring cell which has been imaged with the same laser intensity over time identical to the cell, which has been bleached to induce FRAP. Make sure the whole cell or cell compartment of interest is imaged and therefore illuminated. Use the Slice submenu in the Select toolbar of the image window to display the image right after the bleach event. This makes it easy to chose the ROI for analysis. The region should be slightly smaller than the original region that has been bleached. The latter is listed in the Mean of ROIs list in the image window. In the ROI Mean Display three ROIs are defined. See Additional Display Mode in Time Series, page 4-312 for further details. Fig. 4-140 Image window displaying the first bleached image of a images series • Open the Kinetic dialogue and chose the image series for analysis. • Mark the checkboxes for background and reference region. • Press Select (Click) for the Background Region, than click into the ROI number 2 (Background region) in the image. The value of the mean intensity of this region will be subtracted from the intensity values within the ROI to be analyzed. • Press Select (Click) for the Reference Region. The intensity values of the ROI to be analyzed will be corrected for changes in intensity calculated for the reference region. The correction is done for each time point taking the actual intensity difference in the reference region into account. • If no other option is chosen the remaining ROI or ROIs are used for analysis. Each ROI is analyzed separately. • Chose the Kinetic Model in the pull down list, then press Apply. − The result of the fit is displayed in the table. The result can be copied to the clipboard (Copy Results) or saved as a text file (Save Results). The following values are calculated and shown: − The final signal intensity in the analyzed ROIs following recovery I0 (of the fitted curve). 4-148 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Process Menu Carl Zeiss − The amplitude of the fitted curve (which equals the mobile fraction) I1 mobile fraction. − The fitted parameter T1 (seconds). − The rate constant for the exchange of molecules between the bleached region and the surrounding area K (per second). − The part of the immobile fraction of the protein I delta immobile fraction. Show Table opens a further table beneath the results. It shows all intensity values being corrected for background intensity and intensity loss of the reference region. The values can be saved as a text file (Save Table) or copied into Excel via clipboard (Copy Table). The data can be normalized optionally when marking the checkbox Normalize in the Kinetic Analysis Diagram toolbar. The calculation of the parameters is based on the same ROIs unless other ROIs or moved ROIs are selected again. The Kinetic Display is always available once the analysis has been performed. The analysis is not stored with the image. Note that this modeling is a very basic approach to your experiment. It offers a first hint on the possible presence of only one or, in case of a bad fit, more than one mobile fractions of the labeled protein within the cell or cell compartment examined. The half time of the recovery can be calculated using the following formula: Fig. 4-141 t half = ln 0.5 − T1 Image window displaying the analysis of FRAP data using a mono exponential fit If the analysis is done using the double exponential fit the fitted curve displays the mean of the fitted values for the two different mobile fractions. The table shows the following additional parameters: 03/06 B 45-0019 e 4-149 OPERATION Process Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan − The amplitude of the two curves, displayed as one (which equals each part of the mobile fractions) I1 and I2. − The fitted parameters T1 and T2 (seconds) for each mobile fraction. − The rate constant for the exchange of molecules between the bleached region and the surrounding area K1 and K2 (per second) for each mobile fraction. Fig. 4-142 Image window displaying the analysis of FRAP data using a double exponential fit The raw data of the experiment can be exported for further analysis using the Mean ROI display mode and, within this dialogue, the table display of the results. Note that this modeling is also a very basic approach to your experiment. It offers a first hint on the possible presence of two mobile fractions of the labeled protein within the cell or cell compartment examined. Please refer to relevant scientific literature or the website of the EAMNET (http://www.embl.de/eamnet) for further information on how to set up and perform FRAP experiments. A schematic curve marking the data points that are calculated performing the Kinetic Analysis is shown in Fig. 4-143. Please note, that the naming of the data points is not consistent with the information on the website. 4-150 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-143 03/06 OPERATION Process Menu Carl Zeiss Schematic FRAP curve with marks at the relevant data points. I0, I1 and I delta which are calculated performing the Kinetic Analysis B 45-0019 e 4-151 OPERATION 3D View Menu Carl Zeiss 4.7 LSM 5 LIVE LSM 5 LIVE DuoScan 3D View Menu The 3D View functions serve to record and play back series of images for 3D display of microscopic structures. • In the Main menu toolbar, click on 3D View. − This opens another, subordinate toolbar in the Main menu. Fig. 4-144 3D View menu 4.7.1 3D DepthCod (Color Coded Depth Map) By means of the Depth Coding function, the depth information contained in a sequence can be colored with the colors of the rainbow, in which case "blue " stands for front and "red" stands for rear. A stack of images must be available. 4.7.1.1 Open / Close the Depth Coding Window • Click on the DepthCod button in the 3D View subordinate toolbar of the Main menu. Fig. 4-145 Depth Coding window − This opens the Depth Coding window. • Click on the Close button to quit the window. 4-152 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.7.1.2 OPERATION 3D View Menu Carl Zeiss Source Panel In the Source panel, the image source is selected. The currently selected image is displayed in the display box of the image selection box. Proceed as follows to select an image via the image selection box: • Click on the arrow button. The image selection box will be opened and all the currently loaded images are displayed in a minimized form. • Click on the required image, which will then be shown in the display field of the image selection box and be available for the following operation. The Click into window button enables you to select the opened image directly: • Click on the Click into window button first and then double-click on the relevant Image Display window. The selected image will then be shown in the display box of the image selection box. Select the channels to be processed via the Channel selection box: • Click on the arrow button to open the selection box. Click on the required channel to activate it. 4.7.1.3 Depth Coding Panel • On the Depth Coding panel you can set the desired parameters. Activate the Scale Bar if you want a color scale to be check box shown. Fig. 4-146 (1) Depth Coding tab Depth Coding tab Mode Front View: The image is viewed from the front / above when this option is activated. Mode Rear View: The image is viewed from the rear / below when this option is activated. Threshold: All brightness values below the Threshold (range: 0 to 255) are ignored or treated like 0 when determining the depth and the display. Contrast: Defines the factor with which the contrast of the overlaid series affects the contrast of the depth-coded color. Brightness: Defines the factor with which the brightness of the overlaid series affects the brightness of the depth-coded color. Display Scale Bar: Displays a colored scale in the image. Display Grey level: The depth information is displayed in gray levels. 03/06 B 45-0019 e 4-153 OPERATION 3D View Menu Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Transparency tab Mode Maximum: The color is defined by the z position of the brightness value. Mode Transparent: The transparent projection is built up from the rear to the front. The color is defined by the Z position at which the original was last higher than or equal to Threshold. Mode Keep Maximum: Activating this option modifies the specification governing calculation of the projection. Threshold: Pixel value at which the ramp rises (variable from 0 to 100 %). Ramp: Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise). Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %; 0 corresponds to the bottom edge in the diagram). Fig. 4-147 4-154 Transparency tab B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.7.1.4 OPERATION 3D View Menu Carl Zeiss Preview Panel The Preview function permits you to regard the influence of parameter changes in an Image Display window. • After finding the optimum adjustment using the Preview function, you have to generate the final version of the image using the Apply button. − The system then generates a color-coded depth map for the selected channel. 4.7.2 Fig. 4-148 Depth Coding image Fig. 4-149 Projection window Projection By means of the Projection function, one single projection or a series of projections can be calculated after rotation of the data package about the X, Y or Z axis. A stack of images must be available. 4.7.2.1 Open / Close the Projection Window • Click on the Projection button in the 3D View subordinate toolbar of the Main menu. − This opens the Projection window. • Click on the Close button to quit the window. 4.7.2.2 Source Panel • Select the image for the projection operation from the image selection box. 03/06 B 45-0019 e 4-155 OPERATION 3D View Menu Carl Zeiss 4.7.2.3 LSM 5 LIVE LSM 5 LIVE DuoScan Projection Panel • On the Projection panel, set the parameters needed for the animation: Turning Axis, First Angle, Number Projections and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab. (1) Projection tab Turning Axis X/Y/Z: Selects the axis about which the data package is to be rotated. First Angle: Rotation angle in degrees. Number Projections: Number of projections for a sequence (variable from 0 to 100). Difference Angle: Angle increment of a sequence. The number keys permit the direct selection of preset values for Number Images and Difference Angle. If the Panorama button is pressed, a panorama sequence of the image series is computed. Fig. 4-150 4-156 Projection tab B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION 3D View Menu Carl Zeiss Transparency tab Mode Maximum: The color is defined by the z position of the brightness value. Mode Transparent: The transparent projection is built up from the rear to the front. The color is defined by the z position at which the original was last higher than or equal to Threshold. Mode Keep Maximum: Activating this option modifies the specification governing calculation of the projection. Threshold: Pixel value at which the ramp rises (variable from 0 to 100 %). Ramp: Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise). Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %; 0 % corresponds to the bottom edge in the diagram). Brightness: The image can be brightened again by modifying the value (from 0.2 to 5). Fig. 4-151 4.7.2.4 Transparency tab Preview Panel The Preview function permits you to regard the influence of parameter changes in an Image Display window. Fig. 4-152 Preview panel The Slice slider enables you to select the slice which shall be displayed in the Preview Image Display window. 03/06 B 45-0019 e 4-157 OPERATION 3D View Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • After finding the optimum adjustment using the Preview function, you have to generate the final version of the image using the Apply button. − The projection appears. The computation can be followed in the image or by the progress bar. The computed 3D sequence can be animated with the Anim button in the Select toolbar. In addition, the Animate window appears, in which you can influence the direction and speed of 3D image rotation (see Animate, page 4-239). Fig. 4-153 Projection image You can browse through the rotation sequence manually with the Slice button in the Select toolbar and the Slice slider. • To view the computed 3D sequence as a gallery on the screen, click on the Gallery button in the Display toolbar. Fig. 4-154 4-158 Projection image (Gallery) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.7.3 OPERATION 3D View Menu Carl Zeiss Stereo Stereoscopic images can be generated in a variety of ways by means of the Stereo function. A stack of images must be available. 4.7.3.1 Open / Close the Stereo Images Window • Click on the Stereo button in the 3D View subordinate toolbar of the Main menu. − This opens the Stereo Images window. • Click on the Close button to quit the window. 4.7.3.2 Fig. 4-155 Source Panel Stereo Images window • Select the image for the projection operation from the image selection box. • Select the channel to be used from the Channel selection box. 03/06 B 45-0019 e 4-159 OPERATION 3D View Menu Carl Zeiss 4.7.3.3 LSM 5 LIVE LSM 5 LIVE DuoScan Stereo Images Panel • In the Stereo Images panel, set the parameters needed for stereoscopic viewing: Mode, Basic Angle, Right Left Angle, Number Images and Difference Angle in the Projection tab and the Mode parameters in the Transparency tab. (1) Projection tab Mode This displays a stereo image for red / green anaglyph observation using red / Red / Green Image: green spectacles. Mode Split Images: This displays a pair of stereo images for observation through a stereoscope. Colored stereo images are also possible. Basic Angle: Direction angle at which the specimen is viewed; 0° from the front, 180° from the rear. Right Left Angle: Angle between right and left (red and green) image. Number Images: Number of 3D images (slices). Difference Angle: Angle increment of a sequence. Fig. 4-156 Projection tab The number keys permit the direct selection of preset values for Number Images and Difference Angle. If the Panorama button is pressed, a panorama sequence of the image series is computed. 4-160 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION 3D View Menu Carl Zeiss Transparency tab Mode Maximum: The color is defined by the z position of the brightness value. Mode Transparent: The transparent projection is built up from the rear to the front. The color is defined by the z position at which the original was last higher than or equal to Threshold. Mode Keep Maximum: Activating this option modifies the specification governing calculation of the projection. Threshold: Pixel value at which the ramp rises (variable from 0 to 100 %). Ramp: Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise). Maximum Opacity: Degree of visibility at the top corner of the ramp (variable from 0 to 100 %; 0 corresponds to the bottom edge in the diagram). Brightness: The image can be brightened again by modifying the value (from 0.2 to 5). Fig. 4-157 03/06 Transparency tab B 45-0019 e 4-161 OPERATION 3D View Menu Carl Zeiss 4.7.3.4 Fig. 4-158 LSM 5 LIVE LSM 5 LIVE DuoScan Preview Panel The Preview function permits you to regard the influence of parameter changes in an Image Display window. Preview panel The Slice slider enables you to select the slice which shall be displayed in the Preview Image Display window. • To start computation of the stereoscopic image, click on the Apply button. − The image is built up twice (once each for the red and green colors), resulting in a stereoscopic image. The stereoscopic effect can only be seen with the aid of red / green 3D goggles. The red lens is to be used for the right eye and the green lens for the left eye. The presentation can be modified by selecting the Split Images (Mode) option in the Projection tab of the Stereo Images panel. Fig. 4-159 Fig. 4-160 4-162 • By clicking on the Apply button, the two stereo pairs are presented side by side and can be viewed without red / green 3D goggles. Stereoscopic image (red / green) Stereoscopic image (split) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.7.4 OPERATION 3D View Menu Carl Zeiss 3D DeConVolution (DCV) The 3D Deconvolution option is used for the resolution enhancement of fluorescence image stacks. When a three-dimensional object is reproduced by an optical system the resulting image of the object does not correspond exactly to the object's actual form. The image of the object is "distorted" as it passes through the optical system. In physical terms the actual object is convolved by the optical system's Point Spread Function (PSF). Fig. 4-161 Point Spread Function (PSF) The Point Spread Function describes how the light of a point object is distorted by the optical system. This "convolution" makes the image appear grainy and structures in the image seem blurred. This effect is most prominent in the axial (Z-)direction as each lens is optimized for the two-dimensional image of the object. If the PSF is known it is possible to use mathematical algorithms to undo this distortion. The image of the object is deconvolved using the PSF and the actual form is reconstructed: Fig. 4-162 Point Spread Function (PSF) The effect of 3D deconvolution can be demonstrated impressively on objects with a known form. As a rule fluorescent beads are used for this purpose. The following figure shows the 3D deconvolution of an image stack with a fluorescent bead with a diameter of 1 µm. 03/06 B 45-0019 e 4-163 OPERATION 3D View Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan As the resolution of an optical system is significantly lower in the axial direction than in the lateral (X/Y-)direction, the greatest improvement in resolution can be achieved in the Z-direction. The Z Stack must meet the following requirements: − At least two-fold oversampling in xyz (z: half of optimal interval button) − High signal-to-noise ratio − Detector gain < 500 V Calculation is either made for one channel of the opened image which must first be selected accordingly, or for all channels of a stack. Fig. 4-163 Image of a fluorescent bead with a diameter of 1µm before deconvolution (A,B) and after deconvolution (C,D) Calculation is started via Apply and can be stopped using the ESC key, if required. 4.7.4.1 Open / Close the 3D Deconvolution Window • Click on the DCV button in the Process subordinate toolbar of the Main menu. − This opens the 3D Deconvolution window. • Click on the Close button to quit the window. Fig. 4-164 4-164 3D Deconvolution window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.7.4.2 OPERATION 3D View Menu Carl Zeiss Source Panel The image to be processed is selected in the Source panel. The currently selected image is shown in the display box of the image selection box. Proceed as follows to select a image via the image selection box: Fig. 4-165 Source panel • Click on the arrow button. The image selection box will be opened and all the currently loaded images will be displayed in a minimized form. • Click on the required image, which will then appear in the display box of the image selection box and has been selected for the interpolation procedure. You can also use the Click into window button for image selection. 4.7.4.3 Deconvolution Panel The Deconvolution panel contains the two tabs Method and PSF. (1) Method tab The Method tab permits selection between the calculation methods Nearest Neighbour, Inverse and Iterative. (a) Fig. 4-166 Nearest Neighbor Method tab The Nearest Neighbor method is the simplest and fastest algorithm which in principle corresponds to a 3D sharpness filter. (b) Inverse Filter The regularized inverse filter generally achieves better results than the Nearest Neighbor algorithm. It is well suited to process several image stacks for a pre-selection of images for the use of the iterative highend methods. (c) Constrained Iterative The best image quality is achieved using the Constrained Iterative Maximum Likelihood Algorithm. Increasing the resolution in the image, especially in the Z-direction, is only possible with this method. Due to the complex mathematical method, depending on the image size and the PC being used the calculation can take up to several hours. In the Inverse method, the Restoration Effect slider permits the noise-to-signal ratio to be selected between the settings Weak (low noise) and Strong (pronounced noise). 03/06 B 45-0019 e 4-165 OPERATION 3D View Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Activation of the Auto detect check box will start a routine for the automatic determination of the noise level in the entire image part of the Z Stack (not available in the Nearest Neighbour method). If Auto detect is enabled, the Restoration Effect slider is disabled. The Iterative method permits (in addition to the parameters of the Inverse method) the maximum number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on the set image improvement (delta between last but one and last cycle in %), no matter whether the value under Maximum Iterations has been achieved or not. The Nearest Neighbour method permits entry of the Number of Neighbours and the Sharpness in Focus value in addition to the Restoration Effect. (2) PSF tab In the 3D Deconvolution option a theoretical point spread function (PSF) is calculated from the systems settings (objective data, wavelengths, aperture size). The PSF data are displayed in the PSF tab. In the case of wavelengths above 700 nm, the NLO button is automatically enabled. The displayed values are always taken over by the system data, but can be edited subsequently for simulation purposes. Fig. 4-167 4-166 PSF tab B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.8 OPERATION Macro Menu Carl Zeiss Macro Menu The macro function permits the recording, running and editing of command sequences and their allocation to buttons in the Macro menu. • In the Main menu toolbar, click on Macro. − This opens another, subordinate toolbar in the Main menu. Using the Macro Button (optional) opens the dialogue for editing and recording Macros based on VBA programming. Using the Visual Button (optional) allows to construct automated work flows using the arrangement of symbols which depict the single steps within a work flow. See Visual Macro Editor, page 4-189 for further details. Fig. 4-168 Macro menu 4.8.1 Macro Language "Visual Basic for Applications", called VBA in the following, is used as the Macro language. This language is well known through its widespread use as Macro language in the "Microsoft Word for Windows" and "Microsoft Excel for Windows" products. Experience with "Microsoft Visual Basic" would also be beneficial for macro-programming of the LSM 5 LIVE. An Integrated Development Environment, called IDE in the following, is available for the editing and debugging of macros. IDE includes an "online help program" where the VBA language is described in detail. Macros are stored in project files. One project file can include several macros. 03/06 B 45-0019 e 4-167 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.2 Macro Control 4.8.2.1 Open / Close the Macro Control Window • Click on the Macro button in the Macro subordinate toolbar of the Main menu. − This opens the Macro Control window. • Click on the Close button to quit the window. 4.8.2.2 Edit Macro Function This function allows you to manage project data. Macros can be recorded, stored, performed, edited and, if required, deleted. • Press the Edit Macro button to switch to the Macro and Recording panels. Fig. 4-169 (1) Macro Control window • Click on the Close button to quit the window. Macros panel New button: Creates a new project. Load button: Opens an existing project. Save button: Stores the project on the hard disk. Save As button: Stores an existing project under a new name. Unload button: Removes the selected macro from the Macros list. Edit button: Allows macros to be edited and debugged. The editor (Microsoft Visual Basic) is automatically located at the beginning of the relevant macro. Run button: Runs a macro. Step button: Opens the editor for line-by-line editing / debugging. Delete button: Deletes the selected macro. Editor button: Opens the editor. Displays the processed area of the macro edited last. 4-168 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Fig. 4-170 Carl Zeiss Macro panel Macros are stored and managed in project files (*.lvb). Before you can record or edit a macro, you have to create a project as follows: • Press the New button to create a project file. − A new project is created and displayed in the Project selection box (e.g.: LSM 150503). The project name is automatically default, but can be edited afterwards. To activate an existing project, proceed as follows: • Press the Load button. − The Open window will be opened. • Select the relevant project file (data extension: *.lvb) from the Macros list box. Click on the Open button. − The project file will be opened and the macros contained in it are displayed in the Macros selection box of the Macro Control window. Recorded macros are stored in main memory first. Before the macros can be assigned to the buttons in the Macro submenu, the project must be stored on the hard disk. Fig. 4-171 Open window • Press the Save button under the project name in the Macro Control window and determine the file name in the Project selection box, if required. 03/06 B 45-0019 e 4-169 OPERATION Macro Menu Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Recording panel Before recording a command sequence, you can enter the name for the macro to be created in the Rec Name input box of the Recording panel. Start button: Starts recording. Cancel button: Cancels the recording procedure. Stop button: Stops recording. Edit On Stop: On stopping the recording procedure, the macro editor is automatically opened at the relevant position. Proceed as follows to record a macro: • Enter a name for the macro to be created under Rec Name in the Recording panel. • Click on the Start button to start recording the macro. • Then perform the operations to be stored, e.g.: − Click on the Find button in the Scan Control window. A Find scan will be performed. − Click on the New button in the Scan Control window. A new Image Display window will be opened. − Click on the Single button in the Scan Control window. A Single scan will be performed. • Then click on the Stop button to end the recording. (Cancel enables you to cancel recording) − If recording was successful, the entered Rec Name will then also be available in the Macros list box of the Macro panel. The new macro is automatically assigned to the current project. It is possible to assign as many macros as required to a project. • Click on the Save button to store the new macro. Fig. 4-172 4-170 Recording panel B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss Proceed as follows to perform a macro: • Select the required macro from the Macros list box of the Recording panel. • Click on the Run button to start performing the macro. Provided that a macro is linked to a button in the Macro subordinate toolbar, you only need to click on this button to perform the macro. Proceed as follows to delete a macro: • Select the required macro from the Macros list box of the Recording panel. • Click on the Delete button. The macro will be removed from the list. Proceed as follows to edit a macro: • Select the required macro from the Macros list box of the Recording panel. • Click on the Edit button. The Microsoft Visual Basic editing window will be opened. • Make the required changes (also see the notes on page 4-172). 4.8.2.3 Assign Macro to Button Function This function permits stored macros to be linked with one button each in the Macro subordinate toolbar. • Press the Assign Macro to Button button to switch to the Define Buttons panel. Define Buttons panel Proceed as follows to link a macro to a button of the Macro subordinate toolbar: • Select the button number from the Button selection box. • Enter the button labelling in the Text editing box. • Select the name of the project file from the Project box using the ... button Fig. 4-173 Macro Control window • Select the macro name from the Macros box. • Press the Apply button to assign the relevant macro to the specified button in the Macro toolbar. 03/06 B 45-0019 e 4-171 Carl Zeiss OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan Delete the linking of a button in the Macro subordinate toolbar Proceed as follows to delete the linking between a button in the Macro subordinate toolbar and a macro: • Select the button number from the Button selection box. • Press the Delete button to delete the linking. 4.8.2.4 Editing and Debugging of Macros The Edit button activates IDE (Integrated Development Environment) which allows macros to be edited and debugged. Under the Help - Contents and Index menu item, IDE contains detailed "online" help on its operation and on the VBA macro language. Therefore, only a few hints are provided in the following: You should activate the required toolbars. We would recommend you to activate the Debug toolbar via the View - Toolbars -Debug menu item. The following buttons in the toolbar can help you when debugging macros: Starts running the command lines. Stops running the command lines. Interrupts processing of the command lines (pause). Sets a breakpoint in the line with the text cursor. Processes a command line and steps into subprocedures. Processes a command line and steps over subprocedures. Exits the subprocedure (step out). Displays the value of the marked expression (Watch). If nothing is marked, the value of the variable above the text cursor is displayed. Activates the Watch window in which values of variables and expressions can be displayed. For this, text is marked in the code window and dragged into the Watch window. Variables can be modified in the Watch window. In the left-hand edge of the code window you will find an arrow beside the current command line. A new current command line can be determined by moving the arrow via the mouse. This makes it possible to skip command lines or to process command lines several times. 4-172 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.3 OPERATION Macro Menu Carl Zeiss Overview of Available Macros Documentation files (*.rtf, *.doc) of advanced macros will be located in the macro directory. Name Description AOTFfitlin.lvb Linearize laser attenuation (AOTF or mechanical) Autofocus.lvb Automatic focusing according to a set configuration Bleach.lvb Bleaching of a rectangular area or a line; combines old macros BleachRectangle.lvb, BleachLine.lvb and Spot.lvb CameraColor.lvb (also Button in Maintain) Color balance of Axiocam HRc Centerv.lvb Centers the field of view around the position marked with the cross tool; ConcatenateImages.lvb Enables the combination of all kinds of images irregardless of the size and data depth to be presented in a time series. Imported images can also be included. Type of concatenation (fitted to the smallest frame or ROI, fixed size or fitted to the largest frame) can be defined by the user. CopyPasteOverlays.lvb Copies actual overlay drawing into the clipboard and pastes the drawing into a selected image window CopyPasteRoi.lvb Copies drawing element of overlay into clipboard and pastes it into other selected windows Distance.lvb Example macro for measurement DivideThroughReferenceImage.lvb - divide complete time series through a single image/part of the series - duplicate a single image or part of a time series to this series. ExcitationFingerprinting.lvb Automated generation of excitation lambda stacks for generating excitation spectra which can be used for linear unmixing to separate overlapping dyes. Any detector can be used, also NDDs. Description as guide tour on the installation CD-ROM or DVD. FastModeSwitch.lvb Store settings from "Scan-Control" and reuse. FileExport.lvb Exports one or more selected images according to the set file format in one go; Exports image intensity values in ASCI format; FRET plus.lvb (Option) Enables the user to perform FRET experiments based on sensitized emission or acceptor photo bleaching (depending on the type of LSM) including commonly accepted analysis algorithms. HotKey.lvb Shift focus with a button and start Single-Scan KSPlastv.lvb KS software macro Kollimatic.lvb Automatically generates the calibration matrix of a LSM 5 LIVE system for optimal confocal aperture and collimator settings Lambdatrans.lvb Time series alternating between lambda and transmission mode 03/06 B 45-0019 e 4-173 Carl Zeiss Name OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan Description LsmHWAdmin.lvb (also Button in Direct service hardware access (password protected) Maintain) LsmHWAdminEx.lvb Calibration service macro (password protected) LsmTime.lvb Triggered Time scan Macro MetaExport.lvb Export of META image files including all channels as tif, bmp, ... ModifySeries.lvb Modifies Z Stacks and Time Stacks like Rotation of the stacks, being mirrored, Conversion of time stacks into z-stacks and vice versa; StitchArtPlus.lvb StitchArt Plus macro (Option) MultiTime.lvb Set up of time series experiments including repeated imaging, bleaching and autofocusing with defined configurations at multiple locations and for various views at each location (Software option) OptimizeGDC.lvb Optimize the max. peak power of fiber coupled Ti:Sa lasers (Release 3.0/3.2) OptimizeGDCV3_2.lvb Optimize_Live.lvb Temporarily adjusts the confocal aperture position of a LSM 5 LIVE without changing the general calibration values Pixel.lvb Displays and stores the mean intensity values of each line of one or more channels of one or more images; data from each line are stored as a txt file in the current folder; Profile.lvb Displays the pixel values along a line RecoveryChameleon.lvb Imitates recovery of chameleon NLO laser in case the laser is falling into CW lasing and does not get to mode lock anymore. Reset_live_servos.lvb Reinitializes all servo motors of a LSM 5 LIVE to overcome possible warmup deviations from default positions Scalebar.lvb Indication of self defined intensity levels assigned to a ROI as scale bar in the image; also attaches tick marks and concentration values to the grayscale/color wedge. ScanfieldTransform.lvb Adjusts LSM 510 or LSM 5 LIVE image match relative to each other or to LSM DuoScan ROI’s SphericObjectiveCorrection.lvb Calibrates the spheric component of objective error by automatic acquisition of a Z stack on a flat surface and a spherical fit of the topographic data. TestGrid.lvb Projects a testgrid into the image window (password protected). TileScanRotation.lvb Defines rotation angle of scanners during tile scan TimeSeriesShutter.Lvb Close laser shutter in time series on a LSM 5 LIVE Trigger.lvb Trigger test During installation, default macros will be installed according to their type either in AIM\, AIM\HWT or AIM\Macros\. Self generated Macros will be in AIM\Macros. 4-174 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss In case of a new installation, old macros will be stored in AIM\Macros\BackupMacros or AIM\Backup\, to avoid problems with identical names of existing and new macros. 4.8.4 Sample Macros The LSM 5 software package includes e. g. the Distance, Profile, Multiple Time Series and StitchArt plus sample macros. They can be easily executed by clicking on the relevant button in the Macros subordinate toolbar. During the execution of a macro, the Stop Macros window is always displayed on the screen. This enables a macro to be stopped any time by pressing the Stop button. The functions of the sample macros are explained below. 4.8.4.1 Distance Macro This macro permits measurement of the distance of a line created in the scan image. • Click on the Distance button in the Macro subordinate toolbar. − An XY scanning image of the specimen is recorded and displayed. At the same time, the Mouse position test window appears on the screen. • Then draw a line over the distance to be measured by clicking and holding down the mouse button. The click of the mouse sets the starting point, releasing the mouse sets the end point of the line. − After release of the mouse button, the length of the line in the scanning image is displayed (in µm). − Any required number of lines can be defined in the image. The previous line is deleted. • A click on the Exit button in the Mouse position test window will end the macro. 4.8.4.2 Profile Macro This macro permits the gray values of a line created in the scanning image to be determined pixel by pixel. • Click on the Profile button in the Macro subordinate toolbar. − An XY scanning image of the specimen is recorded and displayed. The Profile window is shown on the screen. • Then click and hold down the mouse button to draw a line in the scanning image for which the gray values shall be determined. − The current numbers of the pixels of the created line to which the relevant gray value is assigned now appear in the Profile window. − At the same time, the distance of the created line is displayed in µm for checking. − Any required number of lines can be defined in the image. The previous line is deleted. • A click on Cancel will end the macro. 03/06 B 45-0019 e 4-175 OPERATION Macro Menu Carl Zeiss 4.8.4.3 LSM 5 LIVE LSM 5 LIVE DuoScan Multiple Time Series Macro The Multi Time Series program is designed to provide flexible programming of automated time dependent experiments. − A pre-program Cycle Delay − A pre-bleaching functionality (not available) − An autofocus functionality − A number of different, single time series (called a block) either at the same position of a sample or at different positions (if a motorized stage is available) − A time series of a tile scan − A time series of a tiled Z stack − A Z-Stack with triggered acquisition of each single image in the stack The basic programming unit is a single Time Series Block in line, frame or Z-stack mode. In each block the user can define a configuration for the data acquisition (single or multi-track), the number of images and the time interval/delay between images. The user can activate an optional autofocus function before each block, pre-program a time interval before each block (from the beginning of the previous block to the beginning of the current block), and/or execute a bleach track with arbitrarily specified bleach ROI's, laser line(s) and power. Fig. 4-174 Multiple Time Series main dialog box The autofocus function can be executed using a specified single-track configuration, or with the configuration used for imaging (using the first channel of the first track). One can define the z-offset, the distance in the z direction from the position where the autofocus finds the reference feature in focus (position of maximum intensity - e.g. position of the cover slip reflection in the reflected light configuration) to the position, which is moved into focus plane when the image acquisition begins (e.g. into the tissue). The image acquisition is done with the configuration specified for the given block. Autofocus search parameters: Z-offset as well as the Z-range (the distance in the z direction over which the autofocus function searches for the plane of maximum intensity) can be set independently for different stage locations (on the systems with the motorized stage). 4-176 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.5 OPERATION Macro Menu Carl Zeiss Kollimatic VBA Macro to calibrate the LSM 5 LIVE depending on the current system configuration (e.g. objectives, filters,…) Prerequisites: − System Warm-Up (at least 2 hours; for procedure please read the User’s Manual) and environmental conditions according to setup requirements. − “OptimizeLIVE.lvb” Macro: ensure both channel sliders set to 0 − Proper adjustment of collimators and Confocal Slit Aperture (CSA) 1 + 2 (this has to be done by the local Service Representative) − AOTF RGB + V linearized properly with Macro AOTFFitlin.lvb − All used objectives have been stored in the database according their exact order in the nose piece. − Necessary calibration slides are available 1x Bead Slide 1µm for dry objectives, # 000000-1371-460, 1x Bead Slide 200nm for immersion objectives, # 000000-1371-461); to be ordered from Carl Zeiss Jena. Basic Functions of the Macro The KolliMatic macro features two main parts, Basic Calibration and Calibration Matrix: A - Basic Calibration − Needs to be done only once with one of the recommended objectives (should be part of the shipment in order to be able to repeat the Basic Calibration without calibrating all objectives again (see objective list down below) − Is for calibrating both collimators RGB + V − Is for calibrating the Y-position of the Beam Shift Compensator (BSC) for all present NFT positions On currently delivered LSM 5 LIVE Systems, the Basic Calibration is done in the factory (This can be confirmed by checking that “BasicCalibrationLog.txt” is present in C:\AIM\Bin). Objective list (objectives are listed in order of most to least recommended) − Plan Apochromat 20x / 0.75 − Plan Neofluar 40x / 1.3 Oil − C-Apochromat 10x / 0.45W − C-Apochromat 40x / 1.2W corr − Plan Apochromat 5x / 0.16 − Plan Neofluar 10x / 0.3 − Plan Apochromat 63x / 1.4 Oil − C-Apochromat 63x / 1.2W corr − Plan Neofluar 20x / 0.5 03/06 B 45-0019 e 4-177 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan B - Calibration Matrix − Is based on the calibration results of the Basic Calibration − Needs to be performed for each objective separately − Is for calibrating the collimator position of both collimators RGB + V only for the selected Objective depending on: Laser Line, Zoom + EF Filter Position − Is for calibrating the BSC Y-position depending on: Laser Line, Zoom + EF Filter Position On currently delivered LSM 5 LIVE Systems, the Calibration Matrix is set up in the factory for every objective belonging to the system (This is done together with the Basic Calibration). Without running Calibration Matrix, the positions of the collimators and BSC Y should be OK. This is due to the Basic Calibration, but the results are not fully optimized. Installation of the Macro: If not already done, copy the required files to the following destinations: File Name Destination KolliMatic.lvb C:\AIM\Macros PotentialKollimaticConfig.ini C:\AIM\Bin KolliMatic_Instructions.pdf C:\AIM\Macros Starting the Macro: • Start the LSM software and check first, if all prerequisites have been fulfilled (see Paragraph 1. Prerequisites) • Put the appropriate calibration slide (depending on current objective: immersion or non immersion) onto the microscope stage, • Activate all lasers, and using the and focus with the 488 nm line start scanning and focus onto the beads • Put the appropriate calibration slide (depending on current objective: immersion or non immersion) on the microscope stage, start single scan • Use the Macro Menu of the main LSM software to Load and Run the macro KolliMatic (See Fig.4-175) Fig. 4-175 4-178 Macro Control Window of the main LSM Software Please wait a few seconds, the macro will set up beam path and all necessary settings automatically. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss Preparations for using the Calibration Slides: • Follow the steps shown in the macro Sample and Configuration Setup window • Use the arrows or the drop down menu in order to select one track for each available laser line (no need to select several tracks for the same laser line), start with a track for the lowest wavelength laser (i.e. typically the 405 nm laser line.) • Select an area of the bead sample that provides as many beads as possible, at least 4 (fit into the green rectangle) • In Fig. 4-176, the Basic Calibration has been selected (Check box Basic Calibration: Requires selecting preferred objective lens), therefore some more tracks are available • Use the Laser Power as a first option in order to set the intensity, second option is Integration Time • In order to start / stop the scan, just select / deselect the check box Continuous Scan • Focus on the beads • Defocus with the Collimator slider until the beads become line shaped • Focus with the microscope focus drive to get the vertical lines as thin as possible (Fig. 4-177) Fig. 4-176 Figure Sample and Configuration Setup Window of the KolliMatic Macro • Now use the Collimator slider to get a round spot out of the line (Fig. 4-178) • Repeat these steps for a track with the 488 nm laser line • After one track for each laser line has been adjusted, click Next. 03/06 B 45-0019 e 4-179 OPERATION Macro Menu Carl Zeiss 4-180 Fig. 4-177 Beads focused for maximal vertical thinness Fig. 4-178 Beads optimally focused B 45-0019 e LSM 5 LIVE LSM 5 LIVE DuoScan 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss Running the Basic Calibration If you have selected the check box for Basic Calibration, please follow these instructions. In order to run the Calibration Matrix only, move on to paragraph 7. • Make sure that both check boxes have been selected (Fig. 4-179) • Double check if the objective settings (shown at the bottom of the macro window) match the currently used objective • Click the button Calibrate • In order to follow the calibration process, just click into the Message window; a scroll bar will appear • The Basic Calibration takes about 15 min • The displayed curve should resemble a Gaussian profile • After the macro has finished, a message will pop up with the request to restart the LSM software to make the changes effective. Fig. 4-179 Collimator Calibration Window • Press Exit and restart the LSM software 03/06 B 45-0019 e 4-181 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Running the Calibration Matrix If you haven’t selected the check box for Basic Calibration, the “Calibration Matrix” will be selected automatically. In this case, when the Calibration Matrix is running , the Basic Calibration is disabled. • This window provides three check boxes (Fig. 4-180) • The recommended setting is to select Create new calibration file. All previous files will be deleted. Exceptions: No check box checked − Especially after an unexpected interruption of the program, it is possible to continue from the position where the process stopped (saving time). Overwrite Collimator values − The values for the chosen objective will be added or updated. Fig. 4-180 Running the Calibration Matrix − After selecting the Collimator option, the Pinhole Y option will be selected automatically. • Double check if the objective settings (shown at the bottom of the Macro window) match the currently used objective • Click the button Calibrate Meaning of the Graph (Figure): Brown: Focal plane of the CCD Line Detector Red: Focal plane of the collimator Orange: Estimated focal plane The brown and red graphs should be close to this curve. Fig. 4-181 4-182 Graph of Focus Function B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss • In order to follow the calibration process, just simply click into the Message window and; a scroll bar will appear (Fig. 4-182) • The Calibration Matrix takes about 25 min for each objective • After the macro has finished, a message will pop up with a the request to switch the objective lens in order to make the changes effective • If necessary, repeat the procedure for the other objectives • Use the Update Diagrams button in order to check, if an objective has already been calibrated − Select an objective, press the Update Diagrams button Fig. 4-182 − If no graphs appear, this particular objective has yet needs to be calibrated Collimator Calibration window after calibration • Click Exit to finish Meaning of the Graph (Figure): − Each coloured line represents an individual laser line. − Each coloured calibration point represents an emission filter. − Standard graphs are displayed with black points. Meaning of the Colours: − <420 nm: Æ Pink − 421…475 nm: Æ Blue − 476…495 nm: Æ Turquoise − 496…550 nm: Æ Green − 551…590 nm: Æ Yellow − >590 nm: 03/06 Æ Red B 45-0019 e 4-183 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Important Information! If the calibration has been completed successfully with the Calibration Matrix, DO NO LONGER USE THE PINHOLE AND COLLIMATOR FUNCTIONALITY OF THE LSM SOFTWARE IN THE MAINTAIN MENU. The use of the pinhole sliders to raise the quality of the images is well known from the other LSM Systems. Fig. 4-183 On the LSM 5 LIVE, only the Optimize sliders of the “OptimizeLIVE.lvb” macro have to be used (Fig. 4-183) to compensate for misalignments (e.g. temperature influences). Optimize Macro After the Calibration Matrix has optimized a particular objective, the results will be stored automatically in a file as shown below. These files can be found in C:\AIM\BIN. DO NOT CHANGE ANY OF TH VALUES! Fig. 4-184 4-184 Calibration file B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss The calibration results of the Basic Calibration will be saved in a file named “BasicCalibrationLog.txt” (to be found in the BIN-Folder as well). Changes won’t effect the stored values. Fig. 4-185 03/06 B 45-0019 e BasicCalibrationLog file 4-185 OPERATION Macro Menu Carl Zeiss 4.8.6 LSM 5 LIVE LSM 5 LIVE DuoScan Optimize Macro For temporary optimization of the LSM 5 LIVE beampath, try to avoid a modification of the general adjustment in the pinhole dialogue. Use the Optimize slider instead. The general setting of the confocal aperture might still be correct for other scan parameters. Fig. 4-186 Optimize window The Optimize slider can be set independently for each channel. The slider is moved to the one or the other side while scanning continuous. The brightest image result is the optimal setting. This setting doesn’t affect the general adjustment of the LSM 5 LIVE collimator and confocal aperture, but can enhance image brightness efficiently. 4.8.7 ReInit Macro To improve the internal adjustment of default positions of motor driven parts, the Reset Servo macro (ReInit) is available. After one hour, we recommend to start the ReInit macro to reset all default positions for the actual warm up state of the system. Repeat this whenever you experience changes in the system performance. After running the macro, close the window. Fig. 4-187 4-186 Reset window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.8 OPERATION Macro Menu Carl Zeiss Image Match (Scanfield Transformation) Macro Adjusts the Image match between two scanning imaging systems or a manipulation and a scanning imaging system. To adjust the image match, set up a multitracking that creates one channel for each scanning and/or manipulation system. In case of the LSM DuoScan, use a T-PMT to create an image with the DuoScan system. • Start the Image Match (Scanfield Transformation) macro. • Use the Image match calibration sample delivered with the system. This sample has a fluorescent layer and a reflective grid, useful to create images in fluorescent, reflective or transmission light mode. • Start continuous scan, and set laser power and gain to appropriate levels. The point scanning system will be the speed limiting system, so choose a fast scanning speed to get a faster image refresh. • Use unidirectional scan to avoid adjustment errors due to bidirectional image jitters. • Set the matching zoom factors with one of the Match DuoScan zoom/offset or Match LIVE zoom/offset buttons relative to each other. It is recommended to adjust the LSM zoom. Fig. 4-188 Scanfield Transform window These buttons set the two scanheads at a matching scanfield diameter, according to the following table (LSM 5 LIVE = Zoom 1.0): Microscope stand LSM 5 LIVE DuoScan LSM 5 DUO Inverted (port config) (SP/RP) DuoScan = Zoom 1.4142 (RP/SP) LSM 510 = Zoom 1.33978 Upright (port config) (Tube/RP) DuoScan = Zoom 1.4142 (RP/Tube) LSM 510 = Zoom 1.25295 03/06 B 45-0019 e 4-187 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Choose Split image for the image window display. You will see the scanned areas of both scanning systems and an overlay. Fig. 4-189 Split image • Choose LSM (510) or DuoScan to adjust the image match. • While scanning, move the sliders of the Image match macro to get a perfect overlay of the scanning and/or manipulation system relative to each other. • Offset X/Y adjusts the horizontal and vertical image shift • Amplitude adjusts a size difference between the images • Rotation compensates for a tilt in one of the images. Store the final image, and use the ReUse function for future image matching. The Multitrack setup and the zoom factors will then be set automatically. 4-188 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.9 OPERATION Macro Menu Carl Zeiss Visual Macro Editor The Visual Macro Editor is an optional software module which allows the user to program a variety of scanning procedures and image calculations as well as the combination of both. 4.8.9.1 Open / Close the Visual Macro Editor Window • Click on the Visual button in the Macro subordinate toolbar of the Main menu. − This opens the Visual Macro Editor window. • Click on the Fig. 4-190 03/06 button in the upper right side of the window to quit[amy362]. Visual Macro Editor window B 45-0019 e 4-189 Carl Zeiss OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan 4.8.9.2 Function Description The Editor uses predefined action blocks. The action blocks for setting up the work flow of the Macro are categorized according to their properties. The following categories are displayed on the left-hand side of the Visual Macro Editor control window. − Acquisition − Windows − Load/Save − Program Flow − Process − Hardware Control • Click on one of the Category selection buttons will display the available action blocks. The action blocks can be positioned according to their function either in the Program Flow column or the Data Flow column. The color of the connection arrows of the action block defines the possibility to position the block in the program flow column or the data flow column(s). • Click at the action block with the left mouse button (hold down the mouse button) and drag the mouse cursor into the flow column. Release the mouse button. − Action blocks for the program flow show red connection arrows ▼ as well as blue output arrows ▼ to connect to the data flow. − Action blocks for the data flow show blue connection arrows ▼ only. It is not possible to drag an action block for program flow into the data flow or an action button for data flow into the program flow. Action blocks can be removed using the Delete key of the keyboard. Without using the Image Display action block in the data flow column, the result of the scan will not be displayed (see also description of Scan and Time Series action blocks). The work flow is generated by connecting the action blocks using the mouse cursor. • Click onto an output arrow with the left mouse button (hold down the mouse button) and drag the mouse cursor away. − A connecting line appears. • Drag the line to the input arrow of another action block. Only arrows of the same color can be connected. According to the action blocks a set of properties is listed which can be assigned with free or predefined values. A set of defined properties is assigned to one block only. If this block is used more than once the properties can be set differently. Clicking the Read Back button will take over the current settings from the LSM main program for the displayed properties list. • Mark the check boxes next to the parameters individually or, click Use all or Use none to check or uncheck all parameters with one click. • For changing a values click into the Value column. 4-190 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss It is possible to define variables. A variable has a (property) name and a mathematical value. The Assignment action block can be used to assign a new value to a variable when inserted into the program flow using mathematical algorithms. The new value is then used for any subsequent calculation in the program flow where the variable is part of the calculation. • Click the Variable button on the toolbar to display the variable list. Click once more to display the parameter list again. • New variables can be added by typing in name and value in the next free line. Toolbar buttons Run Starts the Macro starting with the firs block in the program flow column. Stop Stops the Macro at any time: Break Interrupts the Macro. Clicking the button again will resume the actual program flow of the Macro: Step Allows to go through the program flow of the Macro stepwise starting with the first block in the program flow column. Each click will perform the action of the actually highlighted block, then jump to the next block which will then be highlighted. Load Use to open a specific Macro and Save to store the Macro. SaveAs allows to store the same Macro under a different name. Variables By clicking the button the list of defined variables is displayed. New ones can be added by typing in name and value in the next free line. Variables are assigned to Macros. For each new Macro a new list of Variables has to be generated. 03/06 B 45-0019 e 4-191 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Acquisition Action Blocks Loads a Beam Path Configuration form the Configuration list. Use the pull down menu in the Property/Value list to select the Configuration that should be used for imaging. Changing between Single Track and Multi Track allows to chose between the different Configuration lists. Multi Track also lists tracks that are used for Emission Fingerprinting or Online Fingerprinting. Depending on the type of track different settings can be loaded optionally. If they are not loaded and the parameters list of the Scan Parameter block does not offer them as being editable, for example detector gain and offset, default values will be used. The list of properties lists all parameters that can be adjusted for the scan procedure. The value for each parameter can be typed in individually. Clicking the Read Back button will take over all current settings from the main software. The check boxes next to the parameters can be marked individually or, clicking Use all or Use none they can be all checked or unchecked with one click. Only the parameters with a mark in the check box will be taken into account and adjusted when the Scan Parameter block is set in the program flow column. This allows to change only some of the parameters, for example the zoom, at a certain position within the program flow as for each block the check boxes can be set. This block performs the scanning of the sample according to the settings chosen with Beam Path and Scan Parameters. It can be linked to the Data flow column. If no connection to the data flow is made (for example: Display Image) the result of the scan will not be displayed. The parameters for bleaching the sample can be loaded from the list of bleach settings. Bleach settings are assembled and stored in the Bleach control window of the main software. This block performs the bleaching of the sample according to the bleach settings that have been defined and stored in the Bleach control of the main software. Use the pull down menu to select the bleach setting from the list. The list of properties lists all parameters that can be adjusted for the acquisition of the time series.The value for each parameter can be typed in individually. Clicking the Read Back button will take over all current settings from the main software. The check boxes next to the parameters can be marked individually or, clicking Use all or Use none they can be all checked or unchecked with one click. Only the parameters with a mark in the check box will be taken into account and adjusted when the Time series block is set in the program flow column. This allows to change only some of the parameters, for example the used trigger, at a certain position within the program flow as for each block the check boxes can be set. This block can be connected to the data flow column. 4-192 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss Windows Action Blocks This block has to be used if the actually scanned image(s) should be displayed. For each Image Display block a new image window is opened. If not saved, the image will be overwritten when the program flow is passing by again. Save / Load Action Blocks The image(s) are stored to a image database. The database taken is either the last one, that was open or the current open one. Clicking Read back updates the database to be used for storing the image(s). In addition some other parameters can be set according to the list of properties displayed. Within the program flow already existing images can be loaded. The database taken is either the last one, that was open or the current open one. Clicking Read back updates the database to be used to load the image(s) from. The images can be loaded by Index or by the name of the image. It is possible to export images choosing a certain file format from the pull down list that appears when highlighting the line File Format in the properties list. Specific parameters are assigned to each file format which can be set for the image export. Images can be imported within the program flow. The image to be loaded can be selected by typing in the file path. The import of single images or a image series is possible. The images to be imported as series must have the same name and must be numbered in ascending order. Program Flow Action Blocks If any action(s) should be repeated a defined number of times, the Repeat block should be set into the loop of the program flow. The number of repeats is set in the properties list. The repeat is only performed using the connection of the right arrow. After finishing the repeats, the program flow connected to the arrow pointing down is performed. Using this block in the program flow allows to assign new values to a variable using mathematical calculations. By passing this block the variable will be transiently changed and the new value can for example be used to assign different numbers to the image name or to increase the time delay during a time series acquisition. The two blocks are used as decision makers. Depending on the value of a predefined variable, the program flow can be directed to one of the two possible directions. The expression of the variable’s value which should be used to decide on the program flow is set for each block in the properties list. It takes the actual transient value of the variable into account. 03/06 B 45-0019 e 4-193 Carl Zeiss OPERATION Macro Menu LSM 5 LIVE LSM 5 LIVE DuoScan This block introduces a delay in the program flow. The delay time is started when the block is reached within the program flow. The block allows to assign a potential error message with a textbox and to decide on the Error where to direct the program flow. If no textbox is assigned the program flow will just continue according to the presence of an error message. Choosing a textbox requires the input of the user to continue with the program flow. With the Release 3.5 this works only for loading images from a database when the images cannot be found because the Index number is not present. In all other cases of an error, the error message will be displayed and the program stopped. Process Action Blocks This block allows to do image calculation using the pixel intensities of the image. The operators for the formulas that can be used are described in the Ratio Dialogue within the Process Menu. The formulas set up in the Image calculation window can be copied and pasted into the properties list of the Visual Macro Editor. For calculations using two different sources the Calculate block with two possible inputs is used. The calculation starts when both inputs have arrived. Within program loops the next calculation will start when the inputs have arrived again in the same sequence as the first time. The operators for the formulas that can be used are described in the Ratio Dialogue within the Process Menu. The formulas set up in the Image calculation window can be copied and pasted into the properties list of the Visual Macro Editor. The input image will be copied to a new image format that can be defined by changing the values of the listed properties. Using this block enables to copy one image into another with the coordinates and settings put into the properties list. The destination image is connected to the left input, the source image is connected to the right input. The subsequently arriving images will be concatenated according to the coordinate setting and treated as one image. A subsequent image display will be updated each time a new image arrives. The second concatenate action block allows to concatenate images from two different acquisition blocks. 4-194 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu Carl Zeiss Hardware Control Action Blocks If a motorized stage is available the movement of the stage can be set up as part of the program flow. The stage can be moved to an absolute position which can also be taken from the actual stage position using the Read back function or typed in individually. Using a relative position will use the current stage position as relating position. The values have to be typed in. The movement of the focus can be set up as part of the program flow. The focus can be moved to an absolute position which can also be taken from the actual focus position using the Read back function or typed in individually. Using a relative position will use the current focus position as relating position. The values have to be typed in. For large travel ranges which exceed half of the objectives working distance a warning can be displayed. 4.8.9.3 Editing a Macro Following Example shows a macro which acquires five subsequent images and moves the stage 10 microns in X and Y following each image. The images are stored and numbered in ascending order. • Arrange and connect the action blocks (Fig. 4-191). Fig. 4-191 Arrangement of action blocks Fig. 4-192 Assignment of the variable • Assign the value 1 to a variable named Index (Fig. 4-192). 03/06 B 45-0019 e 4-195 OPERATION Macro Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click the single action blocks and assign the values to the listed properties (beam path and scan parameters can be chosen according to the sample). Fig. 4-193 Fig. 4-194 Fig. 4-195 Fig. 4-196 4-196 Property list of the Beam Path action block Property list of the Scan Parameter action block Property list of the Append to Database action block Property list of the Move stage action block. • The database the images will be appended to is automatically displayed when highlighting the Append to Database action block. The last one used will be taken. To update this, open the desired database and use the Read back function. Type in a name for the images with or without quotation marks. All images will get the same name then. For numbering the images use quotation marks for the name and add the variable Index, which has been assigned with the value 1. This value or the transiently changed value of the variable will be added to the name of the image. • The movement of the stage is defined highlighting the Move stage action block, choosing the Mode Relative Position in the Property list and typing in the value 10 for X and Y which then will move the stage 10 microns into X and Y direction starting with the current stage position on passing that action block. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Macro Menu • Now the first complete scan has been performed and the image is stored. For the next cycle the variable Index has to get another value to make sure the numbering of the images works. Using the Assignments action block, the Variable Index is chosen and the value assigned is Index + 1. Such the variable will get the transient value 2 for the next cycle, the value 3 for the over next cycle and so on. • The number of repeats can be defined using a Decision block and use the value of the variable as base for the decision. By defining that if the Expression of the variable is larger than 5 and not connecting the output arrow marked with ‘yes’ to any other block, the macro will stop after the acquisition of five images. Alternatively one can use at this part of the program flow the Repeat block and define the number of repeats to be performed, which would be 5 in this case (The first scan is already counted!) 03/06 Carl Zeiss Fig. 4-197 Property list of the Assignments action block Fig. 4-198 Property list of the Decision action block B 45-0019 e 4-197 OPERATION Options Menu Carl Zeiss 4.9 LSM 5 LIVE LSM 5 LIVE DuoScan Options Menu The Options menu permits performance of the following functions: − Display of a current list of dyes with preferred wavelengths for the scanning procedure. − Display / modification of the user-accessible program Settings of the LSM 5 software. • In the Main menu toolbar, click on Options. − This opens another, subordinate toolbar in the Main menu. Fig. 4-199 4-198 Options menu B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.1 OPERATION Options Menu Carl Zeiss Dye DB Function The Dye DB function is for information only and permits access to the database contained in the system, including a list of suitable dyes for fluorescence microscopy. The database contains a comparison of tables of dyes, optimum excitation wavelengths and maxima of emission wavelengths. • Click on the Dye DB button in the Options subordinate toolbar. − The Dye database will be opened and displayed on the screen. • Click on the Close button to exit the Dye database. Fig. 4-200 03/06 Dye database window B 45-0019 e 4-199 OPERATION Options Menu Carl Zeiss 4.9.2 LSM 5 LIVE LSM 5 LIVE DuoScan Settings Function The Settings function permits the individual setting and matching of software settings with regard to the following points: Autosave Recording / Reuse Database General Timeseries Database Table Viewer Scan Mean of ROIs Database Gallery Viewer Temporary Files Import / Export Program Start Scan Information Hardware Image Status Display Image Display Toolbars Print Status Display Save 4.9.2.1 Open / Close the Settings for user : ... Window • Click on the Settings button in the Options subordinate toolbar of the Main menu. − This opens the Settings for user : ... window. • Click on the OK button to quit the window. The last settings will be taken over. Cancel enables you to cancel the procedure, with any changes you made not being taken over. Fig. 4-201 4-200 Settings for user : ... window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.2.2 OPERATION Options Menu Carl Zeiss Autosave This tab permits activation or deactivation of automatic data storage. Only one option can be selected at a time. (1) No Autosave On activation of this option, the Autosave function is switched off. Save and Save As give the same dialogues. (2) Fig. 4-202 Autosave tab Use LSM image database and auto increment image name On activation of this option, newly recorded or modified images are stored by Save automatically and assigned to the name or defined in this function. The image name is automatically created using a base name and a serial number. For this, a base name must be entered in the Base image name input box, and a starting value for the serial number in the Counter value input box. The Database selection box permits selection of the directory in which the data will be stored. (3) Export to Attofluor format On activation of this option, newly recorded or modified images are stored by Save in the Attofluor format. The displayed Experimental directory selection box permits selection of the directory in which the data will be stored. (4) Export to Metafluor format On activation of this option, newly recorded or modified images are stored by Save in a subdirectory in the MetaFluor format. An existing higher layer of folders must be selected for the subdirectory from the Base directory selection box. Furthermore, a name for the subdirectory must be entered in the Subdirectory base name input box. The starting value for the images then created, to which a continuous number is automatically assigned, is set in the Subdirectory counter input box. 03/06 B 45-0019 e 4-201 OPERATION Options Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.2.3 Database General This tab permits the basic starting settings for the use of databases. (1) Fig. 4-203 (2) Start with "Form" On opening of a database, the Form option is displayed first. Database General tab Start with "List" On opening of a database, the List option is displayed first. (3) Start with "Gallery" On opening of a database, the Gallery option is displayed first. (4) Show first record set at opening of database On opening of a database, the first record set is displayed. (5) Show first record set at opening of database On opening of a database, the middle record set is displayed. (6) Show first record set at opening of database On opening of a database, the last record set is displayed. (7) Use separate path for "Create" and "Open" This option permits the path to be changed when the Open or New database function is used. (a) Save most recently used path at exit and reuse at next program start On activation of this option, the path setting last used is automatically selected again in the Open Database or Create New Database window. (b) Use the following path at program start On activation of this option, the path for the Open Database or Create New Database window can be entered directly in the relevant selection box, or selected by clicking on the ... button in the Choose Directory window. This path will then always be set when a database is opened or created. Clicking on the User default path button firmly sets the C:\users\default\ path. 4-202 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.2.4 OPERATION Options Menu Carl Zeiss Database Table Viewer The Database Table Viewer tab permits the definition of the columns for the table display of a database. This only requires the relevant check box to be activated with a click of the mouse. On activation of the Automatic column width calculation option, the column width is calculated automatically. Fig. 4-204 Database Table Viewer tab Fig. 4-205 Database Gallery Viewer tab Fig. 4-206 Import / Export tab On activation of Save and use interactive column width setting, the column width in the database can be matched as required. The individual setting will be retained when the database is closed. 4.9.2.5 Database Gallery Viewer The Database Gallery Viewer tab permits the image information to be displayed in the Gallery mode of the database to be activated by clicking on the relevant check box. 4.9.2.6 Import / Export Use separate path for "Import" or "Export" This option permits the change of the path setting for use of the Import or Export function (File menu). (1) Save most recently used path at exit and reuse at next program start On activation of this option, the path used last is automatically selected again in the Import Images or Export Images and Data window. (2) Use the following path at program start On activation of this option, the path for the Import Images or Export Images and Data window can be entered directly in the relevant selection box, or selected by clicking on the ... button in the Choose Directory window. This path will then always be set when the Import / Export function is used. 03/06 B 45-0019 e 4-203 OPERATION Options Menu Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Clicking on the User default path button firmly sets the C:\users\default\ path. 4.9.2.7 Scan Information This tab permits the setting of which scan information shall be displayed in the Scan Information window (see Window pulldown menu of the Main menu, page 4-222f). Activation / deactivation of the information to be displayed is performed with a click of the mouse. Fig. 4-207 Scan Information tab 4.9.2.8 Image Status Display This tab permits selection of which image information is displayed on opening of an image or on activation of the Info button of the Image Display window. Furthermore, you can determine which information will be displayed in the Image status bar. Fig. 4-208 Image Status Display tab On activation of the Show status display upon opening of a new image display check box, the image information is automatically displayed immediately after opening of the Image Display window (Info button is activated). 4.9.2.9 Print Status Display This tab permits selection of which information is displayed in print preview. On activation of the Print Status Information check box, the status information will be printed. Fig. 4-209 4-204 Print Status Display tab B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.2.10 OPERATION Options Menu Carl Zeiss Recording / Reuse The parameters to be taken into consideration for the use or load of a recording configuration are set in this tab. As an option, you can also determine whether the objective setting shall be taken over when the Reuse function is used. 4.9.2.11 Fig. 4-210 Recording / Reuse tab Fig. 4-211 Timeseries tab Fig. 4-212 Scan Mean of ROIs tab Time Series In the Time series tab, you can determine whether the time for the recording of a time series is set as Cycle Delay or as Time Interval. Cycle Delay is the interval between the end of one scan process and the beginning of the next. Time Interval is the interval between the beginning of one scan process and the beginning of the next. You can select the unit for Mean of ROIs diagrams. 4.9.2.12 Mean of ROIs The Mean of ROIs tab permits the presetting of the Image Display window for the optional MeanROI function (time series) to be changed with regard to scaling and display mode of the intensity time diagrams. (1) Diagram Scaling The following settings are possible by activating one of the option buttons: − Automatic diagram scaling − Fixed time range for diagram time scale; input of the time range in seconds via input box − Fixed number of cycles for diagram time scale; input of the time range in number of cycles via input box 03/06 B 45-0019 e 4-205 OPERATION Options Menu Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Initial diagram types The following settings are possible by activating the relevant option button: − One diagram − Channels diagram − ROIs diagram On activation of the Black graphs black (monochrome). (3) check box, the intensity profiles in the diagram are displayed in Live image (a) Scan the whole image If you activate this check box, the complete live image will be scanned; only the defined ROIs will be scanned if the check box is deactivated. (b) Save the whole time series If you activate this check box, the complete Time Series will be scanned; only the Mean of ROI series will be scanned if the check box is deactivated. 4.9.2.13 Temporary Files The Temporary Files tab permits determination of the directory in which temporary files are stored. (1) Fig. 4-213 Temporary Files tab Use "TEMP" environment variable Temporary files are stored in the TEMP standard directory of the computer's hard disk. (2) Use the following path The directory for temporary files can be selected by clicking on the ... button in the Choose Directory window. 4-206 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.9.2.14 OPERATION Options Menu Carl Zeiss Program Start The Program Start tab permits selection of a track configuration via the Startup configuration selection box, which will be loaded automatically when the software program is started. check On activation of the Don't show logo box, the initial screen with the Zeiss logo will not be displayed after the start of the LSM 5 software. 4.9.2.15 Fig. 4-214 Program Start tab Fig. 4-215 Hardware tab Hardware The Hardware tab allows you to set several hardware defaults at the shutdown of the system. The Lasers off on Exit determine, by activation of check box, that the the Lasers off on Exit lasers are automatically switched off when the LSM 5 software is exited. Allow lasers to cool for five minutes before switching off the system. Light manager activates or deactivates the light microscope stands light manager. Shutter filter wheel activates or deactivates additional laser attenuation by grey filters (LSM 510 only). Simultaneous grab and bleach activates or deactivates the simultaneous grab and bleach option in the bleach menu. This function can create fluorescence artifacts depending on sample properties and confocal aperture setting (it is recommended to close the confocal aperture to 1 airy unit). 4.9.2.16 Image Display The Image Display tab enables you to determine the window toolbars which shall be automatically displayed when an Image Display window is opened. Furthermore, the color mode (color / mono), to which the image display will switch when the Color Palette function is opened / closed, can be determined. (1) Fig. 4-216 Image Display tab Display Windows Toolbars On activation of the relevant check box, the following window toolbars are automatically displayed when an Image Display window is opened: Channels, Zoom, Slice, Overlay. 03/06 B 45-0019 e 4-207 OPERATION Options Menu Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Palettes / Color (a) Switch to "mono" on activation of a palette If this check box is activated, the Mono(chrome) image display mode is switched automatically when a palette is selected in the Color Palette window. (b) Switch to "color" on deactivation of a palette If this check box is activated, the Color image display mode is switched automatically when a palette is deactivated in the Color Palette window. (c) Remember color Palette If this check box previous image. (d) is activated, the consecutive image will be displayed using the same palette as the The background color of images and diagrams can be chosen when clicking on the respective color bar . 4.9.2.17 Save The Save tab permits the presetting for the storage of scanned or processed images to be changed. Activation of one of the three option buttons enables you to determine the database directories to which stored images are assigned: − Image files to subdirectory of the database − Database and image files to the same directory − At "Create Database" automatically create a subdirectory with the same name as the specified database and create the database and image files in that subdirectory Fig. 4-217 Save tab If the Save prompt at closing modified windows check box is activated, you are automatically asked on closing a changed Image Display window whether the image shall be stored. If the Warning before overwrite existing recordsets check box is activated, this question is asked automatically on storing an image under a new name if an image file with this name already exists in the database. If the Remember "Name", "Description" and "Notes" in the save dialog check box is activated, the Name, Description and Notes text boxes of the Save Image and Parameter As window show the text for the image last saved. You can edit the text boxes as required for the new image to be saved. If the Remember "Name", "Description" and "Notes" in the save dialog check box is deactivated, the three text boxes are blank when the Save Image and Parameter As window is opened. 4-208 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 03/06 OPERATION Options Menu B 45-0019 e Carl Zeiss 4-209 OPERATION Maintain Menu Carl Zeiss 4.10 LSM 5 LIVE LSM 5 LIVE DuoScan Maintain Menu The Maintain menu contains additional modules to check and guarantee the interference-free operation of all the software and hardware components of the LSM 5 LIVE. • In the Main menu toolbar, click on Maintain. This opens another subordinate toolbar in the Main menu. Fig. 4-218 Maintain menu 4.10.1 Scanner The Scanner function is used for scanner calibration. The following type of calibrations are available: − Electrical calibration with (unidirectional / bidirectional) − Electrical calibration (unidirectional) Fig. 4-219 Scanner Calibration window 4.10.1.1 Electrical Calibration with Speed Speed 1-10 10-11 Electrical Calibration with Speed 1-10 can be performed unidirectional / bidirectional, but with Speed 11-13 only unidirectional (1) Preliminary notes The electrical calibration has to be performed every 2-3 months. For electrical calibration no laser scanning is performed and for that reason no calibration sample is needed. 4-210 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION Maintain Menu Carl Zeiss Calibration conditions Before the calibration process can be started, the system has to be in operation for at least one hour. (3) Calibration procedure • Click on the Scanner button in the Maintain subordinate toolbar of the Main menu. − This opens the Scanner Calibration window. • Click on the Speed 1-10 (electr.) or Speed 11-13 (electr.) button respectively. • Activate the Display Graphics check box enables to check the progress of the calibration process on the Progress Status bar. − During successful calibration process, the status button is of green color, in case of an error it switches to red. The progress of the calibration process is indicated by the Progress Status bar. The calibration process is completed, when the indicator button is grayed. • Click on the Calibrate button to start the automatic scanner calibration. • Confirm warning information with OK. • Click on the Close button to close the Scanner calibration window. The More function is for servicing purposes only and can only be performed by authorized personnel. Its access is therefore password-protected. 4.10.1.2 Important Notes and Hints The tuning procedure runs automatically to a large extent without any problems. However, several errors can occur. That’s why it is strongly recommended to observe the complete calibration process. If a status error message occurs or the calibration procedure is not finished properly, this can have the following reasons: Non-regularities of the scanner feedback. The ticks on the outer sides of the grid vary about more than 1 tick width between consecutive images (in the middle of the calibration process, the linearity is optimized and the problem starts to occur). • Stop the calibration procedure. • Call the LSM service hotline. If optical calibration comes not to a successful end, please contact your service hotline. 03/06 B 45-0019 e 4-211 OPERATION Maintain Menu Carl Zeiss 4.10.2 LSM 5 LIVE LSM 5 LIVE DuoScan Objective This function permits changed objectives to be activated and the parfocality to be set without having to exit the software. 4.10.2.1 Objective Change • Change the required objective in the nosepiece. • Click on the Objective button in the Maintain subordinate toolbar of the Main menu. − The Objective Control window appears on the screen. The Objective button is activated in accordance with the presetting, and the Objective panel is displayed in the Objective Control window. Fig. 4-220 Objective Control window • Click on the graphical button of the relevant nosepiece mount (Position). − The Change Objective window appears. All available objectives are listed in the Potential Objectives directory of the Change Objective window. • Select the new objective by double clicking from the list in the Potential Objectives directory. • Click on Close to exit the Change Objective window. (1) Add Objective This function permits new objectives to be added to the database. For this, proceed as follows: • Click on the Add Objective button on the Change Objective window. Fig. 4-221 4-212 Change Objective window − The Create new Objective window is opened. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Maintain Menu Carl Zeiss • Enter the data of the new objective in the Create new Objective window, then click on the Apply button. The new objective is stored in the database and included in the Change Objectives window. You can now activate the new objective as a favorite objective using the procedure described above. If you have activated the Non Zeiss check box, objectives from other manufacturers can also be included in the database. (2) Fig. 4-222 Create new Objective window Remove Objective You can only remove objectives in the User Defined Objectives directory. • To remove an objective from the database, select it with a click of the mouse in the Change Objective panel and then click on Remove Objective. The objective is removed from the list. • Click on Close to close the Remove Objective window. (3) Edit Objective You can only edit objectives in the User Defined Objectives directories. • To edit an objective from the database, select it with a click of the mouse in the Change Objective panel and then click on Edit Objective. The Edit user defined Objective panel will open and the parameters of the Objective can be edited. • Click on Close to close the Edit user defined Objective window. 03/06 Fig. 4-223 B 45-0019 e Edit user defined Objective window 4-213 OPERATION Maintain Menu Carl Zeiss 4.10.2.2 LSM 5 LIVE LSM 5 LIVE DuoScan Focus Speed Change • Change the required objective in the nosepiece. • Click on the Objective button in the Maintain subordinate toolbar of the main menu. − The Objective Control window appears on the screen. The Focus Speed has to be activated in the Objective Control window. − The focusing speed of the relevant objective can be selected by using either the slider or the input box in 40 steps. Fig. 4-224 Focus Speed window 4.10.2.3 Parfocality Correction The parfocal setting is performed via screen dialogs in successive panels. • Click on the Parfocal Correction button. − The Parfocal Adjustment panel appears. • Start the setting with the objective of the highest magnification factor (reference objective). Proceed in accordance with the displayed instructions. • Click on Start. − The next dialog is displayed in the Parfocal Adjustment panel. • Focus on your slide object. • Click on the Next step button. Fig. 4-225 4-214 Objective Control window • Perform these steps for each objective. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Maintain Menu Carl Zeiss • Click on the Close button to exit the Objective Control window and accept the settings. 4.10.3 Fig. 4-226 Objective Control window Fig. 4-227 Pinhole and Collimator window Pinhole Adjustment In the Pinhole and Collimator window, the aperture are optimally aligned and adjusted to the used beam path (configuration). The position of the aperture (pixel shift and Ycoordinate) in relation to the detector makes a major contribution to image optimization. In all existing standard configurations, the apertures have already been adjusted at the factory. These settings are taken over for active operation when a standard configuration is loaded. If you want to create a setting that differs from the standard configurations, adjust the aperture as follows. The X-position does not influence brightness, but shifts the whole image position in X-direction. Use the Optimize and Kollimatic functions (see chapter Macro Menu) first, the Pinhole Adjustment is not recommended on the LSM 5 LIVE as a general tool. 03/06 B 45-0019 e 4-215 OPERATION Maintain Menu Carl Zeiss 4.10.3.1 LSM 5 LIVE LSM 5 LIVE DuoScan Open / Close the Pinhole and Collimator Window • Click on the Pinhole button in the Maintain subordinate toolbar of the Main menu. − This opens the Pinhole and Collimator window. • Click on the Close button to quit the window. 4.10.3.2 Pinhole Panel No further software function can be activated and executed during aperture adjustment. Pinhole / Description field: Selection of apertures (PH1 to PH2) to be adjusted via the Pinhole selection box, display of the relevant channel in the Description field. Diameter; Pixel Shift; Position Y slider: Setting of size, pixel shift and Y-position of the aperture in relation to the beam path using the slider or arrow buttons, status display for setting procedure: green for ready and red for busy. Store current Position button: Storage of the current aperture setting. Move to stored Position Aperture setting is reset to the position last stored. button: Adjust Automatically button: Automatic aperture adjustment. Fast Adjust mode check box: If this check box is activated, the aperture adjustment is only performed in a limited area. Used for readjustment. Fig. 4-228 4-216 Pinhole panel B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.10.3.3 OPERATION Maintain Menu Carl Zeiss Collimator panel Collimator Description field: Selection of the collimator (IR / VIS or UV / VIS) to be adjusted via Collimator selection box, display of selected collimator in the Description field. Positions field: Setting of collimator position using the slider or arrow buttons; the display to the right of the slider indicates the current position, status display for setting procedure: green for ready and red for busy. Store Current Position button: Stores the current collimator position. Move To Stored Position button: Sets the collimator to the stored value. Move to Optimal Position button: Starts the automatic collimator adjustment. Available for most common objectives. Fig. 4-229 03/06 Collimator panel B 45-0019 e 4-217 OPERATION Maintain Menu Carl Zeiss 4.10.3.4 LSM 5 LIVE LSM 5 LIVE DuoScan Aperture and collimator Adjustment Adjustment of the LSM 5 LIVE apertures can be performed manually or automatically. If several channels are used to produce the image, all the used apertures must be adjusted separately. (1) Manual aperture and collimator adjustment The position of the aperture relative to the detector in terms of Y coordinate contributes substantially to image optimization. Requirements to make aperture position changes visible immediately: − The image must be scanned by the continuous scan method. − Select a fast scanning speed. − Measurement with Average Number 1 only (no averaging of several measurements). − On the Channel Settings panel (click on Channels button in the Scan Control window), select the aperture size so as to have the best possible image contrast. • Click on the Pinhole button in the Maintain subordinate toolbar. • Select the aperture (pinhole) to be adjusted from the Description list box. • Use the Diameter slider to set the smallest possible size which produces a good, highcontrast image. − This setting changes the aperture size. − The Z Slice display box simultaneously displays the depth resolution corresponding to the aperture size. Image optimization can be effected with the Range Indicator or in the Line-Scan mode. Fig. 4-230 Pinhole panel • Optimize the pixel shift and the aperture position in Y relative to the PMT using the Pixel Shift and Position Y sliders to maximum image brightness. • Click on the Save Current Position button to save the aperture adjustment. • Removing the Position slider in the Collimator panel allows the collimator to be adjusted to maximum image brightness. Optimum collimator adjustment obtained in this way can be stored by clicking on the Save Current Position button. • Click on the Stop button to stop the continuous scan. Please do not make any program manipulations while the automatic aperture adjustment is running (status display is red - busy). 4-218 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) Automatic aperture adjustment OPERATION Maintain Menu and Carl Zeiss collimator The automatic adjustment allows the LSM 5 LIVE apertures to be used with any combination of beam splitters. • Click on the Adjust Automatically button. − The Requirements for window will then appear. Adjustment • Meet the requirements listed in the Requirements for Adjustment window and press the OK button. − Aperture adjustment will then run automatically. The adjusting procedure takes approx. 3 min. Fig. 4-231 Requirements for Adjustment window − The determined data are stored automatically and will be available for all further examinations using the same configuration. • The Move To Stored Position button enables the collimator to be set back to the factoryadjustment. • Activate the Fast Adjust Mode check box for a faster readjustment. A change of the aperture size made manually in the Pinhole panel will not be activated in the Scan Control window. Therefore, changes must always be made in the Channel Settings panel of the Scan Control window. A filter change in Autoadjust is not displayed in the Config. Control window. Please do not make any program manipulations while the automatic aperture adjustment is running (status display is red - busy). 03/06 B 45-0019 e 4-219 OPERATION Maintain Menu Carl Zeiss 4.10.4 LSM 5 LIVE LSM 5 LIVE DuoScan Camera In the Camera Color Adjustment window the user can adjust the white balance and color balance of a connected camera. Clicking the Pic button allows to set the white balance using the mouse cursor in the camera image. Using the arrow buttons to adjust the color balance of the camera. Fig. 4-232 Camera Color Adjustment window 4.10.5 Reboot The Reboot function is for servicing purposes only and may only be performed by authorized personnel. Its access is therefore password-protected. 4.10.6 HW/Admin The HW/Admin function is for servicing purposes and may only be used by authorized service personnel. Its access is therefore password-protected. 4.10.7 Test Grid The TestGrid function is for servicing purposes only and may only be performed by authorized personnel. Its access is therefore password-protected. 4-220 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.11 OPERATION Window Menu Carl Zeiss Window Menu The Window menu includes the additional functions Full Screen, Close All Image Windows, Toolbar and Scan Information which are not available from a toolbar. Fig. 4-233 4.11.1 Window menu Full Screen This function shows the active Image Display window in full screen size. • Activate the image to be shown in full size by clicking on the image content. • Click on the term Window in the menu bar of the Main menu. − The Window menu (pull-down) will be opened. • Click on the Full Screen line. − The image will be displayed in full screen size. • Click in the image to show it again as an Image Display window in normal size. 4.11.2 Close All Image Display Windows This function closes all the opened Image Display windows. • Open the Window menu. • Click on the Close All Image Windows line. − All the opened Image Display windows will be closed. In the Options menu in the function Settings in tab Save at position Save prompt at closing modified windows it can be determined whether a prompt is shown on Closing of All Image Display Windows or not. 03/06 B 45-0019 e 4-221 OPERATION Window Menu Carl Zeiss 4.11.3 LSM 5 LIVE LSM 5 LIVE DuoScan Scan and System Information This function opens the Scan Information window, in which the current scan data are displayed. The extent of the data displayed in the Scan and System Information window depends on the settings made in the Options menu under Settings (see page 4-200). • Open the Window menu. • Click on the Scan Information line. − The Scan and System window will be displayed. Fig. 4-234 4-222 Scan Information window Information button to close the Scan • Click on the Information window. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.12 OPERATION Help Menu Carl Zeiss Help Menu The Help menu permits activation of the Help function and of a window containing information on the installed software version. 4.12.1 Help • Open the Help menu. • Click on the Help line to open the online help. 4.12.2 About • Open the Help menu. • Click on the About line to open the About window. The About window includes important information about the software, such as the software version number, copyright, version numbers of the various program components and firmware, and the Dongle number. • Click on the Close button to close the About window. Fig. 4-235 4.12.3 About LSM 5 LIVE window FRAP Guide FRAP Guide opens up a SW integrated Guide for FRAP experiments. Use the buttons in the Guide to set up your experiment. Follow the steps and the descriptions. You are also referred to relevant literature and the website of the EAMNET (see also Kinetic Analysis Tool (optional), on page 4-146). 03/06 B 45-0019 e 4-223 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13 Display and Analysis of Images 4.13.1 Structure of the Image Display Window The Image Display window shows the image or images when they are − scanned by any scanning function (see Scan control and Time series control) or − loaded from the image database (see Open database-Load) or − imported Import). Fig. 4-236 Image Display window single frame image showing a by the import function (see In addition to show images the Image Display window offers two toolbars for − changing the display parameters and save an image or images (see Select toolbar below) − generating new ways of displaying the data as well as analysis tools (see Display toolbar below). The Image Display window of the LSM 5 software corresponds to the basic structure of other Microsoft ® WINDOWS applications. The Image Display window can be moved as required within the screen, and its vertical, horizontal and diagonal size can be matched to the current requirements (identical to Microsoft ® WINDOWS). The caption at the top of the Image Display window contains the control menu for the Image Display window (identical to Microsoft ® WINDOWS), the name of the displayed image, and the Minimize, Maximize and Close buttons. In the status line at the bottom of the Image Display window, the progress bar of a current scanning procedure and the parameters used for image display are shown and updated when changed. On the left-hand side of the Image Display window, an overview of the scan parameter is displayed, provided that the Info button of the Display toolbar is activated. The Settings function of the Options subordinate toolbar with the Image Display tab some of the functions of the Image Display window toolbars can be activated at the opening of a new Image Display window. It is possible to display the Chan, Zoom, Slice and Overlay image display toolbars immediately on opening an Image Display window. The relevant check boxes to be activated in the Image Display Toolbars tab under Settings (see Options menu). It is also possible to display the scan parameter of an image (Info button) immediately when an Image Display window is opened. The data to be displayed can be defined (see Image Status Display tab under Settings in the Options menu. The set of functions available at the Image Display window toolbars depends on the type of image shown. The LSM 5 software handles the following formats: − frame (single image and Z Stack of images) − frame time series (time series of images and time series with Z Stack of images) 4-224 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss − line time series (time series of lines and time series with Z Stack of lines) − point time series (time series of points) 4.13.2 Display Modes The following display modes are available for the different acquisition modes: Image type Frame Series type Frame Frame Z Stack Time Line Line Time Display functions xy • • • xt • • tx •** • • • •** • • • • • •** • • • • • Split xy •* •* •* Split tx Coded • Ortho Max Cut • Gallery • • Histo • • • Profile • • • Diagr • Mean t*** Select • • • 3D •** •*** •** Topo •** •*** •** Prev • • • • • Info • • • • • 2.5 D * only active in case of multi channel images ** inactive *** optional 03/06 B 45-0019 e 4-225 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan All display functions are exclusive functions. Only one can be active at a given time. To generate different views of the same image set use the Duplicate function in the Process menu. During image acquisition all active display functions can be used. 4.13.3 Select - Chan This function permits to − change the color assignment of channels of images − switch individual channels of a multi channel image on/off − switch to monochrome display of the image instead of color display • Click on the Chan button in the Select toolbar. − The Channels toolbar will be displayed on the right-hand side of the Image Display window. Any changes done with this toolbar are effective immediately. • Click on the Chan button again to remove the Channels toolbar. Fig. 4-237 4-226 Image Display window; Select - Chan B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.3.1 OPERATION Display and Analysis of Images Carl Zeiss Assigning Another Color to a Channel • Click on one of the channels button in the Channels toolbar (e.g.: Ch1). Fig. 4-238 Color selection box − The color selection box with all the currently defined colors will appear. • Click on the required color. − The selected color will be assigned to the current channel, the color selection box is closed and the displayed image is updated. The control box of the channel button (e.g.: Ch1) also shows the selected color. 4.13.3.2 Switching a Channel of a Multi Channel Image off or on • Click on one of the channel buttons in the Channels toolbar (e.g.:Ch1). − The color selection box will appear. • Click on OFF to deactivate the display of the relevant channel. A newly assigned color or a channel switched off is not taken into consideration during the following scanning procedure, since the setting in the Configuration Control window always applies here. 4.13.3.3 Switching to Monochrome Image Display • Click on the Mono button in the Channels toolbar. − The image will then be displayed in shades of gray exclusively. If you click on the button again, the channels will be displayed in color again. If you want to view the channels individually, select the split display by clicking on Split xy button in the Display toolbar. 4.13.3.4 Defining a New Color • Click on the Colors button to open the Channel Colors window. • Define a new color in the same way as in the Configuration Control window (see page 4-51). 03/06 Fig. 4-239 B 45-0019 e Channel Colors window 4-227 OPERATION Display and Analysis of Images Carl Zeiss 4.13.4 LSM 5 LIVE LSM 5 LIVE DuoScan Select - Zoom This function allows to change the zoom factor of an image displayed. Click on Zoom will display the Zoom toolbar. Any changes done with this toolbar are effective immediately. The image can be zoomed by various methods. The zoom function can be performed online. • Click on the Zoom button in the Select toolbar. − The Zoom toolbar will be displayed on the right-hand side of the Image Display window. • Clicking on the Zoom button again will remove the Zoom toolbar. Fig. 4-240 Image Display window; Select - Zoom Zoom-Auto The image is fitted automatically to size of the Image Display window. Zoom-Resize Restores the image to its initial size. Zoom + Enlarges the image by factor 2. Zoom – Reduces the image by factor 2. Zoom 1:1 Restores an image zoomed in any way to its original size. Zoom-Mouse Allows you to enlarge / reduce the zoom factor of an image using the left / right mouse button, provided that the cursor is inside the image. 4-228 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss When active it allows you to zoom all images of a gallery to the same extent without changing the display image format. All Zoom-+, Zoom-–, Zoom 1:1, Zoom-Mouse and All can only be defined when the Zoom-Auto function is deactivated. Slider with display box 4.13.5 The zoom factor can be set by moving the slider. The display box below displays the current zoom factor. Factor 1 corresponds to the original size. Select - Slice This function allows to select and view individual slices from a Z Stack or a time series, when images where acquired in frame mode. The button is grayed, when these conditions are not true. Click on Slice will display the Slice toolbar. Any changes done with this toolbar are effective immediately. • Click on the Slice button in the Select toolbar. − The Slice toolbar is displayed on the right-hand side of the Image Display window. • If you click on the Slice button again, the Slice toolbar is removed. Fig. 4-241 03/06 Image Display window; Select - Slice B 45-0019 e 4-229 OPERATION Display and Analysis of Images Carl Zeiss 4.13.6 LSM 5 LIVE LSM 5 LIVE DuoScan Select - Overlay This function allows to − select from a set of drawing functions such as rectangles and arrows − add a scale bar to the image − use a set of interactive measurement functions for length, angle and size 4.13.6.1 Functional Description The overlay function uses a plane separate from the image plane (the graphics plane) and does therefore not change the content of the image(s). The button is only available if the XY or Split XY Display functions are selected. Otherwise it is grayed. Some of the Display functions such as Ortho or Cut turn the overlay graphics off temporarily. Any changes done with this function are effective immediately. The overlay graphics can be stored together with images and can be retrieved from the LSM 5 image database. • Click on the Overlay button in the Select toolbar. − The Overlay toolbar will be displayed on the right-hand side of the Image Display window. • If you click on the Overlay button again, the Overlay toolbar will be removed. Provided that the display of the overlay elements has not been deactivated by clicking on the Off button, the created elements will still be displayed in the Image Display window even after closing of the Overlay toolbar. Fig. 4-242 4-230 Image Display window; Select - Overlay B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.6.2 OPERATION Display and Analysis of Images Carl Zeiss Buttons in the Overlay Toolbar The following functions can be used on activation of the buttons in the Overlay toolbar: Arrow (selection) button: Activation of the mouse button for selection, resizing or movement of an overlay element in the Image Display window. Resizing: Click on the handle and hold down the mouse button, drag the handle, release the mouse button. Movement: Click on the line and hold down the mouse button, move the entire element, release the mouse button. Line button: Creation of a straight line in the Image Display window. Click and hold down the mouse button, draw a line in any required direction, release the mouse button to end the procedure. Rectangle button: Creation of a rectangle in the Image Display window. Click and hold down the mouse button, draw a rectangle in any required direction, release the mouse button to end the procedure. Closed polyline button: Creation of a closed polyline figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Open polyline button: Creation of an open polyline figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button ends the procedure. Ellipse button: Creation of an ellipse in the Image Display window. The first click sets the center point, the displayed line permits the determination of the first dimension, the second click sets the first dimension, the second dimension and rotation direction can then be determined, the third click sets the second dimension and direction and ends the procedure. Closed free-shape curve button: Creation of a closed Bezier figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Open free-shape curve button: Creation of an open Bezier figure in the Image Display window. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. 03/06 B 45-0019 e 4-231 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Circle button: Creation of a circle in the Image Display window. Clicking and holding down the mouse button sets the center point, drag the diameter and release the mouse button to end the procedure. Line with arrow button: Creation of a line with arrow in the Image Display window. Click and hold down the mouse button, drag the line in any required direction, release the mouse button to end the procedure. Scale button: Creation of a horizontal or vertical scale with default increments in the Image Display window. Click and hold the mouse button for the starting point, drag horizontal or vertical scale, release the mouse button to end the procedure. Gray tones / color shades button: Generates a rectangle with a display of gray tones or color shades in the image. Color shades are displayed if a palette has been loaded, with different colors being assigned to the gray tones. A (Text) button: Creation of a text box in the Image Display window. After clicking on A, the Text window will be displayed, and text can be entered via the keyboard. The Font ... button enables you to select the font style and size in the Font window. The entered text will be displayed in the left upper corner of the Image Display window after clicking on OK and can be moved to the required position using the mouse. The Text window can also be activated with a double-click on a created text box, and the entered text can be edited subsequently. Insert opens up a further window which allows you to annotate coordinates, time and Z-position with either automatic or user definable units and precision. This annotation is updated during image acquisition and can be exported with the image. The annotation can be stamped into already existing images. Recycle bin button: All the overlay elements and dimensions dragged to the scanned image are deleted. If one overlay element was marked before, this element is now deleted from the scanned image. Multiple button: On activation of this button, the overlay function subsequently selected is performed several times in succession, without the need to activate the function button again. This function remains selected until the Multiple button is deactivated again. Measure button: Measurement of the overlay element in the Image Display window. On activation of the Measure button, the selected overlay element and all the elements created afterwards are measured and assigned with a measuring value. The measuring value can be shifted without regard to the overlay element. If of importance, the length and perimeter of a line figure, the area of a closed figure and the inclination angle of a single line will be displayed. On deactivation of the Measure button, the measuring value of the selected element is no longer displayed, and all the elements created afterwards will not be assigned with a measuring value. 4-232 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Off button: Deactivation of the display of overlay elements in the Image Display window (hide overlay). Deactivation of the overlay functions. Line button: This button allows you to determine the line thickness of the area outline. Cut button: The image contents of an overlay element are cut out, and the area will then appear in black. Copy button: The image contents of a closed overlay element are copied to the clipboard. Paste button: The image contents of an overlay element copied to the clipboard are inserted in the current Image Display window and can be positioned anywhere in the image using the mouse. Undo button: The last Cut or Paste action can be undone by clicking on the Undo button. Extract Region button: The region of a Z Stack or 4D-image surrounded by an Overlay element is extracted and can be displayed and stored separately in a new Image Display window. This function is only active if an Overlay element is used, that generates a closed contour. Color selection box: The colors displayed in the Color selection box can be assigned to the overlay elements with a click of the mouse. The currently selected color is displayed in the larger rectangle (left top) of the selection box. A selected color is automatically assigned to the currently selected overlay element and then to all the elements created afterwards. 03/06 B 45-0019 e 4-233 Carl Zeiss OPERATION Display and Analysis of Images 4.13.7 LSM 5 LIVE LSM 5 LIVE DuoScan Select - Contr This function allows to − change the contrast and brightness of an image − change the contrast and brightness of a channel of an image − define interactively a new relationship between the intensities of pixels in the image memory and the displayed values of this pixel intensities on the computer screen Click on Contr will display the Contrast toolbar. Any changes done with this toolbar are effective immediately. Fig. 4-243 Modification done by this function are for display purposes only. To permanently change the contrast and brightness of an image use the function Contrast in the Process menu. Image Display window; Select - Contr • Click on the Contr button in the Select toolbar. − The Brightness and Contrast window will be displayed. • Change brightness and contrast via the sliders in the Brightness and Contrast window. You can adjust each channel individually by activating the channel button (e.g.: Ch1), or influence all channels simultaneously by clicking on All. • Clicking on the Reset button will reset the original setting of brightness and contrast. • Clicking on the Close button will close the Brightness and Contrast window. Further contrast and brightness parameters can be activated or deactivated alternately using the More and Less buttons. Fig. 4-244 Brightness and Contrast window • Click on the More button to display the additional functions. − The Brightness and Contrast window will be enlarged, the labeling of the button changes from More to Less. If you click on Less, the additional functions are no longer displayed. Simultaneously with the setting of brightness and contrast, the intensity values of the image can be set directly in the Intensity Screen via the Ramp, PolyLine, Spline and Gamma functions. The intensity values can also be set either for all channels together or individually. If the image has already been changed using the Contrast and Brightness sliders, this setting difference is displayed in the Intensity Screen by means of the Shape and Result lines. 4-234 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.7.1 OPERATION Display and Analysis of Images Carl Zeiss Ramp The intensity is set via two knots in the Intensity Screen, which allows an intensity line to be created in the form of a ramp. The original line form is reset via Reset. The line form will be retained even when the additional functions are no longer displayed, and on closing the Brightness and Control window. 4.13.7.2 Fig. 4-245 Brightness and Contrast window with activated Ramp function Fig. 4-246 Brightness and Contrast window with activated PolyLine function PolyLine The intensity is set in the Intensity Screen via a freely selectable number of knots, which permits the creation of an intensity line in the form of a polyline. The number of knots can be selected from the Number of Knots selection box. The original line form is reset via Reset. The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed. 03/06 B 45-0019 e 4-235 Carl Zeiss OPERATION Display and Analysis of Images 4.13.7.3 LSM 5 LIVE LSM 5 LIVE DuoScan Spline The intensity is set in the Intensity Screen via a freely selectable number of knots, which permits the creation of an intensity line in the form of a spline. The number of knots can be selected from the Number of Knots selection box. The original line form is reset via Reset. The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed. Fig. 4-247 Brightness and Contrast window with activated Spline function 4.13.7.4 Gamma The intensity is set in the Intensity Screen by varying the gamma curve (clicking and dragging with the mouse) or by moving the Gamma slider. It is possible to set gamma values between 0.1 and 2.0. The original line form is reset via Reset. The line form will be retained even when the additional functions are no longer displayed or when the Brightness and Control window is closed. Fig. 4-248 4-236 Brightness and Contrast window with activated Gamma function B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.8 OPERATION Display and Analysis of Images Carl Zeiss Select - Palette This function allows to − change the palette used for displaying the image(s) − define and save new palettes − delete palettes by removing them Click on Palette will display the Palette toolbar. Any changes done with this toolbar are effective immediately. The standard palettes No palette, Range indicator, Glow Scale and Rainbow are system palettes and can not be deleted. The Range indicator palette is useful to optimize the gain and offset setting of images in the Scan control window before scanning. Palettes are stored and retrieved together with the images when archived in the Image Database. • Click on the Palette button in the Select toolbar. − The Color Palette window will be displayed. • Select the required palette from the Color Palette List panel by clicking on the relevant name. • If you want to deactivate a palette selected before, click on No Palette in the Color Palette List panel. • Click on the Close button to close the Color Palette window. • A changed image can be stored via the Save As function. In the Options menu in the function Settings it is possible to switch to Mono automatically when a palette is activated and to Colour on deactivation of a palette. In addition it is possible to activate / deactivate Mono in the Channel toolbar. Some of the handling functions of the Image Display window toolbars can be activated at the opening of a new Image Display window. 03/06 B 45-0019 e 4-237 OPERATION Display and Analysis of Images Carl Zeiss Fig. 4-249 LSM 5 LIVE LSM 5 LIVE DuoScan Image Display window, Select - Palette; Color Palette and Add Palette to List window 4.13.8.1 Editing and Storing a Palette A palette is edited by moving the knots in the Ramp, Polyline and Spline functions (identical to the setting in the Contrast and Brightness window, see page 4-234f). The palette can be set for all colors together or separately for each color. • Activate the relevant button: Red, Green, Blue or All. Proceed as follows to store an edited palette under a new name: • Click on the Add To List button: the Add Palette To List window will be displayed. • Enter a name for the palette and click on Ok. − The palette will be stored and the name included in the Color Palette List panel. Fig. 4-250 4-238 Color Palette window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.8.2 OPERATION Display and Analysis of Images Carl Zeiss Delete a Palette Proceed as follows to delete a palette: • Click on the name of the palette to be deleted in the Color Palette List panel and then on the Remove button. − The palette will be removed from the list. The standard settings (No Palette, Range Indicator, Glow Scale and Rainbow) cannot be deleted. 4.13.8.3 Import a Palette Proceed as follows to import a palette: • Click on the Import button. The Import Palette window will be opened. • Select the required palette (file extension: *.lut) from the relevant directory and click on Open. − The palette will be imported and displayed in the Color Palette List panel. File with the extension *.lut are LSM 310 / 410 palette files. 4.13.9 Select - Anim This function allows to − animate frames of a Z Stack or a time series − specify animation parameters such as range and animation speed Click on Anim will display the Animate toolbar. Any changes done with this toolbar are effective immediately. When the image(s) displayed in the Image Display window is neither a Z Stack nor a time series this button is grayed and not accessible. Fig. 4-251 Animate window • Click on the Anim button in the Select toolbar of the Image Display window of a stack. − The Animate window will be displayed and the animation started immediately. • Click on the Close button to close the Animate window and to stop the animation. 03/06 B 45-0019 e 4-239 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The animation is controlled via the following function elements: Current Slice slider: Manual movement through the individual slices of a stack by moving the slider, or by entering the slice number in the input box. Slider can be accessed only, when the automatic animation is off. Start slider: The setting of the Start sliders limits the number of slices to be used for the animation. Previous slices are not taken into consideration for the animation. Can be changed during automatic animation. End slider: The setting of the End slider limits the number of slices to be used for the animation. Subsequent slices are not taken into consideration for the animation. Can be changed during automatic animation. Starts the forward motion of the automatic animation. After the last slice has been passed, restart is made at the first slice. Starts backward motion of the automatic animation. After the first slice has been passed, restart is made at the last slice. Starts the combined forward / backward motion of the automatic animation, i.e. when the last slice has been reached, the backward motion is activated, and the forward motion is activated again on reaching the first slice. Stops the automatic animation. Move to the first slice. After each click on this button, backward motion is made by the number of slices set under Increment. After each click on this button, forward motion is made by the number of slices set under Increment. Move to the last slice. Speed1 /Speed2 buttons / sliders: Selection between two speeds, change of the relevant speed via slider or input box. Increment slider: Reduction of the slices to be displayed by selecting an increment n (step width) of slices to be taken into consideration for the animation. If n = 3, for example, only every third slice of the stack will be displayed during the animation. 4-240 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.10 OPERATION Display and Analysis of Images Carl Zeiss Select - Reuse This function allows to − transfer acquisition parameters of an image from the image data base to the Microscope control, Configuration Control, Scan Control and Time Series Control windows and applies those parameters directly on the system. The acquisition parameters of an image are displayed in the Image Display window and can be viewed by using the Info function. In the tab Image Status Display in the Settings function of the Options subordinate toolbar it can be determined what parameters to view with the Info function. The parameters include the following: Frame Size, Speed, Data Depth, Scan Direction, Average, Zoom Rotation, Offset, Aperture Size (confocal aperture), Detector Gain, Amplifier Offset, Amplifier Gain, Excitation, Beam Path and Scan Mode (Line, Frame, Stack, Time Series). However, the required objective must be selected by the user. • Click on the Reuse button. The acquisition parameters of the active image (stack) are applied immediately to the system. In the Options menu in the function Settings with the Recording/Reuse tab, it can be determined whether the objective should also be transferred and set. Setting the microscope objective only works in microscopes with motorized objective revolvers. 03/06 B 45-0019 e 4-241 OPERATION Display and Analysis of Images Carl Zeiss 4.13.11 LSM 5 LIVE LSM 5 LIVE DuoScan Select - Crop This function allows to − interactively define the size and orientation of a rectangular scan area on the image displayed in the Image Display window. − The defined area is displayed together with the Zoom and Offset parameters in the Scan Control window in the Mode submenu. Fig. 4-252 Image Display window; Select - Crop Click on Crop will display the Crop Rectangle in the Image Display window. Any changes done with the Crop Rectangle are setting the parameters immediately. On the next execution of a scan (Find, Fast xy, Single, Continuous in Scan Control or Start T or Start B in Time Series Control) these new scan parameters will be used. To reset the crop function and use default values set Zoom=1 and Offset=0 in the Scan Control window in the Mode submenu. • Click on the Crop button. − The Crop Rectangle will appear on the Image Display window. The Crop Rectangle is controlled via the following functional elements: Offset • Click into the crop rectangle, keep the left mouse button pressed and drag the crop rectangle to the required position. Release the mouse button. Zoom • Click on a corner of the crop rectangle, keep the left mouse button pressed and set the required size. Release the mouse button. Side ratio • Click on any of the intersection points between crossline and crop rectangle, keep the left mouse button pressed and change the side ratio as required. Release the mouse button. 4-242 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.12 OPERATION Display and Analysis of Images Carl Zeiss Select - Copy This function allows to − copy the current displayed image into the clipboard. Click on Copy will be immediately effective. From the clipboard images can be incorporated into other programs such as MS Excel, MS Powerpoint or MS Word. To export image series, use the Export function in the File menu. • Click on the Copy button. − The content of the Image Display window is copied to the clipboard. • Start the clipboard application of WINDOWS. • Select Paste in the Edit menu of the Clipboard application. 4.13.13 Select - Save This function allows to − save the image(s) of the Image Display window into an Image Database − by not showing a dialogue and using the automatic assigned and incremented image name and a predefined existing Image Database − Prerequisite: Autosave is checked in the Settings function with the Autosave tab Click on Save will be immediately effective. When the prerequisite is not met, the Save As dialogue is displayed. In the Options menu in the function Settings with the Autosave tab parameters such as an automatically incremented filename can be determined and the Autosave activated/deactivated. 03/06 B 45-0019 e 4-243 OPERATION Display and Analysis of Images Carl Zeiss 4.13.14 LSM 5 LIVE LSM 5 LIVE DuoScan Select - Save As This function allows to − save the image(s) of the Image Display window into an Image Database − by showing a dialogue − use the defaults as defined in the Settings function with the Save tab Click on the Save As button displays the Save Image and Parameter As window. Changes will be effective on closing this dialogue. In the Options menu in the function Settings with the tab Save default parameters such as Name, Description and Notes can be set. • Click on the Save button. − The Save Image and Parameter As window appears • Enter text for the image name, description, notes or change the user name. • Select the Image Database from the list of databases (MDB) or • Open other Image Databases by selecting Open MDB or • Create new Image Databases by selecting New MDB. 4.13.15 Display - xy This function allows to − display a single image in frame mode − display multi channel images in superimposed mode The settings of Chan, Zoom, Slice, Overlay, Contr and Palette are applied. Click on xy will be immediately effective. 4-244 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.16 OPERATION Display and Analysis of Images Carl Zeiss Display - Split xy This function allows to − display the individual channels of a multi channel image as well as the superimposed image The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply. Click on Split xy will be immediately effective. Fig. 4-253 Image Display window, Split xy display This function is useful to optimize the individual channels in a multi channel image acquisition together with the Range Indicator palette . 03/06 B 45-0019 e 4-245 OPERATION Display and Analysis of Images Carl Zeiss 4.13.17 LSM 5 LIVE LSM 5 LIVE DuoScan Display - Ortho This function allows to − display a Z Stack of images in an orthogonal view − measure distances in three dimensions − generate 2D deconvolution views of the yz and xz plane The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply. Click on Ortho will be immediately effective. • By clicking on the Ortho button section lines appear in the Display toolbar together with orthogonal projections in the image. On the right-hand side, the Orthogonal Sections toolbar is shown. Fig. 4-254 4-246 Image Display window, Ortho display B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.17.1 Ortho - Select Function • By changing the parameters X, Y and Z in the Orthogonal Sections toolbar, the section plane can be positioned at any XYZ coordinate of the Z Stack. The position of section planes can be changed in various ways: • By moving the sliders on the Orthogonal Sections toolbar. − X and Y settings may range from 1 up to the maximum number of pixels scanned (in the example shown: 512). − Z settings may range from 1 to a maximum of n, with n standing for the number of slices produced in the stack. • By directly entering the relevant number value in the X-, Y- or Z-input box and pressing the Enter key. . By • If you move the cursor into the Image Display window, it changes into a crosslines symbol dragging this symbol with the mouse you can position the XZ and YZ section planes to any point of intersection with the XY plane. A click with the left mouse button places the intersection to the desired position. • If you move the crosslines symbol onto the intersection of the red and green section planes, it symbol. If you now press the left mouse button and keep it pressed you can changes into the: reposition both section planes simultaneously. • If you move the crosslines symbol onto the green section plane, it changes into the symbol. If you now press the left mouse button and keep it pressed, you can reposition the (green) XZ section plane. • You can reposition the (red) YZ plane in the same way using the symbol. The result of an orthogonal section is visible at the image margin. − Section of the XZ plane (green line) through the stack: above the XY image. − Section of the YZ plane (red line) through the stack: right of the XY image. − Section of the XY plane (blue, slice plane of the stack): center image. 03/06 B 45-0019 e 4-247 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.17.2 Ortho - Distance Function • Activating the Dist. button permits length measurements in 3D space. • Click on the Mark button to set the first XYZ-point for the measurement of the spatial distance. • Set the second XYZ-point for measurement by moving the X-, Y-, Z-sliders or by moving the green, red and blue lines in the image. − The projections of the spatial distance are shown in the image planes by yellow lines. The actual spatial distance is calculated and shown in µm below the Select, Dist. and Mark buttons, e.g. 3D Distance: 55.60 μm. Fig. 4-255 4-248 Image Display window, Ortho Distance display B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.17.3 Ortho - 2D DeConVolution Function The 2D deconvolution function causes orthogonal projection enhancement through the computed correction of the resolution in the Z-coordinate. Image enhancement is only effective for the two projections of a fluorescence stack in the Ortho display or for an XZ-scan in fluorescence, and is also only computed for this. • Activate the 2D DCV button in the Orthogonal Sections toolbar. If the Fast button is activated, calculation of the 2D-deconvolution (inverse DCV mode) is performed immediately. The 2D DCV settings button can only be activated if a licence for the 3D DCV option has been purchased. Otherwise this button is grayed. • Click on the Settings button. The 2D Deconvolution window is opened. The 2D Deconvolution window contains the Deconvolution panel with the two tabs Method and PSF. (1) Method tab The Method tab enables you to choose between the calculation methods Inverse and Iterative. For more details of explanation of deconvolution and the calculation methods see 3D DeConVolution (page 4-163). In the Inverse method, the Restoration Effect slider can be used to set the signal-to-noise ratio between Weak (low noise) and Strong (pronounced noise). Fig. 4-256 Method tab Activation of the Auto detect check box starts a routine for the automatic determination of the noise level in the entire image part of the Z Stack. If Auto detect is enabled, the Restoration Effect slider is disabled. The Iterative method permits (in addition to the parameters of the Inverse method) the maximum number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on the set image improvement (delta between last but one and last cycle in %), no matter whether the value under Maximum Iterations has been achieved or not. 03/06 B 45-0019 e 4-249 OPERATION Display and Analysis of Images Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan PSF tab (optional with 3D DCV) The objective data are displayed in the PSF tab. In the case of wavelengths above 700 nm, the NLO button is automatically enabled. The displayed values are always taken over by the system data, but can be edited subsequently for simulation purposes. • Select the required method and determine the relevant parameters. Fig. 4-257 PSF tab The deconvolution calculation is performed immediately after the 2D Deconvolution window has been closed, and the image display is updated (on-line). 4.13.18 Display - Cut This function allows to − display a Z Stack of images at a user defined section plane (= cut plane) − improve the image of the section plane by trilinear interpolation The settings of Chan, Zoom, Slice, Contr and Palette are applied. • Clicking on the Cut button in the Display toolbar opens the Cut to the right of the Image Display window. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. • By varying the parameters X, Y, Z, Pitch and Yaw, you can position a section plane of any orientation within the stack volume. • The resulting position of the section plane is shown as a red area below the Trilinear Interpolation button. At the same time, the result is shown in the Image Display window. • A click on the Reset All button restores the original position. Fig. 4-258 4-250 Image Display window, Cut display • A click on the Trilinear Interpolation button will improve the quality of the image by performing a 3D interpolation of the image. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.19 OPERATION Display and Analysis of Images Carl Zeiss Display - Gallery This function allows to − display images (Z Stack, time series, combination of both) side by side in tiled fashion − add data relevant to the images displayed (Z Stack slice distance, time of acquisition or wavelength) − extract a subset of images from the original stack and store the result as a new image The settings of Chan, Zoom, Slice, Contr and Palette apply. Click on Gallery will display the Gallery toolbar. Any changes done with this toolbar are effective immediately. • A click on the Gallery button in the Display toolbar not only produces the gallery itself but also the Gallery toolbar with two buttons: Data button and Subset button. • Clicking on the Data button shows the Z Slice distance, the acquisition time or the wavelength or combinations. • Clicking on the color selection button (below the Data button) will open a color selection window allowing you to choose - at a click of the mouse - in which color the data will be shown in the gallery display. • Clicking on the Subset button opens another window entitled Subset, in which you can select images of the set of images displayed. − A stack consisting of the selected images only is generated and displayed. • Select Start Slice and End Slice via the sliders, the input box or the Click (into window) button. • Enter a value for n in the Every n-th Slice panel. Fig. 4-259 Subset window • If required, activate the Convert 12 bit to 8 bit check box . • Clicking on the Ok button will generate a new subset of images. • Cancel will stop the procedure. 03/06 B 45-0019 e 4-251 Carl Zeiss Fig. 4-260 4-252 OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Image Display window, Gallery display B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.20 OPERATION Display and Analysis of Images Carl Zeiss Display - Histo 4.13.20.1 Display - Histo - Overview This function allows to − display a histogram (distribution of pixel intensities) of an image − show the histogram values in table form − copy table to clipboard or save as text file − analyze the colocalization between two channels − measure area and mean gray value and standard distribution in an area − show separate histograms for each channel in a multi channel image Colocalization is only available in case of a two or multi channel image. The settings of Chan, Zoom, Slice, Contr and Palette are applied. Click on Histo will display the Histogram toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. • Click on the Histo button. The Histogram toolbar will be displayed on the right. Fig. 4-261 Image Display window, Histogram display The Histo button can also be used online during scanning. 03/06 B 45-0019 e 4-253 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Histogram functions Skip Black button Ignore black pixels (gray value 0) in the histogram. Skip White button Ignore white pixels (gray value 255 or 4096) in the histogram. Statistics Displays statistical parameters (Mean Intensity, Standard Deviation, Number of Pixels and Size of Area) in a additional table. Area measurements of very small areas (< 10 pixels) give only approximate values Step input box Set the number of intensity steps which shall be displayed in the diagram. Step 1 corresponds to 256 intensity steps, Step 64 to 4 intensity steps in case of 8 bit images. Reduction is made by averaging. Show Image button Shows the image in the Image Display window beside the histogram. Show Table button The histogram is shown as a table at the bottom left of the Image Display window. Copy Table button The histogram table is copied to the clipboard. Save Table button The histogram table can be stored as a text file (extension *.txt). Area functions Area button Interactive definition of area for size and intensity measurement. Save Values button Copies area values to the clipboard (only available if the Area button is activated). 4-254 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.20.2 Area Function • Click on the Area button in the Histogram toolbar. − The function elements for Area measurement are displayed at the bottom right of the Histogram toolbar. Fig. 4-262 Image Display window, Area Measure display The following function elements are available: Set the number of intensity steps which shall be displayed in the diagram. Step 1 corresponds to 256 intensity steps, Step 64 to 4 intensity steps in case of 8 bit images. Reduction is made by averaging. Threshold low slider with Color selection button: The intensity values below threshold are not displayed. The removed areas are masked in the color selected in the Color selection button. Threshold high slider with Color selection button: The intensity values above threshold are not displayed. The removed areas are masked in the color selected in the Color selection button. Ch1, Ch3 ... buttons: Selection of the channel for which the area measurement is to be performed. Arrow (selection) button: Activation of the mouse button for selection, resizing, or movement of a mask element in the Image Display window. Resize: Click on handle Movement: Click on line and hold the mouse button, move the entire figure, release mouse button. and hold down the mouse button, drag the handle, release mouse button. 03/06 B 45-0019 e 4-255 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Closed polyline button: Creation of a polyline figure in the image. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Recycle bin button: All the mask elements are deleted. If one element was marked before, this element is now deleted from the image. Mask button: Enables the Mask Mode, where the region can be defined with ink. Rectangle button: Creation of a rectangle in the image. Click and hold down the mouse button, drag a rectangle in any direction, release the mouse button to end the procedure. Ellipse button: Creation of an ellipse in the image. The first click sets the center point, the displayed line permits the determination of the first dimension, the second click sets the first dimension, the second dimension and the rotation direction can then be determined; the third click sets the second dimension and the direction and ends the procedure. Line button: Determines the line thickness of the area outline. Flood fill button: Fills the overlay element or the scatter diagram with the color selected under Mask. Closed free-shape curve button: Creation of a closed Bezier figure in the scatter diagram. The first click sets the starting point, each additional click adds a further line, a click with the right mouse button closes the figure and ends the procedure. Circle button: Creation of a circle in the scatter diagram. Clicking and holding down the mouse button sets the center point; drag the diameter and release the mouse button to end the procedure. Color selection button: The colors displayed in the selection box can be assigned to the mask elements with a click of the mouse. The currently selected color is always displayed in the larger rectangle (top left) of the selection box. Clear Mask button: Removes the color filling from an overlay element or from the scatter diagram. • The function can be activated by clicking on one of the geometry buttons, e.g. (polyline). • The figure of interest can be marked in the image by cursor control in conjunction with a mouse click. • If more than one Region is drawn either the mean histogram values for all regions is displayed or only the values of the activated region if it is marked by clicking on the drawing line with the mouse. • Areas can also be excluded from the analysis. By clicking on the Flood fill button (paint jar) and moving the cursor to the area to be excluded causes the remaining area to be computed and the result indicated under Area Measure. • If you specify a top and bottom intensity threshold, the area lying within this intensity interval can be computed. 4-256 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss • Specify the thresholds either with the Threshold low and Threshold high sliders, or with the ▲▼ buttons. 4.13.20.3 Colocalisation Function The Colocalisation function permits interactive analysis of two channels of an image by computing a scatter diagram (colocalisation). • Click on the Colocalisation button. The scatter diagram is created and displayed beside the image. • Click on the xy button of the Display toolbar if you want to return to the original image. How a scatter diagram is generated All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2) from the two source images, the intensity level of pixel P1 is interpreted as X coordinate, and that of pixel P2 as Y coordinate of the scatter diagram. The value of the pixel thus addressed is increased by one every time, up to the maximum number of pixels used. This way, each pixel of the scatter diagram is a value that shows how often a particular pair of pixels has occurred. Differences between the images cause irregular spots in the scatter diagram. Identical images produce a clean diagonal line running from bottom left to top right, because only pixel pairs (0,0), (1,1), (2,2) with the same intensity can occur. Differences between the images cause an irregular distribution in the scatter diagram. Fig. 4-263 03/06 Image Display window, Colocalization display B 45-0019 e 4-257 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Function elements The following function elements are available: Displays the scatter Histogram of the two image channels Adds display of the according data table Adds display of the image Displays a movable crosshair for different areas in the scatter histogram Opens the Intensity Threshold window to sets threshold for colocalisation in the scatter histogram. Set from Image ROIs button: Sets background threshold from ROI (Threshold button) Cut Mask button. Channel Toggle button. Invert Mask button: Inverts the mask or the scatter diagram. Source 1 selection box with Color selection box: Selection of the first channel to be selected via the selection box, assignment of a defined color via the Color selection box. Source 2 selection box with Color selection box: Selection of the second channel to be selected via the selection box, assignment of a defined color via the Color selection box. Mask selection box with Color selection box: Selection of RGB or Overlay display of the mask, assignment of a defined color via the Color selection box. Drawing tools Allows the selection of ROIs in the Histogram and the Image. Save at drawing tools Stores ROIs and threshold settings. 4-258 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Enhanced colocalisation Scatter diagram image display and data table are interactively linked. 03/06 Fig. 4-264 Image Display window, Colocalization display Fig. 4-265 Scatter diagram and threshold with crosshair B 45-0019 e 4-259 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Quantitative Parameters − No. of pixels in image ROI or scatter region − Area / relative area of image ROI or scatter region − Mean intensities / SD within image ROI or scatter region − Colocalization coefficients − Weighted colocalization coefficients − Overlap coefficient after Manders 2 − Correlation coefficients (R and R ) Colocalization coefficients c1 = pixelsCh1,coloc c2 = pixelsCh1,total pixelsCh 2,coloc pixelsCh 2,total − Relative number of colocalizing pixels in channel 1 or 2, respectively, as compared to the total number of pixels above threshold. − Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize) − All pixels above background count irrespective of their intensity. Weighted colocalization coefficients ∑ Ch1 = ∑ Ch1 i,coloc M1 ∑ Ch2 = ∑ Ch2 i,coloc i M2 i,total i i i,total i − Sum of intensities of colocalizing pixels in channel 1 or 2, respectively, as compared to the overall sum of pixel intensities above threshold and in this channel. − Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize) − Bright pixels contribute more than faint pixels Correlation coefficient, Pearson´s correlation coefficient Rp = ∑ (Ch1 i − Ch1aver ) * (Ch 2 i − Ch2 aver ) i ∑ (Ch1 i i − Ch1aver ) 2 * ∑ (Ch 2 i − Ch 2 aver ) 2 i − Provides information on the intensity distribution within the colocalizing region − Value range -1 to +1 -1,+1: all pixels are found on straight line in the scatter diagram 0: pixels in scattergram distribute in a cloud with no preferential direction 4-260 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Overlap coefficient, overlap coefficient after Manders (Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993) r= ∑ Ch1 * Ch2 i i i ∑ (Ch1 ) * ∑ (Ch2 ) 2 i i 2 i i − Another parameter used to quantify colocalization in image pairs − Insensitive to differences in signal intensities between the two channels, photo-bleaching or amplifier settings − Value range 0 – 1 (0: no colocalization, 1:all pixels colocalize) 4.13.21 Display - Profile 4.13.21.1 Function This function allows to − display the intensity distribution of an image along a straight or curved line − show the intensity values in table form and copy table to clipboard or save as text file − show separate profiles for each channel in a multi channel image The settings of Chan, Zoom, Slice, Contr and Palette are applied. Click on Profile will display the Profile toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. • Click on the Profile button. The Profile toolbar will be displayed. − The intensity curves are shown in a graph below the image(s). • On the Profile toolbar you can select the width and color of the profile line. • You can place two markers on the profile line to measure differences in intensities and distances in the XY plane. The Profile button can also be used online during scanning. • Click on the Diagr. in Image button to see an intensity graph superimposed on the image. 03/06 B 45-0019 e 4-261 Carl Zeiss 4-262 OPERATION Display and Analysis of Images Fig. 4-266 Image Display window, Profile display Fig. 4-267 Image Display window, Profile display B 45-0019 e LSM 5 LIVE LSM 5 LIVE DuoScan 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.21.2 Function Elements The Profile toolbar contains the following function elements: Arrow (selection) button: Activates the mouse button for selection, resizing or movement of the profile line in the Image Display window. Resize: Click on handle and hold down the mouse button, move the handle, release mouse button. Movement: Click on line and hold down the mouse button, move the entire line, release mouse button. Line with arrow button :(open arrow): Activates the straight profile drawing mode. Click into the image and hold the mouse button, drag a line in any direction and release the mouse button to end the procedure. Open polyline button: Activates the open polyline drawing mode. The first click into the image sets the starting point, each additional click adds a further line, right mouse click ends the procedure. Open free-shape curve button: Activates the Bezier figure drawing mode. The first click into the image sets the starting point, each additional click adds a point, right mouse click ends the procedure. Line button: This button allows you to determine the line thickness of the profile line. It has no influence on the way the intensity profile is generated. Color selection box: The colors displayed in the Color selection box can be assigned to the overlay profile line with a click of the mouse. The currently selected color is displayed in the larger rectangle (top left) of the selection box. Diagr. In Image button: The profile diagram is displayed in the image along the drawn profile line. Marker 1 button (red): Activates the red marker for movement in the profile diagram; the marker shown as a red line in the diagram can now be moved to the right or left of the diagram using the mouse. The marker in the image display (red circle) follows accordingly. Marker 2 button (blue): Activates the blue marker for movement in the profile diagram; the marker shown as a blue line in the diagram can now be moved to the right or left of the diagram using the mouse. The marker in the image display (blue circle) follows accordingly. 03/06 B 45-0019 e 4-263 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Zoom button: Zoom function for profile diagram. Click and drag a rectangle over the area to be enlarged in the profile diagram; the selected area is enlarged on release of the mouse button. The zoom function can be performed several times. A click with the right mouse button will reset the original size. Reset Zoom button: Resets the zoom factor of the profile diagram to the original size. Show Table button: The profile diagram is displayed as a table at the bottom of the Image Display window. Copy Table button: The profile table is copied to the clipboard. Save Table button: The profile table can be stored as a text file (extension *.txt). 4-264 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.21.3 Display - Mean This function allows to − display the intensity time diagram (mean intensity in user defined ROIs over time) − display the intensity time diagram for volumes (3D ROIs) within a Z-Stack over time − use frame time series and frame Z Stack time series as input − show the intensity values in table form and copy table to clipboard or save as text file − show separate diagrams for each channel in a multi channel image The settings of Chan, Zoom, Slice, Contr and Palette apply. Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. To use a similar functionality while scanning use the optional Mean of ROI function with the Time series control. • Click on the Mean ROI button. − The Mean of ROIs image display toolbar will be displayed on the right. The used ROIs become visible in the image, and the Intensity-Time diagram is shown on the left of the Image Display window. Fig. 4-268 03/06 Image Display window, Mean ROI display for time series in single plane B 45-0019 e 4-265 OPERATION Display and Analysis of Images Carl Zeiss Fig. 4-269 4.13.22 LSM 5 LIVE LSM 5 LIVE DuoScan Image Display window, Mean ROI display for time series of Z Stack Display - 2.5 D This function allows to − display the two-dimensional intensity distribution of an image in an pseudo 3D mode − show the intensity values in profile, grid or filled mode − show separate distribution for each channel in a multi channel image The 2.5 D button can also be used online during scanning. • Click on the 2.5 D button. − The Pseudo 3D toolbar is displayed. The settings of Slice apply. The settings of Chan, Zoom, Contr and Palette are not applied. Click on 2.5 D will display the Pseudo 3D toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. The viewing plane of the Image Display window can be rotated, tilted either directly with the mouse or by the scroll bars on the right-hand side and the bottom of the Image Display window. 4.13.22.1 Direct Setting in the Image • Click in the image and hold down the mouse button. The perspective is changed by moving the mouse button in horizontal or vertical direction. 4-266 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.22.2 Setting via Scrollbars • Move the horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is displayed in the yellow display box. • Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is displayed in the yellow display box. The intensity scale can be varied by the scroll bar on the right-hand side of the Image Display window: • Moving the right vertical scrollbar enables you to expand or to compress the intensity scale of the image, while the expansion of this intensity axis ranges between 10 % and 100 % of the X-scale size. Fig. 4-270 Image Display window, 2.5 D display The Pseudo 3D toolbar contains the following function elements: Profile button Profile display (vertical polygon display). Setting of the Profile Distance between 1 and 20 using the slider. Grid button Grid display (horizontal grid display). Setting of the Grid Distance between 1 and 20 using the slider. Filled button Color diagram (filled 3D diagram). Selection between the Mono, Rainbow and Six Step color palettes. Channel list box Permits the selection of a Channel in a multi channel image. 03/06 B 45-0019 e 4-267 OPERATION Display and Analysis of Images Carl Zeiss 4.13.23 LSM 5 LIVE LSM 5 LIVE DuoScan Display - 3D (Image VisArt) This optional function allows to − reconstruct and display 3 D fluorescence image stacks or time series of frames and stacks from the Image Display window − select from a variety of reconstruction modes The settings of Chan are applied. The settings of Zoom, Slice, Contr and Palette are not applied. Click on 3D will display the 3D toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. The 3D button can also be used online during scanning. • Click on the 3D button. The 3D toolbar will be displayed. Fig. 4-271 Image Display window, 3D display The 3D toolbar contains the following function elements: Shadow projection Front button Shadow rendering front view Back button Shadow rendering back view Any View button Shadow rendering with user defined view 4-268 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Transparency projection Basic button Transparency rendering (voxel based) Advanced button Transparency rendering (voxel based) with textures Surface projection Basic button Surface rendering (voxel based) Advanced button Surface rendering (triangle based) Full Resolution button High accurate surface rendering (triangle based) Appearance button Opens the render properties dialog Series button Renders a series of 3D image stack or 3D / 4D time series, opens the Series render dialog 4.13.23.1 Shadow Projection With a click on Front, the 3D reconstructed image is displayed in a shadow projection where it is illuminated at a 45° angle from the front left. A click on the Back button creates the same projection with illumination from back left. Fig. 4-272 03/06 Image Display window, 3D display, Shadow projection, Front view B 45-0019 e 4-269 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The zoom wheel to the left of the Image Display window allows continuous zooming of the 3D reconstructed image. A click on the Any View button displays the 3D reconstruction image in a shadow projection where the viewing point can be defined. In addition to the zoom setting, the image can be rotated around the three orthogonal axes via the relevant setting wheels. However, the 3D orientation can also be set directly in the Image Display window by clicking, holding and dragging the 3D reconstructed image with the mouse. Fig. 4-273 Image Display window, 3D display, Shadow projection, Any View The following additional buttons are available in the Any View shadow projection mode: • After activation of the Frame button (below the image), a bounding box is drawn around the 3D reconstructed image. Depending on the used mode and hardware configuration, it can take several seconds until the 3D reconstruction is refreshed on the monitor after reorientation. • A click on the Coordinate System button displays a colored coordinate system in the Image Display window, where the X axis is displayed in red color, the Y axis in blue and the Z axis in green. • A click on the Scale button display an X-,Y- and Z-scale in the Image Display window. • A click on the Home button resets the display parameters to the default values. A click on the Any View button displays the 3D reconstruction image in a shadow projection where the viewing point can be defined. In addition to the zoom setting, the image can be rotated around the three orthogonal axes via the relevant setting wheels. 4-270 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss However, the 3D orientation can also be set directly in the Image Display window by clicking, holding and dragging the 3D reconstructed image with the mouse. The following additional buttons are available in the Any View shadow projection mode: • After activation of the Frame button (below the image), a bounding box is drawn around the 3D reconstructed image. Depending on the used mode and hardware configuration, it can take several seconds until the 3D reconstruction is refreshed on the monitor after reorientation. • A click on the Coordinate System button displays a colored coordinate system in the Image Display window, where the X axis is displayed in red color, the Y axis in blue and the Z axis in green. • A click on the Scale button display an X-,Y- and Z-scale in the Image Display window. • A click on the Home button resets the display parameters to the default values. • A click on the Animation button activates the animation mode. The object can be pushed by dragging in the Image Display window and rotates continuously. Any new push with pressed left mouse button changes the rotation direction and speed of the animation. The fastest animation results can be achieved with the advanced surface rendering mode (even without additional graphics cards). 03/06 B 45-0019 e 4-271 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.23.2 Transparency Projection The transparency projection generates a transparent 3D reconstructed image. The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation function) are identical to those of the Any View function of the shadow projection and are operated in the same way. The transparency projections Basic and Advanced are perspective-type 3D reconstructions, with the Advanced projection permitting the perspective impression being varied between parallel and centric projection by changing the View angle. The Advanced projection also offers the possibility of selecting between fast and precise calculation via the Precise / Fast slider (at the bottom right in the 3D toolbar). Of course, the precise calculation method is more time consuming. Fig. 4-274 4-272 Image Display Advanced window, 3D display, B 45-0019 e Transparency projection, 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.23.3 Surface Rendering The surface rendering generates surface rendered 3D reconstructed images. The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation function) are identical to those of the transparency projection and are operated in the same way. The surface projections Basic and Advanced are perspective-type reconstructions of the surface and differ in the fact that the calculation of the 3D information is based on voxels or triangles. The Advanced projection permits the View angle to be varied in order to enhance the perspective impression. It is also possible to select between fast and precise calculation via the Precise / Fast slider (at the bottom right in the 3D toolbar). Of course, the precise calculation method is more time consuming. The Full resolution projection is based on a high precision calculation method for 3D information on the basis of triangles with maximum resolution. Depending on the hardware configuration, it can take several seconds until the surface projection is refreshed on the screen. 03/06 B 45-0019 e 4-273 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.23.4 Appearance (Settings) The Appearance button opens the 3D Rendering window. This window allows settings for Light, Material, Background and Projection properties to be defined for all 3D projection modes. Depending on the selected 3D projection mode, different setting parameters are displayed. In the Shadow projection, the parameter Roughness is also available and can be set between 0 and 1. A default setting is permanently available for all modes. If individual settings for 3D rendering are to be used again, they can be stored and loaded when required. See also Show Processing Parameters, page 4-289. Proceed as follows to save individual settings: • Click on the Add to List button. • Enter a name in the opening Add Shading Model to list window. • Click on OK. Fig. 4-275 3D Rendering window (e.g. Surface Advanced rendering mode) − The settings are saved and the entered name appears in the Shading Model List selection box. • To activate the settings, double-click on the relevant name in the Shading Model List selection box. Settings which are no longer required can be removed. • Select the name of the setting to be deleted with a click of the mouse in the selection box and then click on the Remove button. − The setting is deleted. 4-274 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.23.5 Series The Series button opens the Render Series window. This window allows settings for the axis to be used for rotation of the 3D reconstructed images. • Click on the Series button to open the Render Series window. • Select the requested projection mode by clicking on the relevant option button under Mode. • Depending on the activated mode, directly set the parameters for animation in the Render Series window and the position of the 3D image in the Image Display window (zoom, rotation axes, rendering parameters). • Click on Apply to start the animation The animation is performed in a separate Image Display window, which permits the animation to be saved afterwards. (1) Fig. 4-276 Render Series window (e.g. Turn around X mode) Turn around X and Turn around Y mode In this mode, the image is turned around the X-axis or the Y-axis exclusively. The values for Number of Views, Difference Angle and First Angle can be selected accordingly (see Projection, page 4-155). 03/06 B 45-0019 e 4-275 OPERATION Display and Analysis of Images Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Start and End mode In the Start and End mode, the image is reconstructed between a start position and an end position. The rotation angles for X, Y and Z and the distance (zoom) can be determined using the sliders. The value for Number of View can be varied. Fig. 4-277 Render Series window (e.g. Start and End mode) (3) Position List mode In the Position List mode, the image is reconstructed between any required number of interim positions to be determined individually. The rotation angles for X, Y and Z and the zoom can be determined directly in the image. Every required interim position is included in the list of the Render Series window with a click on the Add Position button. Clear List permits the contents of the list to be deleted. The value for Number of View can be varied. • Click on the Apply button calculates a spline along all the defined positions from the list and starts an animation along this spline track in space. Fig. 4-278 4-276 Render Series window (e.g. Position List mode) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (4) OPERATION Display and Analysis of Images Carl Zeiss Time series When the input images is a Z Stack time series, the reconstructed images are generated for each time point. • If you click on the right mouse button in the main display window of Image VisArt two options appear: − Render properties − Renderer This functionality helps to find the optimal performance when working with different hardware renderers and to speed up some advanced rendering modes.} Fig. 4-279 03/06 B 45-0019 e 3D Renderer window 4-277 OPERATION Display and Analysis of Images Carl Zeiss 4.13.24 LSM 5 LIVE LSM 5 LIVE DuoScan Display - Topography for LSM This optional function allows to − process, display and measure topographic information. − use frame Z Stacks − and frame Z Stacks over time The Topo function is mainly used for applications in material research and industry. The settings of Chan and Zoom are applied. The settings of Slice and Contr are not applied. The Palette settings are applied in some 3D display modes. Click on Topo will display the Topography toolbar. All changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. • Click on the Topo button. The Topography toolbar is displayed. − The topography of a Z Stack is displayed in the Image Display box of the Image Display window. The parameter used at the last exit of the Topo function are applied. Fig. 4-280 4-278 Image Display window, Topography display B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss The Topography toolbar contains the following function elements: Channels buttons The selection of a channel to be used. Generate buttons The selection of the mode of calculation of the topography image (maximum, center of gravity, First /Last) Threshold buttons The selection of thresholds (intensity, height, Auto Z) to be used. Filter buttons The definition of filter procedures (geometrical, frequency cut-off filters) for smoothing, separation of roughness or waviness. Geometry buttons Inversion, various fit procedures, hole filling. Display buttons 2D (Intensity, Height, Gradient); Iso-Lines (z Map, Intensity, Black) 3D (Profiles, Grid, Filled, Shaded). Measure buttons Diagrams (Profile, z Distribution, Bearing area ratio plot, Slope distribution); Roughness parameters ; Volume parameters. 4.13.24.1 Channel Selection • Select the channel to be viewed using the relevant button (e. g.: Ch1). 4.13.24.2 Generate Topography The three buttons provided in the Generate button bar allow you to generate the topography in different ways: Maximum • Click on the Maximum button to calculate the topography surface by finding the maximum intensity value. If the optical section with the highest intensity value is found, the intensity values of both neighboring slices are also taken into account, so that a 3 point maximum fit is calculated. • In case it happens, that the maximum possible intensity value is present in more than one optical slice for a given pixel (saturation), the mid section of all saturated intensity slices is chosen as a reasonable approach. Center • Click on the Center button to calculate the topography surface by using the center of gravity of all summed up intensities of the stack for a given xy print. This mode provides better result for smooth surfaces of low intensity or nearly transparent surfaces. The receiver gain and offset has to be properly tuned and MarkFirst- MarkLastpositions of the stack should be located approximately in the same distance from the real surface. 03/06 B 45-0019 e 4-279 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan First / Last • Click on the First button to calculate the topography surface by using the first slice coming from the top, where the intensity reaches the value defined by the lower intensity threshold. This mode provides better result for surfaces of semitransparent materials with inclusions of higher reflectivity or transparent multilayers with subsurface layers of higher signal intensity. Extended First / Last Mode 1. Definition of an intensity (I) threshold. 2. Starting from top / bottom of a stack to find I = 400. 3. Search of a local maximum one FWHM of actual Z PSF forwards / backwards. 4. Search of the next local maximum one FWHM forwards / backwards from the last max until you have not found any new local maximum. 5. Last local maximum is taken as surface point. Fill Holes procedure • Intensity of a missing pixel of a hole has to be interpolated by the distance-weighted intensity of all surrounding pixels. • Fill hole algorithm is optimized for short calculation times. 4.13.24.3 Topography Thresholds Intensity threshold Click on the Intensity button to calculate the topography surface by using the lower and the upper intensity thresholds for image display. Use of this function is recommended to find the real surface in the case of images with pronounced noise. All image pixels with intensity less or higher than the thresholds set are ignored for the surface calculation. • Click on the Intensity button to select the intensity thresholds for the surface generation. The Intensity Threshold window appears. • Set the lower and upper intensity thresholds using the appropriate sliders. Fig. 4-281 4-280 Intensity Threshold window • Click on Close to Threshold window. B 45-0019 e close the Intensity 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Height threshold Click on the Height button to calculate the topography surface by using the lower and the upper height thresholds for image display. Use of this function is recommended to get rid of unwanted peaks and valleys taken into account for parameter calculation. All topographic data with height values less or higher than the thresholds set are ignored for the display and parameter calculation. This threshold applies both for 2D as well as for 3D topography display modes. Fig. 4-282 Z Threshold window • Click on the Height button to select the intensity thresholds for the surface generation. The Z Threshold window appears. • Set the lower and upper intensity thresholds using the appropriate sliders. • Click on Close to close the Z Threshold window. Auto Z By clicking on the Auto Z button the surface topography is displayed in the Image Display window in that way that it is automatically normalized to the lowest and highest Z value of the 3D topography. • Click on the Auto Z button. The topography is automatically normalized with respect to the highest and lowest Z value. 4.13.24.4 Processing by Filtering (1) Topography smoothing The three buttons in the Filter button bar allow activation / deactivation of the filter functions for surface smoothing. None button No filter for input data. Median / Gauss / Aver. button Smoothing of z data using a low-pass Median, Gauss or average filter. Clicking on this button opens a selection box, where the number of neighboring pixels to be used for filtering can be specified: 1st row: small smoothing via Median/Gauss filter (Median; 3 x 3; 5 x 5; 7 x 7) 2nd row: medium smoothing via Average (9 x 9; 11 x 11; 15 x 15) 3rd row: pronounced smoothing via Average (25 x 25; 35 x 35; 45 x 45) 03/06 B 45-0019 e 4-281 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan • To investigate the effects of various filter modes, select one of the 3D display modes (Profiles, Grid, Filled or Shaded) from the Display button bar. • Click on the Median sub button to set the smoothing of the integrated Median filter. Or • Click on a Gauss or Average sub button and select the required degree of smoothing from the selection box with a click of the mouse. FFT button: This function performs a Fast Fourier Transformation (FFT) in the frequency range, applies highpass or lowpass filtering in the frequency range and performs the inverse FFT. • Click on the FFT button, the FFT Filter window opens. • Click on the arrow in the filter Type select box to choose an adequate filter function: − Gauss Lowpass − Gauss Highpass − Butterworth Lowpass − Butterworth Highpass • Select a position of the Cut off slider to display either the lower frequencies (waviness) with the lowpass filters or the higher frequencies (roughness) with the highpass filters. • The Cut off frequencies ranges from 1/1000 of the X dimension of the stack to four times of the X dimension of the stack. The dimensions of the filtering is given in units of μm. • Select a position of the Degree slider. The filter functions can be calculated from 1st order to 5th order accuracy. • Click on the Close button closes the FFT Filter window. 4-282 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (2) OPERATION Display and Analysis of Images Carl Zeiss Changing the topography geometry The three buttons in the Geometry button bar allow the surface geometry to be changed. Inverse button Inverse surface. Depths change to heights, and vice versa. Fit button The following fit modes can be set: 1) No Fit 2) Plane The topography is tilted in such a way that the mean deviation value plane is calculated. 3) Manu Tilt 4) 3Point Tilt 5) Sphere fit correction A spherical form is eliminated, determination of micro roughness on spherical surfaces can be performed. 6) Cylinder fit correction A cylinder form is eliminated, determination of micro roughness on cylindrical surfaces can be performed. You can display the exact values of the Cylinder / Sphere fit by opening a context menu in the Image Display box with a click of the right mouse button and selecting the Show processing parameter function. Manu Tilt button Clicking on the Manu Tilt button opens the Manual Tilt window (Fig. 4-283) for manual tilt correction. In the manual tilt window three types of arrow buttons are present: − One arrow changes pitch and yaw in 0.001° steps − Two arrow changes pitch and yaw in 0.01° steps − Three arrow changes pitch and yaw in 0.1° steps • Click on the Inverse button for the inverse display of the topography. Clicking again will reset the normal display. • Correct the tilt via the Fit or Manu Tilt functions. Fig. 4-283 03/06 Manual Tilt window B 45-0019 e 4-283 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.24.5 Display Modes The three buttons in the Display button bar allow stacks to be displayed in the 2D, Iso-Lines or 3D display mode. (1) 2D modes The following 2D modes can be set: Intensity button: Display of projection of all intensities of the stack (black-and-white display). Height button: Height coded color map with color bar in a separate parameter window on the right hand side. Gradient button: Display of height gradient (slope), averaged pixelwise over all neighbors (black-and-white display). • Click on the 2D button in the Display button bar. − The 2D display mode selected last is activated. At the same time, an additional button bar is displayed beside the 2D button permitting selection of the required 2D display mode. • Select the required 2D display mode with a click of the mouse. (2) 2D Iso-Lines display mode Iso-Lines are lines which connect points of equal height on the topography. The following 2D Iso-Lines display modes can be set: Intensity button: Intensity projection superimposed with colored iso-lines (lines of equal height). Height button: Height function with black iso-lines. Black button: White iso-lines on a black background. • Click on the Iso-Lines button in the Display button bar. − The Iso-Lines display mode selected last is activated. At the same time, an additional button bar is displayed beside the Iso-Lines button permitting selection of the required Iso-Lines display mode. − Below the Measure button bar, the Line Dist. and Line Offset sliders / input boxes are displayed. • Select the required Iso-Line display mode by clicking the left mouse button. 4-284 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss The additional function elements of the Iso-Lines display mode have the following meaning: Line Dist. slider: Changes the distance of the iso-lines. Line Offset slider: Setting of the height level where the Iso-Lines display starts. To apply the topography functions to a small portion of the Z Stack image use the Overlay function (Overlay button) and cut out and store as new topographic evaluation via the Extract Region function. (3) 3D display Topo animations are possible. The following 3D modes can be set: Profiles button: Profile display. Grid button: Grid display. Filled button: Display of color shades. Shaded button: Surface rendering. Can be combined with LUT. Topo animations are possible. • Click on the 3D button in the Display button bar. − The 3D display mode selected last is activated. At the same time, an additional button bar appears beside the 3D button to permit selection of the required 3D display mode. − Below the Measure button bar, the Scaling button bar and the Profile Dist. and Fill Level sliders / input boxes are displayed. − The Image Display box contains one horizontal and two vertical scrollbars for the setting of the image viewing angle. • Select the required 3D display mode with a click of the mouse. The additional function elements of the 3D display mode have the following meaning: Profile Dist. slider: Setting of the distance of profiles and the mesh value of the grid. Fill Level slider: Used to push through a color LUT Look Up Table (e.g.: if the Rainbow palette is used) in the Profiles / Filled display mode. In combination with the Volume button, the filling of the flood function level of the topography can be varied for volume measurements (see the Measurement functions paragraph). 03/06 B 45-0019 e 4-285 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan The image viewing angle is set as follows: Setting directly in the image • Click in the image and hold down the mouse button. The perspective is changed by moving the mouse button in horizontal or vertical direction. Setting via scrollbars • Move the horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is displayed in the yellow display box. • Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is displayed in the yellow display box. right vertical scrollbar enables you to expand the image in height or to compress it, • Moving the while the Z-range between 10 % and 100 % of the X-range is scaled. You can set the x, y and Z scales to an identical ratio by opening a context menu in the Image Display box with a click of the right mouse button and selecting the Metric equal ratio function. The displayed boxes for rotation angle and relative scaling percentage value z : x ratio permit the setting of identical perspectives for different images (e.g.: the plot of several topographies). The Profiles and Filled 3D display modes permit a color palette (e.g.: Glowscale, Rainbow or User defined) to be loaded or redefined by pressing the Palette button (see page 4-237). 4-286 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss 4.13.24.6 Context Menu of the 3D Display Mode • Click in the Image Display box with the right mouse button to open the context menu. The context menu for the 3D mode currently selected is displayed. • Click on the required option with the left mouse button to execute the function. (1) Metric equal ratio item This option is available in all of the 3D display modes. Fig. 4-284 Context menu of the 3D display mode (Profiles) Fig. 4-285 Save As window Fig. 4-286 Topography matrix After activation of the function, the x, y and z scales are set to an identical ratio. (2) Export profiles item This option is available in the Profiles and Grid 3D display modes. Use the function to save the Profiles or Grid data as a text file. • Open the context menu with a click of the right mouse button, then click on the option Export ... with the left mouse button. − The Save As window is opened. • Select the directory where you want the text file to be stored, enter a file name and click on Save. A text file containing the topography in the form of an XYZ matrix is generated. (3) Copy profiles to clipboard item All 3D parameters shown right from the main 3D topo display window are copied in the clipboard. 03/06 B 45-0019 e 4-287 OPERATION Display and Analysis of Images Carl Zeiss (4) LSM 5 LIVE LSM 5 LIVE DuoScan Export x,y,z- triples item This option is available in the Profiles and Grid 3D display modes. Use the function to save the Profiles or Grid data as a text file. • Open the context menu with a click of the right mouse button, then click on the option Export ... with the left mouse button. − The Save As window is opened. Fig. 4-287 Save As window • Select the directory where you want the text file to be stored, enter a file name and click on Save. A text file containing the topography in the form of an XYZ table is generated. Fig. 4-288 (5) Topography table Copy x,y,z- triples to clipboard item This option is available in the Profiles and Grid 3D display modes. After selection of this option, the Profiles or Grid data are copied as an XYZ table to the clipboard and can be inserted in other programs using the Paste command. Please make sure that the amount of exportable data is adequate to the maximum importing size of the following software package. To lower the amount of data points, use the profile distance slider. 4-288 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (6) OPERATION Display and Analysis of Images Carl Zeiss Show processing parameters After selection of the Show processing parameters function, a reporting of the following applied topo processing functionality is displayed on the right-hand side of the Image Display window: − Mode (calculation mode: Max, Center, First) − Threshold (applied intensity threshold) − Filter − Fit (plane, cylinder / sphere parameters) Fig. 4-289 (7) Show processing parameters Ratio of valid data points The ratio of valid data points (means signal intensities within a given intensity threshold) is displayed. (8) Load Calculation Parameters Topo routines can be loaded as tgp-files (TopoGraphic Parameters). (9) Save Calculation Parameters Topo routines can be saved and reloaded as tgp-files. These files include settings for: − reconstruction mode − intensity threshold − filters (including FFT) − tilt angles (manual, 3 point fit) − fit procedures (plane, cylinder, sphere) − inverse − fill holes 03/06 B 45-0019 e 4-289 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan (10) Render properties item This option is only available in the Shaded 3D display mode. Fig. 4-290 Context menu of the 3D display mode (Shaded) Use this function to vary the illumination conditions, reflection properties and projection settings of the topography. You can either select preset Shading Models or use parameters specifically defined as required. The specifically defined parameters can subsequently be stored as a Shading Model and are then available at any time for further use. Shading Models can also be deleted if no longer needed. Load a Shading Model • Open the context menu with a click of the right mouse button, then click on the option Render properties with the left mouse button. − The 3D Rendering window is opened. Fig. 4-291 3D Rendering window • Click on the name of the required model in the Shading Model List. The parameters are immediately set for the current topography. • Click on Close to close the 3D Rendering window again. 4-290 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Defining a specific Shading Model • Open the 3D Rendering window. • Click on the Define button. • Change the parameters of the topography using the appropriate sliders. • Save the settings by clicking on the Add to List button. The Add Shading Model to List window is displayed. • Enter a name for the model and click on OK. The model is included in the Shading Model List. See also Appearance (Settings), page 4-274. Fig. 4-292 3D Rendering window Light panel Determines the properties of illumination on a sample. Distance Goes for diffuse and specular, see visualization. Azimuth See visualization. Rise angle of the "sun". Elongation See visualization. North-south / east-west direction of the "sun". Background Choose background color. Material panel Determines the reflective properties of a sample. Ambient Specular Material properties; how many % of the light component are projected by the material into which spectral ranges. Shininess Goes together with specular light. Shininess equal to 25 % determines diffuse light. 03/06 B 45-0019 e 4-291 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Projection panel Determines the reflective properties of a sample. View angle Determines the perspective, 0.0 parallel projection, central projection Distance Zoom function, zoom in, zoom out Fig. 4-293 Render function visualization aid A zoomed rendering setting permits the zoomed section to be moved via the cursor keys after a click on the 3D window. If a change of the 3D image angle follows, centration is made on the center again. Deleting a Shading Model • Select the model to be deleted in the Shading Model List, then click on the Remove button. The model is deleted. 4-292 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss (11) Renderer item This option is only available in the Shaded 3D display mode. After selection of the Renderer item, the 3D Renderer window appears. It allows the selection of the hardware and software option which shall be used for the 3D graphics calculation. OpenGI - Software The graphics calculation is performed using the installed software. OpenGI - Hardware The graphics calculation is accelerated by using the installed graphics processor. Direct3D – Software / Hardware Rasterizer / Hardware These options can be used for offline versions of the LSM 5 software for PC's with the WINDOWS ® 2000 or XP operating system (not for WINDOWS ® NT). Fig. 4-294 03/06 B 45-0019 e 3D Renderer window 4-293 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.24.7 Measurement Functions The topography measurement functions are activated via the Measure button bar. The measurement functions can be performed in the 2D or 3D display mode. Automated convention in height statistics analysis: Topo Filters None, median, <9x9 FFT High FFT Low >9x9 Data formats Primary profile Roughness Waviness 2D profile Pxx Rxx Wxx n.a. 3D topography PSxx RSxx SWxx n.a. The following measurement functions are available: Diagram button: Diagram display. The Profile, z Histo, Curve of tp and Grad. Histo diagram display modes can be activated via the Diagram button and deactivated via the Off Diagram button. By activation of the Diagram button, an additional button bar is displayed for the selection of the required diagram or for deactivation. The labeling of the Diagram button changes depending on which diagram display mode has been activated. Roughness button: Calculation of the roughness parameters. Volume button: Calculation of the volume parameters. (1) Profile measurement mode in 2D display • Select the required 2D display of the stack via the 2D button. • Click on the Diagram button in the Measure button bar. Click on the Profile button in the button bar displayed afterwards. − The Table and Profile button bars are displayed below the Measure button bar. − A colored arrow (intersection line of the profile) is displayed in the image and the profile diagram appears below the image. • If required, match the size of the Image Display window in order to obtain the complete display of the profile diagram. 4-294 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-295 OPERATION Display and Analysis of Images Carl Zeiss Topography display: 2D - Profile Additional Table / Profile buttons The additional Table / Profile buttons have the following functions: Show button: The profile is displayed in the form of a table at the bottom below of the Image Display window. Copy button: The profile table is copied to the clipboard and can be transferred to other programs (MS Word or MS Excel) via the Paste function. Save button: The profile table can be stored as a text file (ASCII). Arrow (selection) button: Activation of the mouse button for selection, resizing or movement of the intersection line in the image. Click on the handle and hold down the mouse button, drag the handle, Resizing: release the mouse button. Movement: Click on the line and hold down the mouse button, move the entire intersection line, release the mouse button. Line with arrow button (open arrow): Creation of the intersection line to define the position of the profile to be produced in the image. Click and hold the mouse button, drag the line in any required direction, release the mouse button to end the procedure. The profile diagram changes online. Line button: This button allows you to determine the line thickness of the intersection line. Measure button: Activates the Profile measurement mode in the profile diagram. The required tools are displayed to the right of the profile diagram (see Profile measurement mode, page 4-294). z/x=1 button: Sets the z/x ratio in the profile diagram to the value 1. Check: the following creation of a circle using the relevant tool really results in a circle in the profile display. Measured angle values correspond to the actual slope of the line displayed. Color button: Clicking on the Color button opens a color selection box in which the color for the intersection line can be selected with a click of the mouse. 03/06 B 45-0019 e 4-295 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Activating the Profile measurement mode button, the Profile If you click on the window with the tools of the profile measurement mode appears. This window can be moved as required over the entire screen. Fig. 4-296 Tools of the Profile measurement mode Tools of the Profile measurement mode The tools of the Profile measurement mode have the following functions: Zoom button: Zooming of a section of the profile diagram. Click and drag a rectangle over the area to be enlarged in the profile diagram, release the mouse button to enlarge the selected area. The zoom function can be performed several times. A click with the right mouse button resizes the profile. Marker button: Activation of the marker functions for the intersection line. The red and blue marker lines in the profile diagram can now be moved using the mouse. After movement of a marker line in the profile diagram, the relevant marker (red or blue circle) follows along the intersection line in the 2D and Iso-Lines mode. Arrow (selection) button: Activation of the mouse button for selection, resizing or movement of one of the following drawing elements in the profile diagram. Resizing: Click on the handle and hold down the mouse button, move the handle, release the mouse button. Movement: Click on the line and hold down the mouse button, move the entire drawing element, release the mouse button. Inclined Line button: Creation of a straight line in the profile diagram. Display of distance, inclination angle, dxdy and dz. Click and hold down the mouse button, drag the line in any required direction, release the mouse button to end the procedure. Free angle button: Creation of a free angle in the profile diagram. Display of the enclosed angle (max. 180 °). The first click sets the starting point, the second and third clicks define the angle and the end point. Rectangle button: Creation of a rectangle in the profile diagram. Display of distance, area, height and width. Click and hold down the mouse button, drag the rectangle in any required direction, release the mouse button to end the procedure. 4-296 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Open Polyline button: Creation of an open polyline figure in the profile diagram. Display of the length of the line figure. First click sets the starting point, any further click adds another line, click with the right mouse button ends the procedure. Closed Polyline button: Creation of a closed polyline figure in the profile diagram. Display of the perimeter of the figure. First click sets the starting point, each further click adds another line, a click with the right mouse button closes the figure and ends the procedure. Open free-hand curve button: Creation of an open Bezier figure in the profile diagram. Display of the length of the line figure. First click sets the starting point, each further click adds another line, a click with the right mouse button ends the procedure. Closed free-hand curve button: Creation of a closed Bezier figure in the profile diagram. Display of the length of the line figure. First click sets the starting point, each further click adds another line, a click with the right mouse button closes the figure and ends the procedure. Ellipse button: Creation of an ellipse in the profile diagram. Display of the area. First click sets the center point, the displayed line permits the determination of the first dimension, second click sets the first dimension, the second dimension and rotation direction can now be determined, third click sets the second dimension and direction and ends the procedure. Circle button: Creation of a circle in the profile diagram. Display of radius and area. Clicking three times to define 3 points on the profile. A circle fit is automatically applied on the profile. Recycle bin button: Deletes all drawing elements or the one just selected. Line width button: Change of the line width of the drawing elements. Color button: Clicking on the Color button opens a color selection box where the color of the drawing element can be selected with a click of the mouse. x1- button: Resets the zoom factor of the profile diagram to its original size. 03/06 B 45-0019 e 4-297 OPERATION Display and Analysis of Images Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan z Histo measurement mode in 2D display • Click on the Diagram button in the Measure button bar. Click on the z Histo button in the additional button bar now displayed. The lower part of the Image Display box shows the 3D height distribution of the topography. Fig. 4-297 (3) Image Display window, Topography display: 2D - Histo Curve of tp measurement mode in 2D display • Click on the Diagram button in the Measure button bar. Click on the Curve of tp button in the additional button bar now displayed. − The curve of the bearing area ratio as a function of the height is displayed below the image (also see 3D measurement functions, page 4-304). 4-298 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (4) OPERATION Display and Analysis of Images Carl Zeiss Grad. Histo measurement mode in 2D display • Click on the Diagram button in the Measure button bar. Click on the Grad. Histo button in the additional button bar now displayed. The lower part of the Image Display box shows the gradient distribution of the topography. Before creation of the slope diagram, the image should be filtered at least once using a low-pass filter, since otherwise the rough height gradation of the image will result in a comb-shaped histogram. The RootMean-Square Slope (RMS Slope) parameter is calculated and displayed below the chart. The following formula is used for calculation: RDQ = (5) ( ) ( N x N y ⎧⎡ 1 ⎪ z xi , y j − z xi −1 , y j ⋅ ∑ ⋅∑ ⋅ ⎨⎢ ( N x − 1) ⋅ ( N y − 1) i =1 j =1 ⎪⎢ Δx ⎩⎣ ) ⎤⎥ 2 ( ) ( ⎡z x , y − x , y i j i j −1 +⎢ Δy ⎥ ⎢ ⎦ ⎣ ) ⎤⎥ ⎫⎪⎬ 2 ⎥ ⎪ ⎦ ⎭ Roughness measurement mode in 2D display (Profile display) • Click on the Profile button in the Measure button bar. • Click on the Roughn. button in the Measure button bar. − The roughness parameters are calculated and displayed on the left below the image. All roughness parameters calculated from a 2D profile are named with R. − The Copy button is displayed below the right-hand side of the image. This button permits the roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS Word or MS Excel) via the Paste function. 2D Amplitude parameters (Profile Roughness): Mean height z Rc Pc Wc Arithmetic mean deviation Ra Pa Wa Root mean square deviation Rq Pq Wq Asymmetry Skewness Rsk Psk Wsk Sharpness Kurtosis Rku Pku Wku Extremes Highest peak Rp Pp Wp Lowest valley Rv Pv Wv Absolute peak to valley Rt Pt Wt Averaged peak to valley Rz Pz Wz Maximum peak to valley Rmax Pmax Wmax FFT High No, M FFT L Dispersion If chosen filters are 03/06 B 45-0019 e 4-299 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Calculation of roughness parameters The following roughness parameters are calculated (e.g. for a Y-section) − Mean height of all profile height values Rc y 1 − Rc = ⋅ ∑ ⋅ z (x, y j ) N y j =1 N Nx, Ny ... number of pixels in X- or Y-direction Arithmetic mean deviation of all profile height values Ra [ y 1 − Ra = ⋅ ∑ ⋅ z (x, y j ) − Rc N y j =1 N ] − Quadratic mean deviation of all profile height values Rq [ y 1 ⋅ ∑ ⋅ z (x, y j ) − Rc N y j =1 N − Rq = ] 2 − Skewness of the distribution of all profile height values RSK N RSK y 1 = ⋅ ∑ ⋅ z 3 ( x, y j ) N y ⋅ Rq3 j =1 − Kurtosis of the distribution of all profile height values RKU N R KU y 1 = ⋅ ⋅ z 4 ( x, y j ) ∑ 4 N y ⋅ Rq j =1 − Maximum peak height RP RP = z max − Rc − Maximum valley depth RV RV = Rc − z min − Maximum roughness depth Rt (= Peak to Valley / PV) − S t = z max − z min maximum height difference of the overall topography along a profile. 4-300 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Classification of topography in 5 equal area elements (rectangles in the 2D mode) − average roughness depth Rz: − Rz = zmax 1 − zmin 1 + zmax 2 − zmin 2 + zmax 3 − zmin 3 + zmax 4 − zmin 4 + zmax 5 − zmin 5 5 Averaging of Rt-values of all the 5 single area elements. When combined, both parameters provide information about the homogeneity of the surface. Big differences are indicative of pronounced inclination of the overall area or of spikes. Developed Surface Area Ratio: Σ (surface areaij) / Σ (projected areaij) The percentage of the 3D surface area (sum off all triangles formed by adjacent data points) to the 2D surface area produced by projecting the 3D surface onto the threshold plane. − maximum roughness depth Rmax: − Rmax = Max ( zmax 1 − zmin 1 , zmax 2 − zmin 2 , zmax 3 − zmin 3 , zmax 4 − zmin 4 , zmax 5 − zmin 5 ) maximum of Rt-values of all the 25 single area elements. Both the roughness parameters and the z-histogram can be changed by using filters! (6) Roughness measurement mode in 3D display • Click on the Roughn. button in the Measure button bar. − The roughness parameters are calculated and displayed on the left below the image. All roughness parameters calculated from a 3D topography are named with S. − The Copy button is displayed below the right-hand side of the image. This button permits the roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS Word or MS Excel) via the Paste function. 3D Amplitude parameters (Topography Roughness): Mean height z RSc PSc WSc Arithmetic mean deviation RSa PSa WSa Root mean square deviation RSq PSq WSq Asymmetry Skewness RSsk PSsk WSsk Sharpness Kurtosis RSku PSku WSku Extremes Highest peak RSp PSp WSp Lowest valley RSv PSv WSv Absolute peak to valley RSt PSt WSt Averaged peak to valley RSz PSz WSz Maximum peak to valley RSmax PSmax WSmax FFT High No, M FFT L Dispersion If chosen filters are: 03/06 B 45-0019 e 4-301 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Calculation of roughness parameters The following roughness parameters are calculated: − Mean height of all surface height values Sc − RS c = 1 ⋅ Nx ⋅ Ny Nx Ny ∑ ∑ ⋅ z (x , y ) ⋅ i =1 i Nx, Ny ... number of pixels in X- or Y-direction j j =1 − Arithmetic mean deviation of all surface height values RSa − RS a = 1 ⋅ Nx ⋅ Ny Ny ∑ ∑ ⋅ [z (x , y ) − RS ] Nx ⋅ i =1 i j c j =1 − Quadratic mean deviation of all surface height values RSq − RS q = 1 ⋅ Nx ⋅ Ny Ny ∑ ∑ ⋅ [z (x , y ) − RS ] Nx i =1 ⋅ 2 i j c j =1 − Skewness of the distribution of all surface height values RSSK RS SK 1 = ⋅ N x ⋅ N y ⋅ RS q3 Nx Ny i =1 j =1 ∑ ⋅∑ ⋅ z (x , y ) 3 i j − Kurtosis of the distribution of all surface height values SKU RS KU = 1 ⋅ N x ⋅ N y ⋅ RS q4 Nx Ny ∑ ∑ ⋅ z (x , y ) i =1 ⋅ 4 i j j =1 − Maximum peak height RSP RS P = z max − RS c − Maximum valley depth SV RSV = RS c − z min − Maximum roughness depth RSt (= Peak to Valley / PV) − RS t = z max − z min maximum height difference of the overall topography. 4-302 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Classification of topography in 25 equal area elements (rectangles in the 2D mode) − average roughness depth Sz: − RS z = z max 1 − z min1 + z max 2 − z min 2 + ⋅ ⋅ ⋅ + z max 25 − z min 25 25 Averaging of Rt-values of all the 25 single area elements. When combined, both parameters provide information about the homogeneity of the surface. Big differences are indicative of pronounced inclination of the overall area or of spikes. − maximum roughness depth RSmax: − RS max = Max ( z max 1 − z min1 , z max 2 − z min 2 , ⋅ ⋅ ⋅ , z max 25 − z min 25 ) maximum of Rt-values of all the 25 single area elements. Both the roughness parameters and the z-histogram will be influenced by the use of filters! 03/06 B 45-0019 e 4-303 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.24.8 3D Measurement Functions (1) Volume measurement mode (Flood function) • Use the 3D button to select the required 3D display of the stack. • Click on the Volume button in the Measure button bar. − The volume parameters are calculated and displayed below the image. − The Copy button is displayed below the right-hand side of the image. This button permits the volume values to be copied to the clipboard and imported to other programs (e.g.: MS Word or MS Excel) via the Paste function. • Setting the Fill Level slider enables you to change the height level of the topography. The portion of the topography lying below the set height level is filled with "water" (blue color) and the volume parameters are calculated online only for the projecting part of the topography. To use the Fill Level function, load the Profiles 3D display mode containing the Glowscale palette, or activate No Palette to obtain optimum display. • If the Diagram function Curve of tp is also activated, a red marker line shows the position of the height level in the percentage of contact area curve. Fig. 4-298 4-304 Image Display window, Topography display: 3D - Volume B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss Parameters The following parameters are calculated: Z: height level (selectable with the Z-Threshold and Fill Level sliders). The setting of this value influences the following parameters. Vm (z): material volume above chosen height level Vv (z): void volume below chosen height level Smr (z): material volume ratio S mr ( z ) = Svr (z): Vm ( z ) Vm ( zmin ) void volume ratio Svr ( z ) = Vv ( z ) Vv ( zmax ) Au: surface bearing area of the topography at Z (= projection area of those parts which are situated above chosen height level) Smr: surface bearing area ratio of the topography at Z percentage of contact area (= Au / (x * y) * 100 %) Sda: true surface = sum of all triangles formed by adjacent data points of the surface reconstruction Sdr: developed surface area ratio: Σ (surface areaij) - Σ (projected areaij) / Σ (projected areaij) * 100 % projected area = x * y The percentage of the 3D surface area (sum of all triangles formed by adjacent data points of the surface reconstruction) to the 2D surface area produced by projecting the 3D surface onto the threshold plane. absolute flat surface ⇒ is equal to base plane (Sdr = 0 %) The increase by which the 3D surface is larger than the basic plane (e. g. 625 % is a 3D surface which is about 6.25 times larger than the projected basic plane) 03/06 B 45-0019 e 4-305 OPERATION Display and Analysis of Images Carl Zeiss (2) LSM 5 LIVE LSM 5 LIVE DuoScan Profile measurement mode in 3D display This function is performed in the same way as in the 2D display mode, with the following exceptions: The buttons and and . Furthermore, the Position slider and are replaced with the buttons the input box (information of the position of the intersection line in pixels) are displayed below the Table and Profile button bar. Changing the Z-Threshold also results in a change in the profile. In the 3D image, a red marker line shows the y- and x-position of the displayed profile diagram. Fig. 4-299 Image Display window, Topography display: 3D - Profile • The position of the marker line (profile intersection line) can be changed by moving the Position slider in x or y. • Press the x- or y-button to select the required intersection plane. (3) z Histo measurement mode in 3D display This function is performed in the same way as in the 2D display mode. 4-306 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan (4) OPERATION Display and Analysis of Images Carl Zeiss Curve of tp measurement mode in 3D display This function is performed in the same way as in the 2D display mode. Before determination of the tp bearing portion, individual peaks (noise, steep slopes) must be eliminated. The Median filter or a 3x3 longpass filter can be used for this purpose. Fig. 4-300 Image Display window, Topography display: 3D – Curve of tp Shifting the two cursor crosses permits two bearing portions to be given in percent (e.g. Smr1 = 10 %; Smr2 = 90 %) as default values for which the height difference Rdc is determined automatically. (5) Grad. Histo measurement mode in 3D display This function is performed in the same way as in the 2D display mode. (6) Roughness measurement mode in 3D display This function is performed in the same way as in the 2D display mode. 03/06 B 45-0019 e 4-307 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.24.9 Export Data − multiple profiles (Rel. 3.2) − single profile − parameters − topography as matrix − topography as triples 4.13.24.10 Topo ReUse Topo routines can be saved and reloaded as tgp-files (TopoGraphic Parameters). These files include settings for: − reconstruction mode, − intensity threshold, − filters (including FFT), − tilt angles (manual, 3 point fit), − fit procedures (plane, cylinder, sphere), − inverse and − fill holes. 4-308 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.25 OPERATION Display and Analysis of Images Carl Zeiss Display - Prev. This function allows to − compose images, graphs and text for printing − use any image format − change fonts and line width in graphs via context sensitive menus The settings of Chan, Zoom, Slice, Contr and Palette apply. In the Options menu in the function Settings with the tab Print Status Display parameters are determined and the Print Status Information is activated/deactivated. Click on Prev will display the Preview window and the Print toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. Fig. 4-301 03/06 Image Display window, Prev display with Assembly of image, intensity profile and scan info B 45-0019 e 4-309 OPERATION Display and Analysis of Images Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.25.1 Context Menu for Scan Information Text The context menu (right mouse button) allows to vary the output of the scan info. • Click with the right mouse button. A context menu with the options Color and Font is displayed. • In the Color menu, you can select a different type color for the scan info, in the Font menu a different type font and type style. 4.13.25.2 Context Menu for Topography Images When transferring a topography to the print preview, you can change the size and shape of type and scale lines for the 3D graphics and profile measurement results. (1) Context menu for 3D graphics • Click on the right mouse button. A context menu with the options Font enlargement and Line width enlargement is displayed. • You can change the type size in the Font enlargement menu and the line width of the scales in the Line width enlargement menu. (2) Context menu for Profile measurement function • Click on the right mouse button. A context menu with the options Scaling font enlargement, Marker font enlargement and Overlay font enlargement is displayed. • You can change the type font in the Scaling font enlargement menu, the size of the marker table in the Marker font enlargement menu and the type size of the red measurement results in the Overlay font enlargement. 4.13.25.3 Arranging and Printing the Print Preview • Click on the Arrange button for optimum layout of image size and position relative to the textual information. • A layout generated with Prev. (Preview) can be printed by clicking on the Print button in the Print toolbar. • Clicking on the Setup button opens the Print Setup window, in which you can specify print parameters. Fig. 4-302 4-310 Print Setup window • Click on the slider to change the zoom value of the selected items. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.13.26 OPERATION Display and Analysis of Images Carl Zeiss Display - Info This function allows to − display the parameters used during image acquisition of the image(s) displayed in the Image Display window − use any image format − remove the info display The settings of Chan, Zoom, Slice, Contr and Palette are not relevant for this function. In the Options menu in the function Settings with the tab Image Status Display parameters to shown are determined. Click on Info will show the parameters. Click again to hide the info display. Fig. 4-303 03/06 Image Display window, Info display B 45-0019 e 4-311 OPERATION Display and Analysis of Images Carl Zeiss 4.13.27 LSM 5 LIVE LSM 5 LIVE DuoScan Additional Display Mode in Time Series 4.13.27.1 Display - Mean This function allows to − display the intensity time diagram (mean intensity in user defined ROIs over time) − display the intensity time diagram for volumes (3D ROIs) within a Z-Stack over time − use frame time series and frame Z Stack time series as input − show the intensity values in table form and copy table to clipboard or save as text file − show separate diagrams for each channel in a multi channel image The settings of Chan, Zoom, Slice, Contr and Palette apply. Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed. To use a similar functionality while scanning use the optional Mean of ROI function with the Time series control. • Click on the Mean ROI button. − The Mean of ROIs image display toolbar will be displayed on the right. The used ROIs become visible in the image, and the Intensity-Time diagram is shown on the left of the Image Display window. Fig. 4-304 4-312 Image Display window, Mean ROI display for time series in single plane B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Display and Analysis of Images Carl Zeiss The Mean of ROIs toolbar contains the following function elements: ROIs selection box: Display of the ROIs used during scanning of the time series and of the other ROIs available in the system. Add button: Opens the Add ROI List window for the storage of changed or newly defined ROIs under a new name. Remove button: Deletes the selected ROI from the ROIs selection box. ROI data: Display of the data of the ROI selected from the ROIs selection box. On deactivation of the check box of a ROI, its intensity values from the Intensity-Time diagram are not displayed. Arrow button: Activation of the mouse button for resizing or movement of the ROI in the Image Display window. Bezier button: Activates the Bezier figure drawing mode. The first click sets the starting point, each additional click adds a further line, a double-click on the starting point closes the figure and ends the procedure. Circle button: Activates the circle drawing mode. Clicking and holding down the mouse button sets the center point; drag the diameter and release the mouse button to end the procedure. Recycle bin button: All the ROIs to the image are deleted. Rectangle button: Activates the rectangle drawing mode. Click and hold down the mouse button, drag the rectangle in any direction, release the mouse button to end the procedure. Ellipse button: Activates the ellipse drawing mode. The first click sets the center point, the displayed line permits the determination of the first dimension, the second click sets the first dimension, the second dimension and the rotation direction can then be determined; the third click sets the second dimension and the direction and ends the procedure. 03/06 B 45-0019 e 4-313 Carl Zeiss OPERATION Display and Analysis of Images LSM 5 LIVE LSM 5 LIVE DuoScan Polyline button: Activates polyline drawing mode. The first click sets the starting point, each additional click adds a further line, a doubleclick on the starting point closes the figure and ends the procedure. Line button: This button allows you to determine the line thickness of the ROI outline. Color / Auto button: One color from the list of colors can be assigned to all ROIs. When Auto is pressed, the outlines of all ROIs are automatically colored differently. Buttons for diagram display: − 1 button: Intensity values for ROIs and channels are shown in one diagram. − Chan button: Intensity values are shown separately for each channel. − ROI button: Intensity values are shown separately for each ROI. − Mono button: Change between color and monochrome display of the intensity time diagrams. − Area button: Display of the area of the ROI in the intensity time diagram, depending on the set threshold values. Area measurements of very small areas (< 10 pixels) give only approximate values. − Mean button: Display of the mean values of the relevant ROI in the intensity time diagram. Ch1 / Ch2 / Ch4 button: Selection of the channel to be used. Threshold low slider: The intensity values below threshold are not displayed for the Area function. Threshold high slider: The intensity values above threshold are not displayed for the Area function. Buttons for Table functions: − Copy Table button: The table of intensity values is copied to the clipboard. − Show Table button: The table of intensity values is displayed on the bottom left of the Image Display window. − Save Table button: The table of intensity values can be stored as a text file. 4-314 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.14 Image Optimization 4.14.1 Single Channel OPERATION Image Optimization Carl Zeiss Described below is the example of the acquisition of an image, using an excitation wavelength of 532 nm and a fluorescence emission range above 550 nm. Let the specimen be a thin section through a stem of Convallaria majalis (Lily-of-the-Valley). The description applies to the use of the Axio Imager.Z1 microscope, and analogously also to the Axiovert 200 M. 4.14.1.1 Requirements The suitable laser is switched on. The specimen has been positioned and focused for scanning. • The LSM button has to be activated either in the LSM software or on the LSM button on the LSM tube itself. • Click on the Config button in the Acquire subordinate toolbar of the Main menu. − This opens the Configuration Control window. • Click on the Single Track button. ChL1 icon and assign a color to • Click on the Channel 1 in the Channel Color Selection window. Activate channel 1 via check box. • Click on the icon of emission filter 1 (before ChL1) and select the LP 550 filter. • If required, deactivate all the other channels (ChL2) via check box. 03/06 Fig. 4-305 B 45-0019 e Configuration Control window 4-315 OPERATION Image Optimization Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan icon, activate the 532 nm • Click on the laser line by clicking on Line Active . If required, deactivate other laser lines which are not needed. • Use the Transmission slider to set the laser intensity to approx. 30 % at first. The Beam Path and Channel Assignment panel displays the current configuration loaded. Fig. 4-306 The set laser intensity must be subsequently optimized for the current situation via the Transmission slider. Excitation panel For overlaying fluorescence and transmitted-light images, activate a second channel without a laser line but the Halogen illumination of the light microscope as the transmitted image light source. Choose the BP 675-725 in the emission filter menu and swing in the according red filter at the condensor Of course, all other transmitted light applications like − phase contrast − differential interference contrast (DIC) − polarization contrast (Pol) − darkfield can also be performed. 4-316 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Image Optimization Carl Zeiss • In the Main menu click on the Scan button in the Acquire subordinate toolbar. − This opens the Scan Control window. • Click on the Mode button. • For a frame scan, click on the Frame button. • On the Objective Lens Image Size & Line Step Factor panel, select Objective and Frame size for the scan (e.g. X 512 / Y 512 scan). • Enter a Exposure Time of 5 to 10 frames per second, for example, to start with. • Start with the following settings on the Pixel Depth, Scan Direction & Scan Average panel: Data depth: Scan direction: Average: 8 bits unidirectional Number: 1 • On the Zoom, Rotation & Offset panel, set a zoom of 1. Using the Fast XY button is a convenient way of creating an overview scan. Fig. 4-307 03/06 B 45-0019 e Scan Control window (Mode) 4-317 OPERATION Image Optimization Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Click on the Channels button. − This displays the preset parameters of the configuration loaded. • Click on the Find button. Make sure to position the slider correctly. Then scan while the slider is in the LSM position. − This starts the scanning process. − The image is seen to build up gradually in a new window. Function Find produces images of different brightness for different scan speeds. As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the light output. More often than not, the screen image is dull and needs subsequent optimization. Fig. 4-308 Scan Control window (Channels) Fig. 4-309 4-318 Image Display window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.14.1.2 OPERATION Image Optimization Carl Zeiss Confocal Aperture / Detector Gain / Ampl. Offset • In the Scan Control window, click on the Cont. button (see Fig. 4-308). − This starts a continuous scan. • Use the Pinhole slider (confocal aperture) to set the aperture size in the Scan Control window under Channels. − The aperture size should be so small that there is still enough variation for the setting of the detector gain and that sufficient image information is still available. 1 Airy is a good value to enable a confocal fluorescence XY-image to be obtained. − A small aperture size will increase the depth of focus, but reduce the light intensity received by the CCD detector. Fig. 4-310 Image Display window with confocal ROI Fig. 4-311 Color Palette window − The influence of the aperture size on image creation is shown by the example in Fig. 4-310. The entire image was first scanned with too large a aperture size. The aperture size was then optimized for a defined ROI. This considerably improved the display of the specimen structures. • Click on the Palette button in the Select image processing toolbar. − This opens the Color Palette window. • In the Color Palette List panel, click on the Range Indicator item. − The scanned image appears in a false-color presentation. 03/06 B 45-0019 e 4-319 OPERATION Image Optimization Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan If the image is too bright, it appears red on the screen. Fig. 4-312 Image Display window If the image is not bright enough, it appears blue on the screen. • On the Channel Settings panel of the Scan Control window, set the CCD detector gain with the Detector Gain slider. − The image should not have more than a trace of red. − This adjustment is very sensitive. Try using the left and right arrows to make the adjustment instead of dragging the slider bar. Fig. 4-313 4-320 Image Display window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan OPERATION Image Optimization Carl Zeiss • To adjust the black level (background), use the Ampl. Offset slider so that areas without picture content just show a trace of blue. • In the Color Palette List panel of the Color Palette window, click on No Palette. − This deselects the Range Indicator and activates the new presentation. • In the Scan Control window, click on the Stop button. − This stops the continuous scan. If you use the Range Indicator for image optimization, it may happen that the ranges marked in the Range Indicator will vary when the channel color is changed. 4.14.1.3 Fig. 4-314 Image Display window Fig. 4-315 Scan Control window Exposure Time, Scan Average and Pixel Depth The signal-to-noise ratio can be substantially improved by reducing the scanning speed to an acceptable level and averaging over several scans (i.e. with an average Number greater than 1 for the Mean average Method in the Scan Control window). • Use the Exposure Time slider in the Objective Lens, Image Size and Line & Step Factor panel to set the slowest acceptable exposure time speed. − The corresponding value for Frames per Second is shown below the slider. • In the Number text box of the Pixel Depth, Scan Direction & Scan Average panel enter the number of measurements to be averaged. Image optimization can be effected much faster if you select a smaller frame, since less data have to be processed. The greater the number of averages selected for Mean average Method, the better the image quality will be; the scanning time will be prolonged accordingly. 03/06 B 45-0019 e 4-321 OPERATION Image Optimization Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 4.14.2 Multiple-channel 4.14.2.1 Requirements • The suitable lasers are on. • The specimen has been positioned and focused for scanning. • The LSM button has to be activated either in the LSM software or on the LSM button on the LSM tube itself. In the following example, 2 Channels shall be activated for the scanning procedure: one for 488 nm using emission filter BP 500-525 and one for 532 nm with LP 550. NFT 532 is used as the secondary dichroic beam splitter. • In the Acquire subordinate toolbar, click on the Config button. − This opens the Configuration Control window. Fig. 4-316 Configuration Control window • Click on the Single Track button. • Activate (in the same way as for the single channel, see page 4-315) channel 2 (ChL2), the indicated emission filters and the main and secondary dichroic beam splitter for the scanning procedure. − The configuration loaded is displayed in the Beam Path and Channel Assignment panel. • Click on the Scan button in the Acquire subordinate toolbar of the Main menu. − This opens the Scan Control window. • In the Scan Control window, set the parameters in the same way as described for single-channel presentation. • Click on the Find button in the Scan Control window. − This starts the scanning process. The scanned image appears in a separate window. 4-322 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan Fig. 4-317 OPERATION Image Optimization Carl Zeiss Scan control and Image Display windows As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the light output. More often than not, the screen image is dull and needs subsequent optimization. • Click on the Channels button in the Scan Control window. − This opens the Channel Settings and Excitation of Track panels. − The channels used are color-highlighted. 4.14.2.2 Image Optimization The image optimization processes − setting of aperture size − Detector Gain / Ampl. Offset − Scanning speed and Average must be carried out separately for each channel used (see Single Channel, page 4-315). For the optimum setting of the single channels, Split xy-display must be selected in the Image Display window to enable the direct viewing of the separate images of the relevant channels. • Click on the Cont. button in the Scan Control window. − This starts a continuous scan. • Click on the Split xy button in the Image Display window toolbar. − This displays the separate images scanned in the channels and the composite (overlay) image. 03/06 B 45-0019 e 4-323 OPERATION Image Optimization Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan • Now click on the ChL1 button in the Channel Settings panel to optimize Channel 1. Optimization is performed in the same way as for the single channel and can be monitored online in the relevant separate image of the channel. • Then optimize the second channel by clicking on the relevant button (ChL2) in the Channel Settings panel of the Scan Control window. Fig. 4-318 Scan Control and Image Display windows Now effect image optimization as explained for the single-channel mode, but separately for each channel. • Now click on the xy button of the Display toolbar. − The composite scan image of two channels is presented in a common window. Image optimization can be effected much faster if you select a smaller frame, since less data have to be processed. 4-324 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.15 OPERATION Shut-Down Procedure Carl Zeiss Shut-Down Procedure Never shut down the computer by its main switch while your LSM program is still active, or else you will lose the currently set operating parameters and the images just scanned. In the Settings for user dialog window, which can be activated with the Options / Settings buttons, activate Laser off or Exit in the Shutdown tab. The lasers will then automatically be switched off when you exit the LSM program. 4.15.1 Exiting the LSM Program • Close all open windows of the LSM program by clicking on the closing icon of each window. in the top right corner − This closes the respective window and removes the respective icons from the taskbar. − After all dialog windows have been closed, the LSM 5 LIVE Switchboard window appears. Fig. 4-319 LSM 5 LIVE Switchboard menu • Click on the Exit button. − This terminates the LSM program. − The monitor screen shows the desktop of the WINDOWS XP operating system. 03/06 B 45-0019 e 4-325 OPERATION Shut-Down Procedure Carl Zeiss 4.15.2 LSM 5 LIVE LSM 5 LIVE DuoScan Shut Down the WINDOWS Operating System • Move the cursor to the bottom margin of the screen. − This opens the taskbar containing the Start button. • Click on the Start button of the taskbar. − This opens a pop-up menu. • Click on the Shut Down item. − This opens the Shut Down Windows window, in which you can select between Shut down, Restart and Login. • Unless already set by default, click on Shut down the computer? • Click on the Yes button. About 20 seconds after WINDOWS XP has been run down, and your computer turns off. 4.15.3 Turning Power Off Please bear in mind that a cooling phase of at least 5 minutes is required between switching off of the laser via the software and switching off of the entire system via the REMOTE CONTROL main switch or the Power Supply switch of the Enterprise UV laser. Set the REMOTE CONTROL switches “System/PC” and “Components” or the Main switch on the System Electronic Rack and the power supply switch of the Ar Laser to position "OFF" after 5 minutes. This puts your LSM 510 microscope system, including the computer, off power. 4.15.4 Turning off the HBO 100 • Switch off the HBO 103 via the toggle switch of the HBO 100 power supply. 4-326 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.16 OPERATION Software and Hardware Options Carl Zeiss Software and Hardware Options This section describes optional software and hardware configurations. Depending on your configuration, the content of dialogue and function may differ. 4.16.1 Software The following software packages for Release 4.0 are available: − Software "Physiology" − Software "Topography" − Software "Macro Recorder and Editor" − Software "3D for LSM" − Software "Multiple Time Series" − Software "Image VisArt" − Software "Deconvolution" − Software "StitchArt plus" − Software “Visual Macro Editor” − Software “FRAP” If your configuration does not include any of the SW packages "Physiology", “FRAP” or FRET, the following functions are not available: − Mean of ROI button in the Image Display window If your configuration does not include the "Physiology " software package, the following functions are not available: − Mean of ROI scan button in Time Series Control − Ion Concentration button in the Process Menu If your configuration does not include the “Visual Macro Editor” software package, the following functions are not available: − VME button in the Macro Menu If your configuration does not include the "Topography " software package, the following functions are not available: − Topo button in the Image Display window after acquisition of image stacks If your configuration does not include the "Macro Recorder and Editor" software package, the following functions are not available: − New, Save and Save as buttons in the Macro Control window − Edit, Step, Delete, Editor buttons in the Macro Control window 03/06 B 45-0019 e 4-327 Carl Zeiss OPERATION Software and Hardware Options LSM 5 LIVE LSM 5 LIVE DuoScan If your configuration does not include the "3D for LSM" software package, the following separate application is not available: − 3D for LSM If your configuration does not include the "Multiple Time Series" software package, the following function is not available: − Macro: "Advanced Time Series" If your configuration does not include the "Image VisArt" software package, the following function is not available: − 3D button in the Image Display window If your configuration does not include the "Deconvolution" software package, the following functions are not available − DCV Settings button in the Ortho function of the Image Display window − DCV button in the Process menu If your configuration does not include the "StitchArt plus" software package, the following function is not available: − Macro: "StitchArt plus" 4-328 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 4.16.2 OPERATION Software and Hardware Options Carl Zeiss Hardware Depending on whether the following hardware components are available or not, the content of the screens may differ: − Piezo objective focusing device − X-Y scanning stage DC 4 × 4 or DC 100 × 90, each with MCU 28 − Stands: Axio Imager.Z1, Axiovert 200 M − Depending on the configuration the scan head equipment may differ in filters, beam splitters and the number of photomultiplier − Transmitted-light PMT − Monitor diode If your configuration does not include the Piezo objective focusing device, the following functions are not available: − Hyperfine Z Sectioning in the Z Stack function in the Scan Control window − HRZ parameters in the Stage and Focus Control window If your configuration does not include the X-Y scanning stages DC 4 × 4 or DC 100 × 90, each with MCU 28, the following functions are not available: − Stage Position and Tile Scan functions in the Stage and Focus Control window Depending on the used microscope stand: Axio Imager.Z1 or Axiovert 200 M, the following dialogue and available functions may differ: − Context and accessibility of the Microscope Control window If your configuration does not include an AxioCam, the following functions are not available: − Camera in the Config Control window, Scan Control window 03/06 B 45-0019 e 4-329 Carl Zeiss 4.17 OPERATION LSM 5 LIVE Courses on "How to Operate the System in an Optimized Way" LSM 5 LIVE DuoScan Courses on "How to Operate the System in an Optimized Way" Carl Zeiss is offering training courses on how to operate the system in an optimized way. − Courses are held in our application center in Jena, Germany. − Courses are held in English or German language, respectively. Check out: www.zeiss.de/lsm for the latest dates and ask your Zeiss representative for a quotation on courses. 4-330 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 5 TOOLS Contents Carl Zeiss TOOLS CONTENTS Page 5 Tools.................................................................................................................................5-2 5.1 Change Filters ....................................................................................................................5-2 5.2 Stand Select .......................................................................................................................5-4 5.3 LSM Image Browser ...........................................................................................................5-5 5.4 LSM Image Examiner..........................................................................................................5-6 03/06 B 45-0019 e 5-1 TOOLS Change Filters Carl Zeiss 5 TOOLS 5.1 Change Filters LSM 5 LIVE LSM 5 LIVE Duo Scan The Change Filters tool is used to update the filter data in the software after a change of filters in the reflector turret of the microscope, the emission filters and the beam splitters of the LSM 5 LIVE. All filter data are updated in the Emission Filters & Beam Splitter Control window. After activation of the appropriate button (Emission Filters, Filter Cubes Stand or Beam Splitters LSM), the special input mask for the selected filter type is displayed. Since the procedures of updating or entering a new filter type are identical for all types, only the updating of filters in the reflector turret (Filter Cubes Stand) will be described in the following. • Close the LSM 5 LIVE software program. • Insert the new filter module in the reflector turret. • Double-click on the Change Filters icon on the desktop. − The Emission Filter & Beam Splitter Control window appears on the screen. The name of the currently used database is displayed in the System Database box, with the filter type being indicated below for checking purposes. • Click on the Filter Cubes Stand button. The Filter Cubes Stand panel is displayed. − The Filter Cubes Stand panel shows the Filter-Wheel No. and the filter positions available. Use the Name and ID selection boxes to enter the filters installed in the individual positions of the filter wheel. • Open the Name (or ID) selection box of the relevant filter position and select the new filter set from the list. • Click on the Store button to accept the new settings. • Click on the Close button to close the Emission Filter & Beam Splitter Control window. All available filter sets have to registered in the filter list (see Edit Filter List function, next page). Edit Filter List The Edit Filter List function permits updating of the filter data in the software after a change of filters on the stand. • Close the LSM 5 LIVE software program. • Double-click on the Change Filters icon on the desktop. Fig. 5-1 Edit Filter/Beam Splitter List window • Click on the Edit Filter List button in the Emission Filter & Beam Splitter Control window. − The Edit Filter/Beam Splitter List window is opened. This window permits a list of the most frequently used filter sets to be compiled. • Click on the arrow button in the Filtername list box to open it. • Select the filter set which shall be included in the list. 5-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan TOOLS Change Filters Carl Zeiss • Click on the Apply button. The selected filter set is included and displayed in the list (below the Sumary list box). This filter set is now also available in the Name selection boxes of the Filter Cubes Stand panel and can be assigned to a filter wheel position. To remove a filter set which is no longer needed from the list, proceed as follows: • Click on the name of the filter set concerned in the list box of the Edit Filter/Beam Splitter List window. • Click on the Remove button. The filter set is deleted from the list and is then no longer available in the Filter Cubes Stand panel of the Emission Filter & Beam Splitter Control window. Add New This function permits new filter sets to be added to the database. For this, proceed as follows: • Click on the Add New button on the Edit Filter/Beam Splitter List window. − The Add New window is opened. Filter/Beam Splitter • Enter the data of the new filter set in the Filter Cubes Stand Description panel, then click on the Apply button. The new filter set is stored in the database and included in the New Filter Cubes Stand panel. You can now activate the filter for a filter wheel position using the procedure described above. If you have activated the Non Zeiss check box, filter sets from other manufacturers can also be included in the database. Fig. 5-2 Edit Filter/Beam Splitter List window • To remove an new filter set from the database, select it with a click of the mouse in the New Filter Cubes Stand panel and then click on Remove. • Click on Close to close the Add New Filter/Beam Splitter window. • Click on Close to close the Edit Filter/Beam Splitter List window. • Click on the Store button to accept the new settings. • Click on the Close button to close the Emission Filter & Beam Splitter Control window. When you start the LSM 5 LIVE software, the filter data are updated. 03/06 B 45-0019 e 5-3 TOOLS Stand Select Carl Zeiss 5.2 Fig. 5-3 Select Stand Database ... window LSM 5 LIVE LSM 5 LIVE Duo Scan Stand Select The Stand Select tool permits a new or updated database to be assigned to the LSM 5 LIVE software program. This function should preferably be performed by authorized service personnel. If this is not possible, proceed as follows: • Close the LSM 5 LIVE software program and double-click on the Stand Select icon on the desktop. − The Select Stand Database window appears on the screen. The currently used database is displayed in the Database box. • Click on the Browse button to activate the new database. − The Open window appears on the screen. • Select the directory where the new database is stored. • Click on the name of the database (file extension: *.mdb) and then on the Open button. Fig. 5-4 Open window − The Open window is closed and the name of the new database appears in the Database box. • Click on the Permanent button. The Select Name window appears. • Select the relevant stand icon from the Icon list box and click on OK. The Select Name window is closed and the desktop icon is updated. • Then click on the OK button in the Select Stand Database ... window to accept the new settings and to close the window. (Clicking on Cancel will cancel the procedure.) Fig. 5-5 5-4 Select Name window − After the next restart of the LSM 5 LIVE software program, the new database will be automatically read in. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 5.3 TOOLS LSM Image Browser Carl Zeiss LSM Image Browser The LSM Image Browser permits images to be loaded, imported, exported and printed quickly without having to open the LSM 5 LIVE software. The LSM Image Browser can be used without dongle. When images are opened, image processing functions of the LSM 5 LIVE software are available to a limited extent (Chan, Zoom, Contr, Palette, Copy, Save, Save As, xy, Split xy, Prev, Info). • Click on the LSM Image Browser icon on the desktop of the PC. The Zeiss LSM Image Browser main menu is opened. Fig. 5-6 Zeiss LSM Image Browser main menu The following function buttons are available: New button Opens a new database. Open button Opens an existing database. Save button Saves the current image. Save As button Saves the current image under a new name. Import button Imports images. Export button Exports images. Full Screen button The current image is displayed on the full screen. Deactivation of the function with a click of the mouse. Multi Print button Several images are printed on one page. RAM button Use of the RAM memory for image display. DISK button Use of the hard disk as storage medium for image display. Exit button The Zeiss LSM Image Browser main menu is closed. The functions New, Open, Save, Save As, Import, Export and Multi Print correspond to those of the Expert Mode of the LSM 5 LIVE software and have already been described in chapter 4. 03/06 B 45-0019 e 5-5 TOOLS LSM Image Examiner Carl Zeiss 5.4 LSM 5 LIVE LSM 5 LIVE Duo Scan LSM Image Examiner The LSM Image Examiner can be used without having to open the LSM 5 LIVE software. However, this requires the installation of the relevant dongle. The LSM Image Examiner provides all the functions of the LSM Image Browser, plus the 3D functions and selected Process functions of the Expert Mode of the LSM 5 LIVE. When images are opened, a large scope of the image processing functions of the LSM 5 LIVE software is available (for further details see chapter 4). • Click on the LSM Image Examiner icon on the desktop of the PC. The Zeiss LSM Image Examiner main menu is opened. Fig. 5-7 Zeiss LSM Image Examiner main menu In addition to the buttons of the LSM Image Browser mentioned above, the following function buttons are available in the lower row of the Zeiss LSM Image Examiner main menu: Projection button One single projection or a series of projections can be calculated after rotation of the data package about the X, Y or Z axis. DepthCod button The depth information contained in a sequence can be colored with the colors of the rainbow. Stereo button Stereoscopic images can be generated. Ratio button Permits two channels to be linked into a new channel by the creation of a ratio. Copy button Permits one channel each of an existing image to be copied and stored as a new image. Filter button Permits the subsequent processing of scanned images via the integrated filters. Interpolate button Permits the continuous contrast and brightness change in a stack or time series through interpolation between the starting and end values. Shift button Produces a congruent image with relation to the pixels of the various channels. Duplicate button Permits images (including Z Stacks and Time Series) to be duplicated completely. Contrast button Permits the subsequent modification of contrast and brightness of the stored image. These functions correspond to those of the Expert Mode of the LSM 5 LIVE software and have already been described in chapter 4. 5-6 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 6 3D FOR LSM Contents Carl Zeiss 3D FOR LSM CONTENTS Page 6 3D for LSM .......................................................................................................................6-2 6.1 6.1.1 6.1.2 6.1.3 Overview and Explanations.................................................................................................6-2 The Image Sequence ..........................................................................................................6-2 The Image Properties..........................................................................................................6-3 Memory Usage...................................................................................................................6-3 6.2 6.2.1 6.2.2 6.2.3 User Interface.....................................................................................................................6-3 Introduction .......................................................................................................................6-3 Main Window ....................................................................................................................6-4 Display Window .................................................................................................................6-7 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 Functions .........................................................................................................................6-11 Functions in the File Menu ...............................................................................................6-11 Functions in the Edit Menu...............................................................................................6-14 Functions in the Process Menu .........................................................................................6-17 Functions in the View Menu.............................................................................................6-43 Functions in the Measurement Menu ...............................................................................6-50 03/06 B 45-0019 e 6-1 3D FOR LSM Overview and Explanations Carl Zeiss 6 3D FOR LSM 6.1 Overview and Explanations (0, 0, 0) 6.1.1 Voxel Single slice with single channel Single slice with multiple channels Image sequence Multichannel Positive rotation directions of the axes Z X LSM 5 LIVE LSM 5 LIVE DuoScan The Image Sequence The "3D for LSM" handles image sequences generated by the Zeiss LSM software. This can be three-dimensional image data or a time sequence of two-dimensional images (slices). Each slice (as well as the sequence) can consist of up to eight channels. An image sequence consists of a series of individual (2D) images and has a name that designates the entire sequence. In general an image sequence is handled as a single object in the system. Individual channels or slices can be addressed. The following terms and definitions apply for the "3D for LSM" software. − An image sequence is a number of individual sequential images (usually called slices in the dialog boxes), the spacing between which is equal. − Image sequences can contain up to 12 bit of image data (per channel). Y Fig. 6-1 − A sequence (slice) can consist of up to eight channels. − The maximum size of an image sequence is limited by the provided memory of the operating system. − A voxel is the smallest element of an image sequence (the equivalent of a pixel in a 2D image). All voxels in a given image sequence are the same size. − The coordinate system originates in the left upper front corner of the image sequence. This point has the coordinates 0, 0, 0. − All angles are positive for rotations to the right in the direction of the positive coordinate axis (righthanded coordinate system). − A slice is an individual image in a sequence of images. The numbering of the slices starts with "1". Image sequences can consist of several channels. Most functions and the Display window are providing buttons to select all or a subset of channels stored in the selected image sequence. The Output image sequence will only get those channels which are selected on the input side. The button selects all channels in the image sequence to be used clicking with the left mouse button on it. Clicking with the left mouse button on any of the number buttons toggles the state of this single channel. Clicking with the right mouse button on any of the number buttons selects this single channel exclusively. All other channels are deselected. 6-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 6.1.2 3D FOR LSM User Interface Carl Zeiss The Image Properties Every image sequence has its own set of properties. They contain the scaling and the scaling units. The scaling and its units are required for 3D reconstruction and measurement. If a sequence of LSM-TIFF images is read in, the image properties are loaded automatically from the file header and allocated to the image properties of the new image sequence. 6.1.3 Memory Usage All images shown in the Gallery are currently loaded in the system memory of the operating system. Some functions need additional temporarily used memory during their execution. If the memory is running low delete some images from the Gallery. If the images are needed afterwards they must be saved to disk first. Normally all functions produce a new result (output) image sequence. In order to save some memory, other image sequences currently presented in the Gallery can be selected as result position. The output image is overwritten by entry execution of a function. 6.2 User Interface 6.2.1 Introduction This section describes the following main components of the system: Main window Main window with the Menu, the Tool bar and Gallery. All general system functions are located here. Gallery Normally several images are required in order to accomplish a particular task. These images are displayed in reduced size to provide an overview and facilitate selection. This area is located just below the Tool bar. Fig. 6-2 Tool bar 03/06 This menu shows all image processing functions. B 45-0019 e 6-3 3D FOR LSM User Interface Carl Zeiss Display window LSM 5 LIVE LSM 5 LIVE DuoScan This window is used to display image sequences. Display window Fig. 6-3 Dialog boxes 6.2.2 Display window All dialog boxes provide three buttons. Pressing the OK button executes the function with the defined parameters and closes the dialog window. Selecting the Cancel button does not execute the function, restores the parameters, and closes the dialog window. Pressing the Apply button executes the function with the defined parameters; the dialog window will stay opened. Main Window The Main window includes: the Menu the Tool bar and the Gallery 6-4 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM User Interface Carl Zeiss File Menu Open Image Opens a file selector dialog to load an image sequence. Save Image As Opens a file selector to save an image or image sequence. Save Display As Saves the currently shown contents of the Display window as a single colour image. Print The printer parameters can be set with this tool. The standard Windows printer dialog is opened. Exit Terminates the application. Edit Menu Copy Copies the contents of the Display window to the clipboard. Edit Channels Allows to add or to remove channels to a single or multichannel image. Delete All Images Deletes all images and image sequences from the memory. Process Menu 03/06 Arithmetics Adds or subtracts the grey values of two image sequences (Add, Subtract). Contrast Enhances the contrast and brightness of an image sequence (Interactive, Automatic, Linearize). Smooth Smoothes an image sequence. Morphology Performs morphological operations on image sequences (Erode, Dilate, Open, Close). Segment Segmentates an image sequence to propose measurement (Interactive, Automatic). Boolean Combines two image sequences by Boolean operations (And, Or, Not, Xor, Mask). B 45-0019 e 6-5 Carl Zeiss 3D FOR LSM User Interface Scrap Selects or deletes objects of a defined size. Fill Holes Fills holes in objects. LSM 5 LIVE LSM 5 LIVE DuoScan View Menu Set Channel Colour The colour and the weight of the single channels can be defined. Properties The properties of the image (e.g. scaling, use laser etc.) are displayed. Render Calculates 3D reconstructions of an image sequence (Surface, Alpha). Measurement Menu Automatic Object Measures geometrical and densitometrical features (General, Object Features, Volume Features, Condition). Windows Menu Arrange All Arranges the windows automatically. Display The current image is displayed in this window. Help Menu Content Opens the help for the software. About 3D for LSM Displays status and release message of the software. Tool Bar This bar provides buttons with iconized images of nearly all functions. Clicking on one of the buttons will open a dialog window to define the function parameters. Selecting an entry from the menu alternatively can activate the same functions. Placing the cursor on a tool bar button will show a short description, if the window is activated. 6-6 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM User Interface Carl Zeiss Gallery The Gallery is used as an overview of the images available in memory and their contents. It is located just below the Tool bar. Each small image represents a sequence. The middle slice of each image sequence is shown. The status bar of each image shows the name. The name might be a number or a string. Every image sequence has its own channel colour assignment (see Display window). When an image is copied the channel colour assignment is copied too. Drag and drop techniques can be applied to copy images or define the function parameters Input and Output using the Gallery thumbnails. • Position the cursor on an image in the Gallery. • Press the left mouse button. • Hold the mouse button down and move the mouse to the destination position. • At the destination release the left mouse button, the destination image will be overwritten. To delete an image, drag it, move it to the wastebasket, and drop it. 6.2.3 Display Window This window is used to display an image sequence, regardless of size or type. To show multiple channel sequences each channel could have its own base colour. The user can set these colours and the weighting for each channel by pressing the corresponding button at the bottom of the window. To display a different image or image sequence, it can be dragged from the Gallery and dropped to the Display window. The image can be displayed in full size (one pixel on the screen represents one pixel of the image) or in a zoomed size. To zoom the display view click and hold down the right mouse button on the window border and resize the window. The aspect ratio of the image will not be changed. Clicking on the button resets the Display window to a full size view of the image (see above). The title bar shows the currently displayed sequence name. The status bar displays the size of the current sequence and the selected slice on the left. On the right the cursor position within the window and the corresponding intensity (grey) value of each channel is shown. The Display window can be closed without any effect to the image processing functions. If no Display window is opened select the entry Display in the Window menu. The scroll bar at the lower right of the window enables to show the images in a sequence. The range reaches from one to the maximum slice provided by the current sequence. To start the automatic animation of an image sequence start the Player tool by clicking on the button . The colour selection for the channels can be activated by clicking on the button image can be displayed as a grey value image by clicking on the button 03/06 B 45-0019 e . A colour . 6-7 3D FOR LSM User Interface Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Player This function plays back the sequential images of an image sequence. Fig. 6-4 The image sequence is displayed in the Display window. The display process is working as a background task; other functions can be executed while the player is running. There are several ways to stop the player: by closing the player window by pushing the red Stop button of the player window (the window remains open) by closing the image window. The Increment parameter specifies whether each sequential image (1) should be displayed or whether some sequential images should be skipped during display. The value 2 skips one image for every sequential image displayed, in other words, it displays only every second image. The parameter Wait Time states the delay in milliseconds between two successive sequential images. The maximum display speed depends mainly on the hardware. The sequential images are always displayed in their entirety, regardless of the set delay. 6-8 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM User Interface Carl Zeiss Control Element of the Player The three arrow shaped controls on the scale show the start slice and the currently displayed sequential image. The values (positions) can be changed using the mouse. Press and hold the left mouse button and move the pointer to the desired position. The set values are shown in the numerical windows at right. Start slice Currently displayed sequential image End slice The buttons in the left group start and stop playback of an image sequence. Reverse playback Forward playback Play forward and then backward again (jojo) Stop playback Pause playback The buttons in the middle group control the settings of the current sequential image. Reset to start slice. Single step backward (1 sequential image each regardless of Increment). Single step forward (1 sequential image each regardless of Increment). Set to end slice. Increment Image increment. Wait Time Displays delay between two images (in milliseconds). 03/06 B 45-0019 e 6-9 3D FOR LSM User Interface Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Set Channel Colour This function sets the colour and weight for the channels. Fig. 6-5 Each image sequence can get its own colour definitions. All functions will inherit the colour definition from the Input sequence to the Output sequence. By default the colours are set to 100 % weighting and the pure base colours (red, green, blue) are defined. The weight can be any value between 0 % and 200 %. The colour can be redefined by clicking on the coloured button on the right of the dialog. The standard Windows colour selection dialog is opened. The solution is done by clicking on one of the colours or by entering appropriate numbers in the corresponding edit boxes. Pressing the OK button will close the colour selection dialog and update the Display window immediately. Only those channels, which are available in the image sequence, can be defined. Parameters: 6-10 Image Image sequence to edit Weight Colour weighting for each channel Colour Base colour for each channel B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions 6.3 Functions 6.3.1 Functions in the File Menu Carl Zeiss Open Image This function reads a Zeiss LSM (*.lsm), Zeiss LSM TIFF (*000.tif) or Carl Zeiss Vision (*0.img) image sequence from a disk or network drive. Fig. 6-6 The individual files of a Zeiss TIFF image sequence are read and saved as an image sequence in image memory. In addition, the image properties are read out of the TIFF files and allocated to the image sequence Input. The directories of the current drive are listed in the Directories list box. Use the Drives list box to choose a different drive. In case of choosing the TIFF-format in the Files of Type box, three number characters are always expected before the dot in the filename extension. The first number must be 000 at the end of the filename. From a complete sequence only this file is listed in the dialog, if "LSM TIF Images (*000.tif)" is selected in the Files of Type box. To view all TIFF files "All TIF Images (*.tif)" in the Files of Type box must be selected. This selection enables to start with a different file than with the very first (named *000.tif) at the end of the filenames three number digits. Currently the Carl Zeiss Vision file format "KE Images (*0.img)" is supported. Two files per channel are saved. 03/06 B 45-0019 e 6-11 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Carl Zeiss Vision image sequences must have a number digit at the end of the base filename. They are used to indicate the different channels in a multichannel sequence. The numbering starts with zero (0). If a sequence is saved in the Carl Zeiss Vision format the numbers are generated automatically. To load such an image sequence "KE Images (*0.img)" in the Files of Type box must be selected. The window incorporates the usual file selection controls. The bottom half displays a selection of the image properties that are stored in the image sequence. Parameters: BaseName Base name of the TIFF files (image sequence) to be loaded. Only the letters before the first number are stated. Input Name of the resulting image in which the image sequence will be saved. Save Image As This function saves an image or image sequence to disk or network drive. Fig. 6-7 All the files in the current directory that have the selected image format are listed in the File Name list box. The directories of the current drive are listed in the Directories list box. Use the Drives list box to choose a different drive. Use the list box Files of Type to select the image format. Currently the LSM image format (*.lsm) and the Carl Zeiss Vision file format "KE Images (*0.img)" is supported. 6-12 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss By choosing the Carl Zeiss Vision file format "KE Images (*0.img)", two files per channel are saved. On one hand the Carl Zeiss Vision type image sequence file, on the other hand the file with the image properties. One pair of files is written per channel. They are numbered automatically, starting with zero. A one number digit is added to the end of the filenames. The two files share the same filename but have different filename extensions (*.img and *.3d). The content of the Gallery is shown in the Input section. The selection of the sequence to save is done by highlighting one of the provided names or by drag and drop from the Gallery. Parameters: Input Name of the image sequence to be saved Filename Name of the file to be used on disk Save Display As This function saves the current Display window contents to a disk or network drive. Fig. 6-8 Before the execution of this function any image or image sequence can be selected to be displayed. From a multichannel sequence any channel status (on or off) combination can be defined. The colours of the shown channels can be set with the function Set Channel Colour. The current zoom factor of the Display window is not taken into account, the image is saved without any zoom. The image is saved as a true colour image with 24-bit resolution. From the Save as Type list box one of the provided formats can be selected. Parameters: None 03/06 B 45-0019 e 6-13 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Print This function prints the current Display window contents. The standard Windows print dialog is opened. Before the execution of this function any image or image sequence can be selected to be displayed. From a multichannel sequence any channel status (on or off) combination can be defined. The colours of the shown channels can be set with the function Set Channel Colour. Parameters: None Exit This function terminates the application completely. All images and image sequences shown in the Gallery will be deleted from the memory. Save those images which might be used for any further processing. Parameters: None 6.3.2 Functions in the Edit Menu Copy This function copies the current Display window contents to the clipboard. No dialog is shown. Before the execution of this function any image or image sequence can be selected to be displayed. From a multichannel sequence any channel status (on or off) combination can be defined. The colours of the shown channels can be set with the function Set Channel Colour. The current zoom factor of the Display window is not taken into account; the image is copied without any zoom. The image is copied as a true colour image with 24-bit resolution. Afterwards the contents can be pasted to any other Windows application. Parameters: None 6-14 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Edit Channels This function allows to add or to remove channels to a single or multichannel image. On the Add Channel tab sheet the channels of (different) Input sequences can be defined to add (combine) channels to an Output sequence. Fig. 6-9 This operation is useful to add a segmented channel (or any other result of a function) to the original image sequence. The selected channels of Input 1 and Input 2 are copied to Output. The maximum number of channels in an image sequence is eight. If the image sequences do not have the same extents Output Size defines which input is taken as a reference. This selection also defines the properties for scaling and units in the output image sequences. Parameters: 03/06 Input 1 First input image sequence Input 2 Second input image sequence Output Output image sequence Output size Defines source image sequence for size, scaling, and units B 45-0019 e 6-15 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan On the Delete Channel tab sheet channels of the Input 1 image sequence can be selected to delete channels. Fig. 6-10 This operation might save time and memory for further processing if not all channels are needed. Only the selected channels of Input 1 are copied to Output. Parameters: Input 1 Input image sequence Output Output image sequence Delete All Images This function deletes all images and image sequences from the memory (Gallery). The function is used whenever a completely new image sequence should be processed. In order to drop the images item by item to the wastebasket all of them can be deleted by a single function. If any image or image sequence is needed for further use save them first. Parameters: None 6-16 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 6.3.3 3D FOR LSM Functions Carl Zeiss Functions in the Process Menu Arithmetics - Add This function adds two image sequences. Fig. 6-11 The Add tab sheet of the Arithmetics dialog window must be selected. If one or both input sequences are multichannel sequence, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right. This function adds the two image sequences Input 1 and Input 2 voxel by voxel and generates the image sequence Output. Note that a resulting grey value may be greater than 255 (4095). The parameter Mode determines how a range overflow is handled: 1 - Wrap No normalization - the grey values are displayed modulo 256 (4096). If the result is greater than 255 (4095), the value 256 (4096) is subtracted from it. 2 - Clip Grey values which exceed 255 (4095) are replaced with 255 (4095). 3 - Normalize The resulting grey value range is scaled to the range 0...255 (0...4095). Parameters: 03/06 Input 1 First input image sequence Input 2 Second input image sequence Output Output image sequence Mode 1 - Wrap 2 - Clip 3 - Normalize B 45-0019 e 6-17 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Arithmetics - Subtract This function subtracts two image sequences. Fig. 6-12 The Subtract tab sheet of the Arithmetics dialog window must be selected. If one or both input sequences are multichannel sequence, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right. This function subtracts the two image sequences Input 1 and Input 2 voxel by voxel and generates the image sequence Output. Note that a resulting grey value may be less than 0. The parameter Mode determines how a range overflow (negative values) is handled. 1 - Wrap No normalization - the grey values are displayed modulo 256 (4096). If the result is less than 0, the value 256 (4096) is added to it. 2 - Clip Negative values are set to 0. 3 - Normalize The resulting grey value range is scaled to the range 0...255 (0...4095). 4 - Shift/Clip 128 (2048) is added to the difference, then negative values are set to 0. Values greater than 255 (4095) are set to 255 (4095). Parameters: 6-18 Input 1 First input image sequence Input 2 Second input image sequence Output Output image sequence Mode 1 - Wrap 2 - Clip 3 - Normalize 4 - Shift/Clip B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Contrast - Interactive This function allows interactive changes of the contrast of an image sequence. Fig. 6-13 The Interactive tab sheet of the Contrast dialog window must be selected. A grey value range of the Input image sequence is scaled to another range in the Output image sequence. Both ranges can be edited interactively. This function is used to achieve a better view of an image sequence, or to scale a range of grey values to single value for a special coding in an image sequence. The function does not improve the result of the linear segmentation function Segment. Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels, or any number can be selected. The Input histogram shows the grey value distribution of the selected channels of the Input image sequence. Output defines the name of the result sequence. It will get only those channels which are chosen by the Input parameter. The buttons labeled with 8 and 12 define the grey value (intensity) resolution in bit. Normally the result will get the same resolution as the Input sequence. A change will be needed if image sequences with different resolutions should be combined. Rising the grey value range to 12 bit will not enhance the display quality or measurement accuracy. The smooth and morphology functions will produce results with finer gradations. If Clip Grey Values is selected, the output grey values are clipped to the Low (L) and High (H) values. If Clip Grey Values is not selected, output grey values beyond the Low and High value range are possible. 03/06 B 45-0019 e 6-19 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from 0 to the maximum, which is either 255 or 4095, depending whether the input is 8 bit or 12 bit. The vertical axis represents the pixel count. The selected range is marked by the borderlines in the histogram. The blue line or L indicates the lower boundary, the red line or H the upper one, C indicates the center of the range. There are three ways to change the range: clicking and dragging the borderlines with the mouse. or using the arrow keys Entering a new value in the appropriate text boxes, clicking on the buttons from the keyboard. To alter the values within the histogram move the mouse pointer over one of the three coloured lines until the shape changes. Press and hold the left mouse button to move the line to a new position. To change the values with the arrow keys click once into the histogram. Using the left or right arrow key by its own will move the whole range. Pressing the Shift key additionally moves the lower boundary, the Control key the upper boundary. The vertical scale of the histogram is set using the scroll bar. The units are percents of the maximum grey value distribution. This setting has no influence on the function. Parameters: 6-20 Input Input image sequence Output Output image sequence Channel Selection of the channel numbers for the Output image after contrast enhancement Clip Grey Values Clipping of grey values to the Low (L) and High (H) output grey values boundaries Input L Lower boundary of grey value range Input Input C Center of grey value range Input Input H Upper boundary of grey value range Input Output L Lower boundary of grey value range Output Output C Center of grey value range Output Output H Upper boundary of grey value range Output B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Contrast - Automatic This function scales the grey values of an image sequence to the maximum possible range. Fig. 6-14 The Automatic tab sheet of the Contrast dialog window must be selected. This function enhances the contrast of an image sequence by spreading the grey value distribution over the maximum possible range. This function is used to achieve a better view of an image. The light and dark grey value ranges with a low share of pixels are excluded from the operation by the parameter Threshold. The Threshold units are in thousandths of the total number of voxels. Using a value of 10 means that the scale interval is set so that 5/1000 of the total number of voxels on the light side, and 5/1000 of the total number of voxels on the dark side of the grey value distribution are excluded. Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels, or any number can be selected. The Input histogram shows the grey value distribution of the selected channels of the Input image sequence. Output defines the name of the result sequence. It will get only those channels which are chosen by the Input parameter. The buttons labeled with 8 and 12 define the grey value (intensity) resolution in bit. Normally the result will get the same resolution as the Input sequence. A change will be needed if image sequences with different resolutions should be combined. Rising the grey value range to 12 bit will not enhance the display quality or measurement accuracy. The smooth and morphology functions will produce results with finer gradations. 03/06 B 45-0019 e 6-21 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The Output histogram shows the resulting histogram. They are not editable. The horizontal axis represents the grey values from 0 to the maximum, which is either 255 or 4095, depending whether the input is 8 bit or 12 bit. The vertical axis represents the pixel count. The vertical scale of the histogram is set using the scroll bar. The units are percentages of the grey value distribution maximum. This setting has no influence on the function. Parameters: 6-22 Input Input image sequence Output Output image sequence Threshold Exclusion value - 0...1000 Input L Lower boundary of grey value range Input Input C Center of grey value range Input Input H Upper boundary of grey value range Input Output L Lower boundary of grey value range Output Output C Center of grey value range Output Output H Upper boundary of grey value range Output B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Contrast – Linearize This function scales a range of grey values of an image sequence to equal area fractions in the histogram. Fig. 6-15 The Linearize tab sheet of the Contrast dialog window must be selected. This function enhances the contrast by linearizing the histogram of the image sequence to equal area fractions in the histogram. The areas (voxel count multiplied by grey value range) of all grey values in the Output histogram are the same. This function is used to achieve a better view of an image sequence. When Skip Black is checked the grey value 0 will not be taken into account for linearization. Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels, or any number can be selected. The Input histogram shows the grey value distribution of the selected channels of the Input image sequence. Output defines the range of the result sequence. It will get only these channels which are chosen by the Input parameter. The grey value (intensity) resolution will be the same as the one from Input. The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from 0 to 255. The vertical axis represents the pixel count. The vertical scale of the histogram is set using the scroll bar. The units are percentages of the grey value distribution maximum. This setting has no influence to the function. 03/06 B 45-0019 e 6-23 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Parameters: Image Input image sequence Output Output image sequence SkipBlack 0 - Grey value black is ignored 1 - Grey value black is taken into account Input L Lower boundary of grey value range Input Input C Center of grey value range Input Input H Upper boundary of grey value range Input Output L Lower boundary of grey value range Output Output C Center of grey value range Output Output H Upper boundary of grey value range Output Smooth (Gauss) This function performs a Gauss filter. Fig. 6-16 The noise in the image sequence is reduced, the edge shape is nearly unchanged, local maxima are leveled, the dynamic range is reduced. Image sequences should be smoothed before they are reconstructed or segmented. For most sequences a Size value of 3 is sufficient enough. If Input is a multichannel sequence, any number and combination of channels can be selected. Output will only get the selected channels as results. The grey value of every pixel is substituted by a weighted average of its surrounding neighbors. The neighbors are defined by a cube. The affected pixel is the central pixel of the filter cube. The weighted filter cube is approximated by a binomial distribution. The size of the filter cube is set using the Size scroll bar. Even numbers are set to the next odd value. The Size defines the strength of the smoothing. Parameters: 6-24 Input Input image sequence Output Output image sequence Size Filter size (3...31, only odd numbers) B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Morphology The following four functions perform basic operations of mathematical morphology on image sequences. Fig. 6-17 As generalization of the morphology of two-dimensional images to three dimensions the structural elements are small volumina. Literature Bomans, M.; Höhne, K.-H.; Tiede, U.; Riemer, M.: 3D-Segmentation of MR Images of the Head for 3-D Display IEEE Transactions on Medical Imaging 9, 1990, 177-183 Schiemann, T.; Bomans, M.; Tiede, U.; Höhne, K.-H.: Interactive 3D-Segmentation of Tomographic Image Volumes 14. DAGM-Symposium Mustererkennung, Springer-Verlag 1992, 73-80 03/06 B 45-0019 e 6-25 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The input image sequence is analyzed voxel by voxel with a selected shape (Shape). The voxel to be analyzed is always the central voxel of the shape. The shape type determines which neighboring voxels are used to compute the resulting voxel. The following structural elements are available for all morphological operations. They represent approximated spheres with an increasing radius. Sequential image: Volume view: Cross shape Volume view: Cross shape Z-1 Z Z+1 Sequential image: Z-1 Z Z+1 6-26 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Sequential image: Volume view: Carl Zeiss Cube cross shape: created through application of "cube" and "cross" one after the other. Z-2 Z-1 Z Z+1 Z+2 For regions (voxels) that are at the edge of the image sequence, it assumed for erosion that there are white voxels with a grey value of 255 (4095) outside the edge. For dilation, it is assumed that there are black voxels with the grey value 0 outside the image sequence. If the Grey Morphology tickbox is activated, erosion sets the grey value of the central voxel to the minimum of all neighboring voxels affected by the structural element; dilation sets the grey value of the central voxel to the maximum. If the Grey Morphology tickbox is not activated, the neighboring voxels are only distinguished by grey value 0 and non-0. For erosion the central voxel is set to 0 if any of the neighbors is 0. It is set to 255 (4095) if any neighbor is not 0. For dilation the central voxel is set to 255 (4095) if any of the neighbors is not 0. It is set to 0 if all neighbors are 0. Erosion reduces the size of bright regions, separates thin connections between them, and makes small regions disappear. Dilation, on the other hand, makes bright regions of the image grow in size, fills gaps, and smoothes small contour details. 03/06 B 45-0019 e 6-27 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan The result of erosion and dilation is called opening. On the one hand, this maintains to some extent the original size of the regions while not losing the smoothing effect of erosion on the image. This name stands for the operation of reducing convex bulges in the contour of the region. Thin connections between regions are eliminated, broken borders between regions are connected, and small regions disappear. The opposite operation (first dilation, then erosion) is called closing. Concave bulges in the contours of regions are filled in; connections are formed between adjacent regions. The following example illustrates the operations "Open" and "Close" in two dimensions: Open = Erosion + Dilation Fig. 6-18 Close = Dilation + Erosion Fig. 6-19 The "cube cross" shape was used for the operations shown. 6-28 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Morphology - Erode This function erodes structures in an image sequence. Fig. 6-20 In the Morphology dialog window, the tab sheet Erode must be selected. Erosion makes bright regions smaller on a dark background. It also results in separation of thin connections between regions. Small regions disappear entirely. If Input is a multichannel sequence any number and combination of channels can be selected. Output will only get the selected channels as results. The Input image sequence is eroded Count times with the shape Shape. The Count scroll bar determines the number of recursive operations. The following shapes (numbered 1 to 3 from left to right) are available: If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result Output will be a binary sequence. Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only 1 - All grey value shades are taken into account 03/06 B 45-0019 e 6-29 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Morphology - Dilate This function dilates structures in an image sequence. Fig. 6-21 In the Morphology dialog window, the tab sheet Dilate must be selected. Dilation makes bright regions larger on a dark background. It also results in the filling of gaps and smoothing of small contour details. If Input is a multichannel sequence any number and combination of channels can be selected. Output will only get the selected channels as results. The Input sequential image is dilated Count times with the shape Shape. The Count scroll bar determines the number of recursive operations. The following shapes (numbered 1 to 3 from left to right) are available: If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result Output will be a binary sequence. Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only 1 - All grey value shades are taken into account 6-30 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Morphology - Open This function carries out an opening. Fig. 6-22 In the Morphology dialog window, the tab sheet Open must be selected. This function carries out an erosion followed by a dilation. For the most part, the opening maintains the original size of the regions. Thin connections between regions and small regions themselves disappear. Convex bulges in the contours of the regions are reduced. The opening is applied to the grey value image sequence Input Count times with the shape Shape. If Input is a multichannel sequence any number and combination of channels can be selected. Output will only get the selected channels as results. The Count scroll bar determines the number of recursive operations. The following shapes (numbered 1 to 3 from left to right) are available: If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result Output will be a binary sequence. Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only 1 - All grey value shades are taken into account 03/06 B 45-0019 e 6-31 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Morphology - Close This function carries out a closing. Fig. 6-23 In the Morphology dialog window, the tab sheet Close must be selected. This function carries out a dilation followed by an erosion. For the most part, the closing maintains the original size of the regions. Connections are formed between adjacent regions; gaps and bright concave bulges in the contours of regions are filled in. The closing is applied Count times to the grey value image sequence Input with the shape Shape. If Input is a multichannel sequence any number and combination of channels can be selected. Output will only get the selected channels as results. The Count scroll bar determines the number of recursive operations. The following shapes (numbered 1 to 3 from left to right) are available: If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result Output will be a binary sequence. Parameters: Input Input image sequence Output Resulting image sequence Shape Shape used 1 - cross 2 - cube 3 - cube cross Count Number of recursive operations Grey Morphology 0 - Distinguish between 0 and non 0 only 1 - All grey value shades are taken into account 6-32 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Segment - Interactive This function carries out a grey value segmentation by means of thresholding. Fig. 6-24 The Interactive tab sheet of Segment dialog window must be selected. Segmentation is especially used to generate binary regions. These are required for the measurement. Two threshold values determine which grey value range of the Input image sequence is preserved and/or deleted in the Output image sequence. Only one channel of a multichannel sequence can be selected as Input. Output will always be a single channel sequence. The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the histogram. This setting has no influence on the function. The thresholds Low and High are determined either by moving the borderlines in the grey value histogram or by the scroll bars underneath. Furthermore, the values for Low, Center and High can be set through entry in the corresponding fields. To move the lower (L) and upper (H) thresholds at the same time, move the vertical line in the grey value histogram or set the scroll bar (C). The Green and Blue/Red option buttons of the parameter Colour determine whether the voxels within (Green) or outside (Blue/Red) of the grey value interval [L, H] are displayed with the corresponding colour. If Green is selected, the voxels within the selected interval are highlighted in green. The rest of the image retains its original grey values. The voxels with the grey values Low and Low+1 are displayed in blue. The voxels with the grey values High and High-1 are displayed in red. 03/06 B 45-0019 e 6-33 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan If Blue/Red is selected, the voxels with grey values within the interval Low, High remain unchanged. Voxels with grey values less than Low are highlighted in blue; those with grey values higher than High are highlighted in red. If the Invert option is selected, the grey values outside the defined interval will be segmented. If the option Binary is selected, then all grey values in the range from Low to High will be set to white (grey value 255) in the Output image sequence, while all others will be set to black (grey value 0). If the option is not selected, the grey values within the selected interval remain unchanged, while those outside the range will be set to black. The measurement function accepts both results without any difference in the results. Parameters: 6-34 Input Input image sequence Output Resulting image sequence Colour Green - Selected interval is displayed in green Blue/Red Grey values below the selected interval are displayed in blue, grey values above in red Binary 0 - Selected voxels retain the original grey value 1 - Selected voxels are set to grey value 255, the rest to grey value 0 Invert 0 - Grey values inside the selected interval are segmented 1 - Grey values outside the selected interval are segmented L Low grey value threshold C Center of threshold interval H High grey value threshold B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Segment - Automatic The function carries out an automatic grey value segmentation by means of thresholding. Fig. 6-25 The Automatic tab sheet of the Segment dialog window must be selected. Segmentation is especially used to generate binary regions. These are required for the measurement. The function calculates the two strongest local minimums in the histogram of the Input image sequence. These values are used for the discrimination. Only one channel of a multichannel sequence can be selected as Input. Output will always be a single channel sequence. The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the histogram. This setting has no influence on the function. The Green and Blue/Red option buttons of the parameter Colour determine whether the voxels within (Green) or outside (Blue/Red) of the grey value interval [L, H] are displayed with the corresponding colour. If Green is selected, the voxels within the selected interval are highlighted in green. The rest of the image retains its original grey values. The voxels with the grey values Low and Low+1 are displayed in blue. The voxels with the grey values High and High-1 are displayed in red. If Blue/Red is selected, the voxels with grey values within the interval Low, High remain unchanged. Voxels with grey values less than Low are highlighted in blue; those with grey values higher than High are highlighted in red. If the Invert option is selected, the grey values outside the defined interval will be segmented. If the option Binary is selected, then all grey values in the range from Low to High will be set to white (grey value 255 (4095)) in the Output image sequence, while all others will be set to black (grey value 0). If the option is not selected, the grey values within the selected interval remain unchanged, while those outside the range will be set to black. 03/06 B 45-0019 e 6-35 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan Parameters: 6-36 Input Input image sequence Output Resulting image sequence Colour Green - Selected interval is displayed in green Blue/Red - Grey values below the selected interval are displayed in blue, grey values above in red Binary 0 - Selected voxels retain the original grey value 1 - Selected voxels are set to grey value 255 (4095), the rest to grey value 0 Invert 0 - Grey values inside the selected interval are segmented 1 - Grey values outside the selected interval are segmented L Low grey value threshold C Center of threshold interval H High grey value threshold B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Boolean - And This function carries out a bit-by-bit And calculation for the image sequences Input 1 and Input 2. Fig. 6-26 The And tab sheet of the Boolean dialog window must be selected. This function is especially well suited for masking images. If one or both input sequences are multichannel sequences, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right. Parameters: Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence Boolean - Or This function carries out a bit-by-bit Or calculation for the images Input 1 and Input 2. Fig. 6-27 The Or tab sheet of the Boolean dialog window must be selected. This function can be used to combine binary masks or regions. 03/06 B 45-0019 e 6-37 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan If one or both input sequences are multichannel sequences, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right. Parameters: Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence Boolean - Xor This function carries out a bit-by-bit Xor calculation for the images Input 1 and Input 2. Fig. 6-28 The Xor option button of the Function option group in the Boolean dialog window must be selected. This function can be used to combine binary masks or regions. If one or both input sequences are multichannel sequences, any number or combination can be selected. The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from left to right. Parameters: 6-38 Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Boolean - Not This function carries out a bit-by-bit negation of an image. Fig. 6-29 The Not tab sheet of the Boolean dialog window must be selected. If Input is a multichannel sequence any number or combination can be selected. Parameters: 03/06 Input Input image sequence Output Resulting image sequence B 45-0019 e 6-39 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Boolean - Mask This function masks a grey value image sequence. Fig. 6-30 The Mask tab sheet of the Boolean dialog window must be selected. This function modifies the Output image sequence depending on the mask image sequence used. If the grey value in Input 2 is higher than 0, then the voxel values are copied from Input 1 to the image sequence Output. If the grey value of the voxel is 0, then the voxel value of the Output image sequence is taken over. If one or both input sequences are multichannel sequences, any number or combination can be selected. The number of selected channels for Input 2 must be 1 or the same as for Input 2. They will be combined from left to right. Parameters: 6-40 Input 1 First input image sequence Input 2 Second input image sequence Output Resulting image sequence B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Scrap This function deletes or selects objects in a specified size range. Fig. 6-31 The operation deletes or selects objects on the basis of their total volume in voxels. Objects with a volume within the range MinVolume to MaxVolume are effected. To delete objects outside the range, the parameter Select must be active. If the parameter is not activated objects outside the defined volume range are deleted. Parameters: 03/06 Input Input image sequence Output Output image sequence MinVolume Minimum object size MaxVolume Maximum object size Select 0 - Select the objects outside the size range 1 - Select the regions within the size range B 45-0019 e 6-41 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Fill Holes This function fills holes in all objects. Fig. 6-32 All holes in objects are filled by this operation. Holes are structures, which have a grey value of 0 and are surrounded completely by voxels with a grey value not equal to 0. It is assumed that regions outside the image are black. Holes, which touch the image border, are retained. Parameters: 6-42 Input Input image sequence Output Output image sequence B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 6.3.4 3D FOR LSM Functions Carl Zeiss Functions in the View Menu Render - Surface This function displays an image sequence according to the gradient shading method. Fig. 6-33 The Surface tab sheet of the Render dialog window must be selected. 03/06 B 45-0019 e 6-43 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan Method The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all channels can be selected for the reconstruction. Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the button New will generate a new name (number). The size of the sequential images in Output is determined by the size of the sequential images in Input. Number of Views determines the number of reconstructions which should be computed. The radio buttons Start and End define which angle settings are currently shown. A definition for the angle End is only necessary if Number of Views is higher than 1. If this is true the result sequence will get views from the Start to the End angle definition. The other reconstructions are determined through the linearly interpolated intermediate angles. The direction of view is determined from the angles as follows: The angle Angle Z determines the rotation of the direction of view on the Z-axis. The angle Angle Y determines the rotation of the direction of view on the Y-axis that has been rotated by the angle Angle Z. The angle Angle X determines the rotation of the direction of view on an X-axis that is rotated by Angle Z and Angle Y. Channel defines if the following parameters are valid for All or just for one. Defining the thresholds for the channels independently is useful if the grey value boundaries of the objects differ too much in the different channels. The thresholds Grey Low and Grey High define the grey value range of the objects. The parameter Aperture is a measure of the size of the highlights. Small values generate large highlights. Large values generate small highlights (similar to a spot). Use the parameter Reflection to control the ratio of diffuse and reflective brightness components, i.e., the overall basic brightness compared with the highlights. When the value of Reflection is low, the highlights predominate; when the values are high, the region appears to be uniformly illuminated and the highlights are not so pronounced. When Auto Update is selected, the reconstruction is updated automatically whenever a parameter is modified (except Input, Output, or Number of Views). Show Cube defines whether a wire frame cube is shown in the Display window or not. Application This method can be applied, if the structures in the Input sequence can be segmented by grey value thresholding. Because the gradient is calculated for every pixel, the Output appears in very fine detail. Noisy Input sequences must be smoothed (function Smooth) before rendering, otherwise the surface appears rough. Parameters: Input Input image sequence Output Resulting image sequence Number of Views Number of reconstructions to be calculated 6-44 Angle X Angle of rotation on the X-axis, start position Angle Y Angle of rotation on the Y-axis, start position Angle Z Angle of rotation on the Z-axis, start position Channel All - The following parameters are valid for all channels X - The following parameters are valid for the selected channel only Grey Low Low grey value threshold of the region to be displayed B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Grey High High grey value threshold of the region to be displayed Aperture Measure of the extent of the highlights Reflection Weight of the defuse brightness components in comparison to the highlights Auto Update 0 - Function execution is performed on OK or Apply 1 - Function execution for the current angle is performed on any parameter change Show Cube 0 - The wire frame cube is not shown 1 - The wire frame cube is shown in the Display window Render - Surface: Method Description This method displays the surface of structures in the Input sequence shaded as if a light illuminated it. The position of the light is behind the view point with parallel rays in the direction of the sequence. The input sequence is segmented into object and background by grey value thresholding: object voxels are within the grey value range Grey Low to Grey High. Each Output pixel corresponds to a point at the surface at which the ray in view direction through the Output pixels hits the surface. All rays are parallel. The surface normal required for shading in this gradient renderer is the grey value gradient in the Input volume at the surface voxel position. It is not the geometric surface normal. The grey value gradient is determined from the grey values in a 3x3x3 cube around the surface voxel by averaging e.g. the xgradient in y- and z-direction [4]. There is no depth cueing (far objects would appear darker). The illumination model is a Phong model [1] (surface normal is determined for each Output pixel) with diffuse reflection and specular reflection. Diffuse reflection means that the surface reflects light with equal intensity in all directions. The brightness of a given surface patch depends not on the viewdirection, but only on the angle between light and surface normal. Specular reflection is observed on shiny surfaces as a highlight. The light is reflected as from a mirror. The maximum intensity is observed when the view direction is the one of the mirrored light direction. 03/06 B 45-0019 e 6-45 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Render - Alpha This function displays an image sequence according to the alpha rendering method. Fig. 6-34 The Alpha tab sheet of the Render dialog window must be selected. One or more reconstructions of the input image sequence are computed according to the alpha rendering method. This type of reconstruction should be used if there is no possibility to segment the structures in the image sequence and also if the objective is to make deeply layered structures visible. 6-46 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Method The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all channels can be selected for the reconstruction. Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the button New will generate a new name (number). The size of the sequential images in Output is determined by the size of the sequential images in Input. Number of Views determines the number of reconstructions which should be computed. The radio buttons Start and End define which angle settings are currently shown. A definition for the angle End is only necessary if Number of Views is higher than 1. If this is true the result sequence will get views from the Start to the End angle definition. The other reconstructions are determined through the linearly interpolated intermediate angles. The direction of view is determined from the angles as follows: The angle Angle Z determines the rotation of the direction of view on the Z-axis. The angle Angle Y determines the rotation of the direction of view on the Y-axis that has been rotated by the angle Angle Z. The angle Angle X determines the rotation of the direction of view on an X-axis that is rotated by Angle Z and Angle Y. Channel defines if the following parameters are valid for All or just for one. Defining the opacity for the channels independently is useful when the brightness and contrast of the channels differ too much. Threshold defines the range with no opacity. It is completely transparent. The range starts at grey value 0. The length of slope is defined by Ramp. The maximum opacity value is set with the parameter Max. Opacity. This range ends at the maximum grey value. The Opacity Table shows the grey value histogram of Input with the opacity definition as a red line. When Auto Update is selected, the reconstruction is updated automatically whenever a parameter is modified (except Input, Output, or Number of Views). Show Cube defines whether a wire frame cube is shown in the Display window or not. Application 1. This method can be applied, if the structures in the Input sequence are unsharp so that objects are poorly defined by their grey value. 2. In this case, the Opacity Table is defined as a ramp. Low grey values have weight 0 to suppress the background voxels. The opacity rises with increasing grey values, depending on the parameter Ramp. The value of Max. Opacity defines the weight of the high grey values. High grey values above a threshold have weight 255 to show the "object" voxels unsuppressed. Of course a smooth step can be used. 3. The result is a display with inside structures shining through. A 3D impression can be obtained by rendering with several view directions. 4. In contrast to this, a voxel renderer like the gradient renderer would display only the surface of objects that are defined by grey value-thresholds. This surface would appear shaded as if illuminated by a light. 5. The method can also be applied to visualize pronounced structures within other enclosing structures, if the structures have different grey value ranges. 6. In this case, the Opacity Table is defined as a step. Low grey values (background) have weight 0. High grey values (inside structures) have maximum weight. 03/06 B 45-0019 e 6-47 Carl Zeiss 3D FOR LSM Functions LSM 5 LIVE LSM 5 LIVE DuoScan Parameters: Input Input image sequence Output Resulting image sequence Number of Views Number of reconstructions to be calculated 6-48 Angle X Angle of rotation on the X-axis, start position Angle Y Angle of rotation on the Y-axis, start position Angle Z Angle of rotation on the Z-axis, start position Channel All - The following parameters are valid for all channels X - The following parameters are valid for the selected channel only Threshold Grey value where the opacity starts rising Ramp Length of the opacity slope Max. Opacity Maximum opacity value Opacity Table Maximum opacity value Auto Update 0 - Function execution is performed on OK or Apply 1 - Function execution is performed on any parameter change Show Cube 0 - The wire frame cube is not shown 1 - The wire frame cube is shown in the Display window B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Render - Alpha: Method Description Each Output pixel is a weighted sum of the Input voxels along a ray in view direction through the Input sequence. Each Input voxel has an opacity value, dependent only on its grey value. The opacity values are defined by the parameters Threshold, Ramp, and Max. Opacity. Accumulation of pixels proceeds along the ray from back to front, i.e. from far pixels to near pixels. If a new pixel is added, it increases the result intensity by its grey value weighted by the opacity value, and attenuates the previously accumulated intensity according to the opacity value. Full intensity stops accumulation. This calculation must be repeated for each pixel of the ray to generate one Output pixel. Then for each Output pixel to produce a 2D Output image for the selected view-angle. Then for each view-angle to produce an output sequence for Number of Views different view angles. Render - References [1] J.D. Foley,A.van Dam, S. K. Feiner, J.F.Hughes, Computer Graphics: Principles and Practice, Addison Wesley, Reading, MA, 1990. [2] M. Levoy, Display of Surfaces from Volume Data, IEEE Computer Graphics & Applications, May 1988, 29-37. [3] J. Ylä-Jääski, F.Klein, O. Kübler, Fast Direct Display of Volume Data for Medical Diagnosis, VGIP:Graphical Models and Image Processing 53,1991,7-18. [4] K.H. Höhne, R. Bernstein, Shading 3D-Images from CT Using Gray-Level Gradients, IEEE Transactions on Medical Imaging, 5, 1986, 45-47. [5] D.Gordon, R.A. Reynolds, Image Space Shading of 3-Dimensional Objects, CVGIP 29, 1985, 361-376. 03/06 B 45-0019 e 6-49 3D FOR LSM Functions Carl Zeiss 6.3.5 LSM 5 LIVE LSM 5 LIVE DuoScan Functions in the Measurement Menu Measurement Concept Measurement is based on regions (objects) in three-dimensional space. Segmenting an image sequence generates these. The image segmentation process produces a mask image that defines the region. A region is a group of voxels that touch at the surfaces or at the edges, but not at the corners (18 voxel neighborhood). This is illustrated by the following example. The voxels marked black in sequential image Z-1, Z, Z+1 all belong to the same region as the grey central voxel in sequential image Z. The volume view shows the neighborhood interrelationships as a 3D projection. Sequential image: Volume view: Z-1 Z Z+1 Fig. 6-35 6-50 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Measurement Process The measurement process consists of three steps: region definition, checking of the validity of the regions, and feature calculation. Region definition: - Automatically from the mask image Region validation check depends on: - Minimum volume - Measurement condition Feature calculation depends on - Shape of the region - Densitometric value distribution of the region - Feature parameters Image Region generator Region Region filter Valid region Data Measurement Image sequence Minimum volume Feature name Measurement condition Feature parameter Fig. 6-36 All regions found are checked according to certain conditions. The voxel volume of each region must be equal to or greater than MinVolume. The measurement condition must be fulfilled. Only those regions that meet all the conditions are valid for the measurement. The region can be measured or labeled. Measurement is a process that produces data. Labeling is a process that generates an image volume. 03/06 B 45-0019 e 6-51 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Automatic Object Measurement – Object Features A measurement feature describes a region characterized by a number (e.g. volume, area or a densitometrical statistic). The features can be selected on the Object Features and Volume Features tab sheets. Fig. 6-37 The scalings and units are taken automatically from the assigned sequence. The measurement features can be selected individually for each measurement. The object features generate a result value for every single object. 6-52 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss The dialog shows two lists. One shows the Available Features as groups (on the left). The other one shows the Selected Features. Double-clicking on items of the left list will add the Selected Features to the right list. Double-clicking on an item of the right list will remove this item from the list. Selected Features can also be transferred by clicking on the button in the middle (<< / >>) of the dialog. The combo box above the right list represents predefined feature lists. Selecting one of the entries will fill the right list with these features; previously selected features will be overwritten. The button Select All will copy all features to the list of selected features. The button Remove All will clear the list of selected features. Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog. Parameters: Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remove All Remove all object features from the selected features list The following sections describe all measurement features which are defined in the system. 03/06 B 45-0019 e 6-53 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Object Features (geometric) If Object Features are selected, one set of measurement data is calculated for each object. Group Name Name Description Volume Volume Volume of the object. Volume Filled VolumeF Volume of the filled object. Ellipsoid EllipseA Length of the main axis of the ellipsoid with the same geometrical moment of inertia as the object. EllipseB Length of the middle axis of the ellipsoid with the same geometrical moment of inertia as the object. EllipseC Length of the minor axis of the ellipsoid with the same geometrical moment of inertia as the object. EllipseAF Length of the main axis of the ellipse with the same geometric moment of inertia as the filled object. EllipseBF Length of the middle axis of the ellipse with the same geometric moment of inertia as the filled object. EllipseCF Length of the minor axis of the ellipse with the same geometric moment of inertia as the filled object. Surface Area SurfArea Surface area of the object. Surface Area Filled SurfAreaF Surface area of the filled object. Sphere Diameter Dsphere Diameter of the sphere with the same volume. Ellipsoid filled 6 * VOLUMEF / π Sphere Form Factor Fsphere Form factor of the object. 6⋅ π ⋅ Number of Holes 6-54 Nparts VOLUMEF SURFAREAF3 Number of holes within an object. B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Object Features (densitometric) Group Name Name Description Mean Densitometric MeanD Densitometric mean value of an object. Standard Deviation Densitometric StdD Standard deviation of the densitometric values of an object. Minimum Densitometric MinD Minimum grey value of an object. Maximum Densitometric MaxD Maximum grey value of an object. Automatic Object Measurement - Volume Features A measurement feature describes a region characterized by a number (e.g. volume, area, or a densitometrical statistic). The features can be selected on the Object Features and Volume Features tab sheets. Fig. 6-38 The measurement features can be selected individually for each measurement. The object features generate a result value for every single object. 03/06 B 45-0019 e 6-55 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan The dialog shows two lists. One shows the Available Features as groups (on the left). The other one shows the Selected Features. Double-clicking on items of the left list will add the Selected Features to the right list. Double-clicking on an item of the right list will remove this item from the list. Selected Features can also be transferred by clicking on the button in the middle (<< / >>) of the dialog. The combo box above the right list represents predefined feature lists. Selecting one of the entries will fill the right list with these features; previously selected features will be overwritten. The button Select All will copy all features to the list of selected features. The button Remove All will clear the list of selected features. Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog. Parameters: Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remove All Remove all object features from the selected features list Volume Features (geometric) The volume-related measurement generates one measured value per image sequence. The following table contains the predefined volume characteristics. Group Name Name Description Count VolCount Number of regions measured. Volume VolVolume Total volume of all regions. Volume Percentage VolVolumeP Total volume of all regions, in relation to the volume of the image sequence. Volume Features (densitometric) Group Name Name Description Surface Area VolSurfArea Total surface area of all regions. Mean Densitometric VolMeanD Mean grey value of all regions. Standard Deviation VolStdD Densitometric Grey value standard deviation of all regions. Minimum Densitometric VolMinD Minimum grey value in the image sequence. Maximum Densitometric VolMaxD Maximum grey value in the image sequence. 6-56 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss Automatic Object Measurement - Condition The measurement conditions are used to limit the objects to be evaluated (e.g. only objects with defined minimum value). All objects are tested against the defined conditions. If the conditions are fulfilled the feature values are written to the data table. Fig. 6-39 To define the following parameter select the Condition tab sheet of the Automatic Object Measurement dialog window. The list on the very left at the dialog shows all the measurement Features. The second list provides the comparison Operators and the next Numbers to define a value. This gives the possibility to compose an expression to test a feature value against a constant value. The fields above the lists will show the composed (selected) string. Clicking on the desired list entry does the selection. The button with the „>>„ characters adds this string to the List of Conditions. All lines of the List of conditions are combined with the AND expression automatically. To remove a condition line double-click on it. The parameter Minimum Volume defines the minimum voxel volume for the measurement. This is an easy way to eliminate very small regions caused by noisy sequences and segmentation process. The button Remove All will clear the list of defined conditions. Clicking on the Apply button will execute the measurement process and switch to the General tab sheet of the dialog. Parameters: Feature Operator Number List of conditions Remove All Minimum Volume 03/06 List of available object features List of available condition operators List of numbers to compose the value Defined condition list Remove all entries from the List of conditions Minimum object volume in voxel B 45-0019 e 6-57 3D FOR LSM Functions Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Automatic Object Measurement - General This function carries out an automatic measurement and labeling. Measured Object Features Measured Volume Features Fig. 6-40 The regions must be defined by an image sequence Mask Image (the objects must be separated from one another by black voxels with the grey value 0). This sequence is generated with the function Segment. If it is a multichannel sequence a single channel has to be chosen. The image Dens Image is needed for the measurement of the densitometric features. Image sequence properties like scaling and unit are taken from Dens Image. A single channel of this sequence (if it is multichannel) must be selected with the buttons to the right of the parameter. The measurement results can be stored to database files. These files are tab delimited ASCII files which can be easily imported to major Windows programs like text processing or spreat sheet application. Writing database files are independently supported for object and volume features. Activating the corresponding check boxes enables it. The name of the database is defined with the field Database. The files will be located in the subdirectory DATA of the main installation directory. The filename extension TXT will be added automatically. If the check box Label is activated a single channel sequence will be generated. It contains all the measured objects, each object is coloured homogeneous but in different colours. To copy all measurement values to the clipboard activate the check box Clipboard. 6-58 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 3D FOR LSM Functions Carl Zeiss A single object of interest can be visualized. Clicking on a specific row in the data grid chooses the object. By selecting a row in the data grid a new image is created with the object of interest visualized. The visualization depends on the settings in the Object Visualisation field. If Render is chosen, the object of interest is displayed with the Surface Rendering method. If Mask is chosen, the object is labelled in a pseudo colour in a new image stack. Parameters: 03/06 Mask Image Single channel mask image sequence that defines the objects Dens Image Image sequence for densitometric measurement and property source Object Stores measurement values of objects, including database filename Volume Stores volume measurement values of objects, including database filename Label Generates an image sequence with all objects labelled in different pseudo colours Clipboard Measurement values are automatically written to the clipboard B 45-0019 e 6-59 LSM 5 LIVE LSM 5 LIVE DuoScan CHAPTER 7 ANNEX Contents Carl Zeiss ANNEX CONTENTS Page 7 Annex...............................................................................................................................7-2 7.1 Recommendations for Excitation Laser Lines and Emission Filters of Dyes............................7-2 7.2 7.2.1 7.2.2 Configurations Overview....................................................................................................7-3 LSM 5 LIVE 1 Channel Biomedical Configurations...............................................................7-3 LSM 5 LIVE 2 Channel Biomedical Configurations...............................................................7-4 7.3 Changing Filters in the Scanning Module ...........................................................................7-5 7.4 Detaching / Attaching the Scanning Module from / to Microscope Stands ..........................7-5 7.5 7.5.1 7.5.2 7.5.3 Hints on the Use of the Piezo Fine Focusing Stage ..............................................................7-7 General Description............................................................................................................7-7 Application Fields ...............................................................................................................7-7 Additional Information on the Operation............................................................................7-7 7.6 Piezo Objective Focussing Device ........................................................................................7-9 7.7 Specifications of Trigger-Interface LSM 5 LIVE ..................................................................7-10 7.8 7.8.1 7.8.2 7.8.3 7.8.4 AxioCam High Resolution Digital Cameras .......................................................................7-13 Microscopy Camera AxioCam MRm Rev.2 ........................................................................7-13 High Resolution Microscopy Camera AxioCam HRm Rev.2................................................7-14 High Resolution Microscopy Camera AxioCam HRc ..........................................................7-15 Microscope Camera Port Adapters for the AxioCam .........................................................7-16 7.9 List of Key Words .............................................................................................................7-17 03/06 B 45-0019 e 7-1 ANNEX Recommendations for excitation laser lines and … Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan 7 ANNEX 7.1 Recommendations for Excitation Laser Lines and Emission Filters of Dyes Dye Laser line/HFT Emission/EM DAPI 405 > 420, max. at 461 EBFP 405 > 420, max. at 447 Hoechst 405 > 420, max. at 440 Fluoro-Gold 405 or 440 > 420/475, max. at 536 ECFP 405 or 440 > 420/475, max. at 501 Lucifer Yellow 440 or 488 > 475, max. at 536 EGFP 488 > 505, max. at 507/516 FM 1-43™ 488 > 505, max. at 598 Alexa Fluor 488™ 488 > 505, max. at 520 Calcium Green 488 > 505, max. at 531 Cy2™ 488 > 505, max. at 508 DiO (DiOC18(3)) 488 > 505, max. at 508 Fluo-3 488 > 505, max. at 520 Fluorescein (FITC) 488 > 505, max. at 520 Cy3™ 532 > 530, max. at 566 EYFP 532 > 530, max. at 535 Oregon Green 532 > 530, max. at 535 SYTOX Green 532 > 530, max. at 536 FM 4-46 532 > 560, max. at 640 Alexa Fluor 546™ 532 > 560, max. at 572 Calcium Orange 532 > 560, max. at 575 DiI (DiIC18(3)) 532 > 560, max. at 565 DsRed 532 > 560, max. at 583 Tetramethylrhodamine (TRITC) 532 > 560, max. at 576 Rhodamine B 532 > 560/585, max. at 625 Texas Red™ 532 > 560/585, max. at 620 Alexa Fluor 633™ 633 > 650, max. at 654 Cy5™ 633 > 650, max. at 666 7-2 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX Recommendations for excitation laser lines and … Carl Zeiss Here you can note your specific combinations: Dyes Laser/HFT EM1 NFT EM2 Dyes Laser/HFT EM1 NFT EM2 FITC/Cy3 488/532 BP 500-525 532 LP 550 Example: 7.2 Configurations Overview 7.2.1 LSM 5 LIVE 1 Channel Biomedical Configurations 1 channel, FL VarioOne GB Laser lines 488, 532 (635) nm Emissionfilters channel 1 LP 505 BP 495-525 SK LP 550 BP 550-615 LP 650 SK BP 700-750 03/06 B 45-0019 e 7-3 ANNEX Configurations Overview Carl Zeiss 7.2.2 LSM 5 LIVE LSM 5 LIVE DuoScan LSM 5 LIVE 2 Channel Biomedical Configurations 2 channel, FL VarioTwo VRGB Laser lines (405/440), 488, 532 (635) nm Secondary Plate beam splitter mirror NFT 488 NFT 532 NFT 635 Emission- LP 420 filters LP 505 channel 1 BP 495-525 SK LP 550 BP 560-675 SK LP 650 SK BP 700-750 Emissionfilters channel 2 BP 415-480 SK BP 455-525 BP 495-525 SK BP 550-615 7-4 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 7.3 ANNEX Detaching / Attaching the Scanning Module ... Carl Zeiss Changing Filters in the Scanning Module For optimum investigation of specimens it is useful to employ filter wheels permitting the motorcontrolled change between different filters for narrow-band or broad-band detection depending on the wavelength. The number of filters is limited by the capacity of the filter wheel. The change of the filter wheel as a whole involves complete readjustment. Please ask your LSM service engineer to do this exchange in order to maintain your warranty. 7.4 Detaching / Attaching the Scanning Module from / to Microscope Stands Tool needed: 3 mm Allen key The user can remove the Scanning Module from one microscope and attach it to another within a few minutes. Described below is the change-over from an Axioskop 2 FS MOT to an Axiovert 200 M in sideport configuration. Before the change-over, shut down the system as described in chapter 4 in order to avoid damage to the system and loss of data. • Loosen the three screws (7-1/1) at the Scanning Module (7-1/2) fitted to the Axioskop 2 FS MOT. • Cautiously pull Scanning Module off the Axioskop 2 FS MOT stand. • Attach Scanning Module to the left sideport of the Axiovert 200 M, minding the guide pins (7-1/5), and secure it with the three screws (7-1/1). As the Scanning Module is heavy, weighing about 19.5 kg, it is easier if the changeover is carried out by two persons. • Pull off covering caps (7-1/3) from the CAN-BUS and RS232 interface ports at the rear of the Axiovert 200 M, remove the two cables 457411-9011 (CAN-BUS) and 457411-9012 (RS232) from the Axioskop 2 FS MOT, plug them into the Axiovert 200 M and secure them there. • Switch the LSM 5 LIVE on. • Click on the Stand select icon to update the system database with the new database of the Axiovert 200 M microscope. • Restart the LSM 5 LIVE program. 03/06 B 45-0019 e 7-5 Carl Zeiss ANNEX Detaching / Attaching the Scanning Module ... LSM 5 LIVE LSM 5 LIVE DuoScan 04 08 01 07 02 06 03 05 100 90 O ZER E TIV JEC OB OR CT FLE RE S CU FO Fig. 7-1 7-6 Change-over of the Scanning Module B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX Hints on the Use of the Piezo Fine Focusing Stage 7.5 Hints on the Use of the Piezo Fine Focusing Stage 7.5.1 General Description Carl Zeiss The Piezo fine focusing stage is a compact attachment for the Axio Imager.Z1 and Axiovert 200 M microscope stages, which allows the particularly fast and high-precision fine focusing of the object. The Piezo stage permits fine focusing over a range of 250 µm, with the smallest step width being less than 10 nm, reproducibility better than 40 nm, and the maximum speed amounting to 60 Hz. The stage allows the use of specimens with a weight of less than 100 g. The piezo stage is not used if manual coarse focusing is performed. To position the objective in relation to the optical Z-axis, the standard XY-microscope stage is used. The piezo stage features a mount for standard object carriers of 76 mm x 26 mm x 1 mm and a milledout receptacle for ∅ 36 mm x 1 mm Petri dishes. 7.5.2 Application Fields − High-precision fine focusing and translation of the object along the optical axis. − Fast and high-precision mounting of one-dimensional Z-line sections. − Fast and high-precision mounting of two-dimensional R-Z-longitudinal sections. − Fast and high-precision mounting of XY-Z-Stacks for the three-dimensional reconstruction of the object. − Exact measurement of Point-Spread-Functions for deconvolution. 7.5.3 Additional Information on the Operation The piezo fine-focusing stage is a high-precision, sensitive accessory for the LSM 5 LIVE from Carl Zeiss and must therefore be treated carefully. High mechanical stress, such as the use of specimens weighing more than 100 g or the application of pressure or knocks on the movable stage tongue, can result in damage and therefore in failure of the stage function. To be able to fully utilize the outstanding precision attainable with the fine focusing stage, anything which could interfere with its operation, especially mechanical knocks and impact of the LSM 5 LIVE components, should be avoided. We would recommend you to always use the actively vibration-damped table. The specifications of the stage are obtained only after a heating phase of approx. 30 minutes. Furthermore, the installation conditions for the LSM 5 LIVE system must be observed. The maximum reproducibility (better than 40 nm) for moving to an absolute position in Z is achieved by always moving to the required position from below. 03/06 B 45-0019 e 7-7 Carl Zeiss ANNEX Hints on the Use of the Piezo Fine Focusing Stage LSM 5 LIVE LSM 5 LIVE DuoScan Fine focusing is performed mechanically via an inclined position of the stage tongue. Therefore, the lifting range Z at the location of the image field depends on the position of the piezo stage in relation to the optical axis. This means: if the user shifts the object on the microscope stage to the right via the piezo stage, the lift will be different from the one in the zero position of the stage (max. 250 µm) and also from the one after a shift of the stage to the left. If the LSM 5 LIVE system is equipped with a motorized scanning stage, this shift is read back to •x and the lift is calibrated automatically if the zero position of the piezo stage has been matched to the zero position of the scanning stage via an initialization run. For this, activate the Stage button of the Acquire toolbar. Then position the scanning stage in such a way that the optical axis of the microscope corresponds to the zero position of the piezo stage, i.e. to the center of the specimen holder in the stage tongue. Then perform initialization by pressing the piezo Null button. This step must be repeated after every new start of the system. Also see the notes on the operation of the motorized scanning stages. If the system is equipped with a manual microscope stage, the user has the option of performing the calibration by entering the •x shift in mm via the Calibration slider. The shift is read off from the microscope stages. In the case of the manual AxioImager stage, •x can be read directly from the scale adhered to the front of the stage. In the case of the manual Axiovert 200 M stage, a scale is located on the right of the knob, where the 45 mm •x shift relative to the zero position of the microscope stage can be read off. The •x value is positive for both stages if shift from the zero position is made to the right and negative if the shift is made to the left. On account of the inclined position of the stage tongue, the object is also shifted laterally during the fine focusing motion. This lateral shift is negligibly small if, as recommended by us, specimen carriers with thickness 1.0 mm are used exclusively. Otherwise, the marked lateral shift of the object during fine focusing can result in image distortion. For the same reason, Petri dishes without fixation ring must be used exclusively. The nosepiece of the Axiovert 200 M stand is moved to the load position prior to switching off the LSM 5 LIVE system and the piezo stage is then moved to the lowest position to avoid damage of the objective or object by a possible collision. The user must refocus after start-up of the system. Before an objective change in the Axiovert 200 M or the Axio Imager.Z1 the nosepiece and the microscope stage must be moved to the load position by the user, and then back to the work position to prevent the objectives from hitting the piezo components. This is performed automatically if the objectives are changed menu-controlled via the relevant buttons of the LSM 5 LIVE program. 7-8 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 7.6 ANNEX Piezo Objective Focussing Device Carl Zeiss Piezo Objective Focussing Device For upright stands Axio Imager.Z1, Axio Imager.M1, Axioskop 2 FS MOT Range: 250 µm Minimum step size: 5 nm Speed: Piezo objective focussing device Piezo focussing stage Slices Step size [µm] xz-lines / s xz-lines / s 20 0.5 60 60 Objectives: − W0.8/M27 − Modified Achroplan 40x / 0.8 W with reduced length to compensate for piezo height Installation: • Screw in your microscope objective into Piezo Objective Focusing Device (see Fig. 7-2/1). • Screw the thread-ring into your microscope (see Fig. 7-2/2). • Easy clamp the Piezo Objective Focusing Device on the thread-ring (see Fig. 7-2/3). Fig. 7-2 03/06 B 45-0019 e Installation of the Piezo Objective Focusing Device 7-9 ANNEX Specification of Trigger-Interface LSM 5 LIVE Carl Zeiss 7.7 LSM 5 LIVE LSM 5 LIVE DuoScan Specifications of Trigger-Interface LSM 5 LIVE Application: With the LSM 5 LIVE you can control various actions externally using Trigger-In or force external devices to work at a defined time depending on an action using Trigger-Out during time series. These actions are: Scan-Start / Stop, Bleach, Change of Scan-Interval, end of a countdown, set marker into image or even a mouse-click on a button. Interface: − User Port Adapter on the back of the Real Time Control Unit in the ECU of LSM 5 LIVE: − Connector 2x high density D-Type plug, 1x coax with outer shield Number: − 8x signal IN (all able to generate an interrupt) − 18x signal OUT (10 synchronous, 8 asynchronous relative scan position) − 1x clock Connector: Coax: Triax Lemosa Serie 00, EPL.00.650NLN Pin Signal name Description Signal PCLK_Out PixelClock Output Signal shield GND Signal ground Outer shield Shield Chassis ground 7-10 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX Specification of Trigger-Interface LSM 5 LIVE Carl Zeiss High density D-Type 15pol.: Plug „A“ Pin Signal name Description 1 SyncOUT0 Synchronous output 2 SyncOUT1 Synchronous output 3 SyncOUT2 Synchronous output 4 SyncOUT3 Synchronous output 5 GND Signal ground 6 AsyncOUT0 Asynchronous output 7 AsyncOUT1 Asynchronous output 8 AsyncOUT2 Asynchronous output 9 AsyncOUT3 Asynchronous output 10 GND Signal ground 11 AsyncIN0 input 12 AsyncIN1 input 13 AsyncIN2 input 14 AsyncIN3 input 15 Connector detector* Input / Connector detector High density D-Type 15pol.: Plug „B“ Pin Signal name Description 1 SyncOUT4 Synchronous output 2 SyncOUT5 Synchronous output 3 SyncOUT6 / sync outline ** Synchronous output 4 SyncOUT7 / sync outframe ** Synchronous output 5 GND Signal ground 6 AsyncOUT4 Asynchronous output 7 AsyncOUT5 Asynchronous output 8 AsyncOUT6 Asynchronous output 9 AsyncOUT7 Asynchronous output 10 GND Signal ground 11 AsyncIN4 input 12 AsyncIN5 input 13 AsyncIN6 input 14 AsyncIN7 input 15 Connector detector* Input / Connector detector *) pull to signal ground 03/06 **) depending on internal switch inside real time cintrol unit B 45-0019 e 7-11 ANNEX Specification of Trigger-Interface LSM 5 LIVE Carl Zeiss LSM 5 LIVE LSM 5 LIVE DuoScan Type/Voltage Range: − TTL signal level 3.3 V, CMOS low power consumption − 5.0 V tolerant input/output for interfacing with 5 V logic Load: Output: < 50 mA (internal serial 68 ohm resistor) Input: 4.7 kOhm input impedance (internal 4.7 kOhm pullup to 3.3 V) Trigger pulse description: Signal output: − Low level < 0.4 V, high level > 2.8 V − Slew rate 10 ns/V Signal input: − Low level < 0.8 V, high level > 2.0 V − Falling edge force interrupt − Pulse width to detect signal > 50 ns Caution: − Never apply more than 5 V or negative voltages to avoid any damage. − In and outputs are not galvanically decoupled. − Therefore proper measures for galvanic decoupling of external devices have to be taken (optocoupler etc.). Hint: Use the Zeiss “Trigger Box” (1315-620) for immediate use of the User-Port: − 4 momentary-on switches, 2 BNC connectors including optional signal inversion for input − 8 leds and 2 BNC connectors for output 7-12 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX AxioCam High Resolution Digital Cameras 7.8 AxioCam High Resolution Digital Cameras 7.8.1 Microscopy Camera AxioCam MRm Rev.2 Carl Zeiss High resolution microscopy camera AxioCam MRm Rev. 2 (D) Cat. No 000000-0445-554 Mid Range Monochrome incl. AxioVision AC, digital interface, cable and IR barrier filter BG40 (enclosed) Number of Pixels: 1388 (H) x 1040 (V) = 1.4 Mega pixel Pixel size: 6.45 µm x 6.45 µm Chip size: 8.9 mm x 6.7 mm, equivalent to 2/3" Spectral range: With protection glass limited appr. 350 nm to 1000 nm, With IR barrier filter BG40 appr. 350 nm to 700 nm NIR-Mode: Increase of IR sensitivity Max. Full Well Capacity: Approx. 17.000 e Selectable Resolution: H x V 276 x 208 (Binning 5x5) 346 x 260 (Binning 4x4) 462 x 346 (Binning 3x3) 694 x 520 (Binning 2x2) 1388 x 1040 (single shot) Live frame rates (depending on hardware and software configuration): H x V Binning Factor 1388 x 1040 Frame Rate @20ms 1 7 694 x 520 2 13 462 x 346 3 17 Readout of Sensor Sub Regions("ROI"): Adjustable Digitalization: 12 bits / 18 Mhz pixel clock Dynamic Range: Typical 1700 : 1 ati 10 e readout noise Integration time: 1 ms to 20 s Cooling: One stage Peltier cooling Control signals: TTL output for controlling of external shutters Interface: PCI interface card (32 bit / 5 V) with one cable (5m) for data, control and power supply Optical Interface: C-Mount Thread depth for objectives: max. 5 mm 03/06 B 45-0019 e 7-13 Carl Zeiss ANNEX AxioCam High Resolution Digital Cameras Max. file size per image: About 2.8 MB at 1388 x 1040 @ 1 x 12 bit Operating Systems: Win 2000, Win XP Size / Weight: About 11 cm x 8 cm x 4.5 cm (2.3” x 3.2” x 1.7”) / 370 g Housing: Blue anodized aluminum, with cooling fins, 1/4" photo thread for tripod mount Registration: CE, cUL Power Supply: Via PC Environment conditions: 0° ... +35° Celsius, 10% .... 80% relative air huminity, no condensing, free air circulation required 7.8.2 LSM 5 LIVE LSM 5 LIVE DuoScan High Resolution Microscopy Camera AxioCam HRm Rev.2 Cat. No 000000-0445-553, incl. digital interface and cable High Range Monochrome Number of Pixels: 1388 (H) x 1040 (V) = 1.4 Mega pixel Chip size: 8.9 mm x 6.7 mm, equivalent to 2/3" Spectral range: With BK-7 protection glass up to 1000 nm, with IR barrier filter BG40 limited to about 350 nm to 700 nm Selectable Resolution by Binning or Microscanning H x V 694 x 520 1388 x 1040 2776 x 2080 4164 x 3120 Acquisition Time (s) @ 20 ms exposure 0.07 (13 images / s 0.2 (5 images / s) Dynamic Range: Better than 2000 : 1 @ 8 e readout noise Integration Time: 1 ms to several minutes Cooling: Single stage Peltier cooling Optical Interface: C-Mount Size: about 11 cm x 8 cm x 6.5 cm (2.3" x 3.2" x 2.6") Registration: GS, CE, cUL Power Supply: 12 V DC, 1 A, 230 V/110 V, autodetecting 7-14 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 7.8.3 ANNEX AxioCam High Resolution Digital Cameras Carl Zeiss High Resolution Microscopy Camera AxioCam HRc Cat. No 000000-0412-312, incl. digital interface and cable High Range Color Number of Pixels: 1300 (H) x 1030 (V) = 1.3 Mega pixel Chip size: 8.7 mm x 6.9 mm equivalent to 2/3” Spectral range: Limited by IR barrier filter BG40, about 400 nm to 700 nm Selectable Resolution by Binning or Microscanning H x V Acquisition Time (s) @ 20 ms exposure 432 x 342 1300 x 1030 0.2 (Color interpolation) 1300 x 1030 0.7 (full resolution for color channels) 2600 x 2060 3900 x 3090 0.07 (Color interpolation) Dynamic Range: Typical 2000 : 1 @ 9 e readout noise Integration Time: 1 ms to several minutes Cooling: One stage Peltier cooling Optical Interface: C-Mount Size: about 11 cm x 8 cm x 6.5 cm (2.3" x 3.2" x 2.6") Registration: GS, CE, cUL Power Supply: 12 V DC, 1 A, 230 V/110 V, autodetecting 03/06 B 45-0019 e 7-15 ANNEX AxioCam High Resolution Digital Cameras Carl Zeiss 7.8.4 LSM 5 LIVE LSM 5 LIVE DuoScan Microscope Camera Port Adapters for the AxioCam Adapter Video V200 C 2/3" 0.63x at frontport Axiovert 200 M Cat. No 000000-1071-171 This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the Axiovert 200 M Adapter Video 60 C 2/3" 0.63x Cat. No 000000-1069-414 This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the Axio Imager.Z1 and Axioskop 2 FS MOT. For an additional documentation port to be connected to the camera port of the tubes with interface 60: Double video adapter Cat. No 000000-1058-640 For connection to interface 60, 2 switching positions for switching to 100% mirror or to interface for P&C modules. Adapter Video 44 C 2/3" 0.63x Cat. No 452997-0000-000 This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the Axiovert 200 M SP. No other cameras are supported by the LSM Software! 7-16 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan 7.9 ANNEX List of Key Words List of Key Words Numbers 2.5 D .......................................................4-266 3D ...........................................................4-268 3D for LSM Terms and definitions...............................6-2 3D view ......................................... 4-15, 4-152 A About LSM 5 LIVE....................................4-223 Achrogate .................................................4-55 Acquire............................................ 4-15, 4-37 Add image sequences................................6-17 Alpha rendering method............................6-46 And calculation..........................................6-37 Annex..........................................................7-2 Ampl. gain...............................................4-319 Ampl. offset ............................................4-321 Analysis of images ...................................4-224 Animation ...............................................4-239 Area ........................................................4-254 Automatic object measurement .................6-52 Average.....................................................4-73 Axio Imager control ...................................4-41 Axioskop 2 FS control ................................4-50 Axiovert control .........................................4-46 B Beam path............................. 4-55, 4-57, 4-59, .......................................... 4-60, 4-316, 4-322 Bleach .....................................................4-110 Bleach parameter.....................................4-112 Bleach regions .........................................4-112 C Calculation And .......................................................6-37 Or ..........................................................6-37 Xor ........................................................6-38 Camera color adjustment.........................4-220 Camera detection ......................................4-66 Carl Zeiss vision image sequences ..............6-12 03/06 Carl Zeiss Channel......................................... 4-75, 4-226 Add, remove .................................. 6-5, 6-15 Delete ................................................... 6-16 Channel assignment ..4-55, 4-60, 4-316, 4-322 Channel mode configuration .................... 4-62 Closing ..................................................... 6-32 Collimator adjustment ............................ 4-218 Color palette .......................................... 4-237 Configuration control ............................... 4-51 Contrast ...................................... 4-132, 4-234 Change contrast ................................... 6-19 Enhance................................................ 6-23 Copy ............................................. 4-25, 4-243 Content of display window ................... 6-14 Copy full resolution ................................ 4-146 Copy window contents........................... 4-145 Crop....................................................... 4-242 Cut......................................................... 4-250 D Database .................................................. 4-18 Delete function ..................................... 4-28 Export of images ................................... 4-33 Filter function........................................ 4-26 Form mode ........................................... 4-21 Gallery mode ........................................ 4-23 Import of images................................... 4-32 Load function............................... 4-24, 4-25 Multi print............................................. 4-34 New...................................................... 4-18 Open .................................................... 4-19 Saving an image.................................... 4-29 Table mode........................................... 4-24 Deconvolution ........................................ 4-163 Delete Images .................................................. 6-16 Object in specified size range ................ 6-41 Depth coding.......................................... 4-152 Detaching the Scanning Module ................. 7-5 Detector gain............................... 4-319, 4-320 Dialog boxes............................................... 6-4 Dichroic beam splitters.............................. 4-56 Dilation..........................6-28, 6-30, 6-31, 6-32 Display of images.................................... 4-224 Display window ................................... 6-4, 6-7 Dyes ........................................................... 7-2 B 45-0019 e 7-17 ANNEX LSM 5 LIVE LSM 5 LIVE DuoScan Carl Zeiss E Edit menu....................................................6-5 Emission filter .................................... 4-55, 7-2 Erosion .................6-27, 6-28, 6-29, 6-31, 6-32 Excitation ..................................................4-59 Excitation laser lines.....................................7-2 Excitation of bleach track.........................4-113 Exiting the LSM program .........................4-325 Exposure time....................... 4-75, 4-89, 4-317 Render-Surface ......................................6-43 Save Display As ......................................6-13 Save Image As........................................6-12 Scrap .....................................................6-41 Segment-Automatic ...............................6-35 Segment-Interactive ...............................6-33 Set Channel Colour................................6-10 Smooth (Gauss)......................................6-24 Volume Features ....................................6-55 Full screen ...............................................4-221 G F File .................................................. 4-15, 4-17 File menu ....................................................6-5 Fills holes...................................................6-42 Filter.............................................. 4-57, 4-130 Find............................................. 4-318, 4-322 Frame .............................................. 4-68, 4-71 creation of a ..........................................4-78 FRAP Guide .............................................4-223 Functions Add Channel .........................................6-15 Arithmetics-Add.....................................6-17 Arithmetics-Subtract ..............................6-18 Automatic Object Measurement, General6-58 Boolean-And..........................................6-37 Boolean-Mask........................................6-40 Boolean-Not ..........................................6-39 Boolean-Or ............................................6-37 Boolean-Xor...........................................6-38 Condition ..............................................6-57 Contrast-Automatic ...............................6-21 Contrast-Interactive ...............................6-19 Contrast-Linearize ..................................6-23 Copy......................................................6-14 Delete All Images ...................................6-16 Delete Channel ......................................6-16 Edit Channels.........................................6-15 Exit ........................................................6-14 Fill Holes ................................................6-42 Morphology...........................................6-25 Morphology-Close .................................6-32 Morphology-Dilate .................................6-30 Morphology-Erode .................................6-29 Morphology-Open .................................6-31 Object Features ......................................6-52 Open Image ...........................................6-11 Print.......................................................6-14 Render-Alpha.........................................6-46 7-18 Gallery...................................... 4-251, 6-3, 6-7 Gauss filter ................................................6-24 Gradient shading method ..........................6-43 Grey value segmentation ................. 6-33, 6-35 H Help ........................................................4-223 Help menu...................................................6-6 Histogram................................................4-253 Holes to fill ................................................6-42 Hyperfine Z sectioning ...............................4-84 I Image Delete function ......................................4-28 Display and analysis..............................4-224 Export of an ...........................................4-33 Import of an...........................................4-32 Information..........................................4-311 Load an..................................................4-19 Saving an ...............................................4-29 Image acquisition.......................................4-37 Image optimization...................... 4-315, 4-323 Image sequence...........................................6-2 Channels..................................................6-2 Information of the image.........................4-311 Interpolation............................................4-134 Ion concentration ....................................4-141 K Kinetic .....................................................4-146 L Laser attenuation.......................................4-59 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX List of Key Words Laser control..............................................4-38 Line ...........................................................4-68 Creation of a line ...................................4-87 Lowpass filter ..........................................4-130 LSM 5 LIVE switchboard...........................4-325 M Macro................................. 4-16, 4-167, 4-215 editing and debugging.........................4-172 Main components of the system ..................6-3 Main menu................................................4-15 Main window ..............................................6-3 Maintain........................................ 4-16, 4-210 Admin..................................................4-220 Collimator adjustment..........................4-218 Objectives ............................................4-212 Pinhole adjustment ..............................4-215 Reboot.................................................4-220 Test grid...............................................4-220 Mathematical morphology.........................6-25 Mark first/last ............................................4-83 Marker ........................................ 4-100, 4-103 Mean........................................... 4-265, 4-312 Mean ROI ................................................4-106 Measurement concept ...............................6-50 Measurement menu ....................................6-6 Measurement process................................6-51 Median filter............................................4-131 Microscope control ....................................4-40 Mode ........................................................4-71 monochrome image display .....................4-227 Multi track.................................................4-51 Multiple-channel......................................4-322 multitracking ................................... 4-60, 4-61 N Negation of image.....................................6-39 NFT............................................................4-55 Non Descanned .........................................4-67 Opening ................................................... 6-31 Options ......................................... 4-16, 4-198 Dye database ...................................... 4-199 Hardware............................................ 4-329 Settings............................................... 4-200 Software ............................................. 4-327 Or calculation ........................................... 6-37 Orthogonal sections................................ 4-246 Overlay ................................................... 4-230 P Palette .............................. 4-237, 4-319, 4-321 Parfocality............................................... 4-214 Paste ........................................................ 4-25 Paste bitmap........................................... 4-146 Piezo fine focusing stage ............................ 7-7 Piezo objective focussing device.................. 7-9 Pinhole ................................................... 4-319 Pinhole adjustment ................................. 4-215 Pixel depth.............................................. 4-321 Player ......................................................... 6-8 control elements ..................................... 6-9 Preview print .......................................... 4-309 Print .............................................. 4-34, 4-309 Content of display window ................... 6-14 Process .......................................... 4-15, 4-121 add ..................................................... 4-121 channel shift ....................................... 4-135 contrast .............................................. 4-132 copy.................................................... 4-127 duplication.......................................... 4-128 filter.................................................... 4-129 interpolate .......................................... 4-133 multiply............................................... 4-125 ratio.................................................... 4-126 subtract .............................................. 4-124 Process menu ............................................. 6-5 Profile..................................................... 4-261 Projection ............................................... 4-155 Properties ................................................... 6-3 R O Object features Densitometric.........................................6-55 Geometric..............................................6-54 Objective .................................................4-212 03/06 Carl Zeiss Range....................................................... 4-82 Range indicator ........................... 4-319, 4-321 Ratio....................................................... 4-126 Ratio settings................................... 4-64, 4-65 B 45-0019 e 7-19 ANNEX LSM 5 LIVE LSM 5 LIVE DuoScan Carl Zeiss Recording configuration .................. 4-51, 4-60 Refresh......................................................4-25 Region of interest Edit........................................................4-90 Reuse ......................................................4-241 ROI Edit............................................ 4-90, 4-112 Running down the operating system .......4-326 S Save ........................................................4-243 As........................................................4-244 Content of display window ....................6-13 Image .......................................... 4-29, 6-12 Image sequence .....................................6-12 Scaling image ..........................................4-145 Scan average ...........................................4-321 Scan control ..............................................4-68 Scan speed .................................... 4-71, 4-321 Scatter diagram .......................................4-257 Segmentation Automatic..............................................6-35 Interactive ..............................................6-33 Select Object in specified size range .................6-41 Settings ...................................................4-200 Sharpness filter........................................4-130 Shut-down procedure..............................4-325 Single channel .........................................4-315 Single Track...............................................4-51 single tracking ...........................................4-61 Single tracking...........................................4-55 Slice ................................................ 4-229, 6-2 Split xy ............................... 4-78, 4-245, 4-323 Stage.......................................................4-114 Starting the LSM 5 program ......................4-13 Stereo .....................................................4-159 Structures Dilate.....................................................6-30 Erode.....................................................6-29 Subset .....................................................4-251 Subtract image sequences .........................6-18 Switching on the enterprise UV laser .........4-38 Switching on the system ............................4-12 7-20 T Tile scan ..................................................4-118 Time delay .................................................4-98 Time interval..............................................4-98 Time series.................................................4-94 Of a frame ...........................................4-102 Of a Z stack..........................................4-104 With mean ROI ....................................4-106 Toolbar....................................... 4-15, 6-3, 6-6 Topography .............................................4-278 3D measurement functions ..................4-304 Data processing ....................... 4-279, 4-281 Display modes.......................... 4-284, 4-287 Measurement functions .......................4-294 Track .........................................................4-51 Track configuration.......................... 4-51, 4-53 Tracks panel, list of ....................................4-61 Trigger-interface LSM 5 LIVE ......................7-10 Turning power off ...................................4-326 U Unmix......................................................4-137 User-defined filter....................................4-131 V Value Enter new value .....................................6-20 Mask grey value .....................................6-40 Scale a range of grey values ...................6-23 Scale grey value .....................................6-21 Vertical scale of histogram .........................6-20 View menu ..................................................6-6 Visual macro editor...................... 4-177, 4-189 Volume features Densitometric.........................................6-56 Geometric..............................................6-56 W Window ..................................................4-221 full screen ............................................4-221 Windows menu ...........................................6-6 X Xor calculation...........................................6-38 Xy display ................................................4-244 B 45-0019 e 03/06 LSM 5 LIVE LSM 5 LIVE DuoScan ANNEX List of Key Words Carl Zeiss Z Zeiss TIFF image sequence..........................6-11 Z sectioning ...............................................4-81 Z settings.................................................. 4-79 Z stack.......................................................4-79 creation of a ..........................................4-86 Zoom.......................................................4-228 03/06 B 45-0019 e 7-21 LSM 5 LIVE LSM 5 LIVE DuoScan Laser Scanning Microscope Brief Operating Manual Release 4.0 March 2006 Contents Page Starting the System ................................................................................................................3 Setting the microscope...........................................................................................................6 Configuring the beam path and lasers..................................................................................8 Scanning an image ...............................................................................................................11 Storing an image ..................................................................................................................15 Switching off the system .....................................................................................................15 Introduction For your safety! Observe the following instructions: 2 − The LSM 5 LIVE / LSM 5 LIVE DuoScan laser scanning microscope, including its original accessories and compatible accessories from other manufacturers, may only be used for the purposes and microscopy techniques described in this manual (intended use). − In the Operating Manual, read the chapter Safety Instructions carefully before starting operation. − Follow the safety instructions described in the operating manual of the microscope and HBO 100 mercury lamp. 03/06 Starting the System Switching on the LSM system • Set the MAIN SWITCH on the system rack to ON position (Fig. 1). • When set to ON the System/PC switch provides power to the microscope and the computer. This allows to use the microscope and the computer without running the LSM Software (Fig. 1). • To switch on the system completely put the Components switch to ON. Insert the key into the key-operated LASER switch and switch also to ON. Now the complete system is ready to be initialized with the LSM Software. Fig. 1 System rack Fig. 2 HBO 100 power supply Switching on the HBO 100 mercury lamp • Switch on the HBO 100 mercury lamp via the switch of the power supply, see operating manual of the mercury lamp or microscope. 03/06 3 Starting the LSM 5 software program LSM 5 Live • Double click the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5 software program. The LSM 5 LIVE Switchboard window appears on the screen (Fig. 3). Fig. 3 LSM 5 LIVE Switchboard menu • Click on the Scan New Images button in the LSM 5 LIVE Switchboard window. Clicking on this button activates the complete LSM hardware (on-line mode). • Click on the Start button in the LSM 5 LIVE Switchboard window. The LSM 5 LIVE - Main menu appears on the screen. Use of this mode requires to be thoroughly familiar with the exact microscope procedures and interrelations. Main menu (pull-down) Main menu toolbar Subordinate toolbar Fig. 4 4 Main menu 03/06 Creating a database for acquired images • Click on the File button in the Main menu toolbar. The File subordinate toolbar appears in the Main menu. Fig. 5 Main menu with File subordinate toolbar • Click on the New button in the File subordinate toolbar. The Create New Database window appears. • Select drive C: or D: from pull down menu. • Create a new directory if needed. Fig. 6 Fig. 7 Create New Database window Create New Database window Turning on the lasers • Click on the Acquire button in the Main menu to open the Acquire subordinate toolbar. Fig. 8 03/06 Acquire subordinate toolbar 5 • Click on the Laser button to open the Laser Control window. • Select the appropriate Laser Unit by clicking on the name of it. • Click on the On button to switch required laser(s) to on (or standby if required). Fig. 9 Laser Control window Setting the microscope Changing between direct observation or laser scanning The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in the binocular tube of the microscope: • Click on the VIS button to set the microscope for direct observation via the eyepieces of the binocular tube, lasers are off. • Click on the TV button to set the microscope camera observation (if connected) via camera adapter of the binocular tube. • Click on the LSM button to set the microscope screen observation via laser excitation using the LSM 5 LIVE and software evaluation. Setting the microscope and storing the settings • Click on the VIS button for direct observation. • Click on the Micro button in the Acquire subordinate toolbar to open the Microscope Control window of the used microscope. The Microscope Control window appears (Fig. 10). 6 03/06 Selecting an objective • Open the graphical pop-up menu by clicking on the Objective button (Fig. 10). • Click on the objective you want to select. The selected objective will automatically move into the beam path. Focussing the microscope for transmitted light • Open the graphical pop-up menu by clicking on the Transmitted Light button (Fig. 11). • Click on the On button. Set the intensity of the Halogen illuminator using the slider. • Click on Close to close the pop-up menu. • Place specimen on microscope stage. The cover slip must be facing up. • Use the focusing drive of the microscope to focus the required object plane. • Select specimen detail by moving the stage in X and Y using the XY stage fine motion control. Fig. 10 Microscope Control window, e.g.: Axiovert 200 M Fig. 11 Microscope Control window with Transmitted Light pop-up menu Setting the microscope for reflected light • Click on the Reflected Light button to open the shutter of the HBO 100 mercury lamp. • Click on the Reflector button and select the desired filter set by clicking on it. Storing the microscope settings Microscope settings can be stored and up to 8 buttons assigned for fast retrieval and adjustment using the Microscope Settings panel. The Store button permits existing microscope configurations to be stored under any name. The Apply button permits existing microscope configurations to be loaded. stored The Delete button permits existing microscope configurations to be deleted. The Assign button permits the assignment of a microscope configuration to a button. Note: Depending on the microscope configuration, settings must be done manually if necessary. 03/06 7 Configuring the beam path and lasers • Click on the LSM button in the Acquire subordinate toolbar for laser scanning. Choosing the configuration Single Track − Use for single, double and triple labeling; simultaneous scanning only − Advantage: faster image acquisition − Disadvantage: cross talk between channels Multi Track − Use for double and triple labeling; sequential scanning, line by line or frame by frame − Advantage: when one track is active, only one detector and one laser is switched on. This reduces cross talk. − Disadvantage: slower image acquisition • Click on the Config button in the Acquire subordinate toolbar to open the Configuration Control window. Fig. 12 Configuration Control window for Single Track The Configuration Control window appears (Fig. 12). Setting for single track configuration in Channel Mode • Select Channel Mode if necessary (Fig. 12). • Click on the Single Track button in the Configuration Control window (Fig. 12). The Beam Path and Channel Assignment panel of the Configuration Control window displays the selected track configuration which is used for the scan procedure. • You can change the settings of this panel using the following function elements: Activation / deactivation of the excitation wavelengths (check box) and setting of excitation intensities (slider). Open the Laser Control window via the Laser button. Selection of the secondary dichroic beam splitter (NFT) position through selection from the relevant list box. Selection of an emission filter through selection from the relevant list box. Activation / deactivation (via check box) of the selected channel (ChL 1, ChL 2) for the scanning procedure by assigning an existing color icon or defining a new one. 8 03/06 • Select the appropriate filters and activate the channels. • Click the Excitation button to select the laser lines and set the attenuation values (transmission in %) in the displayed window. For the configuration of the beam path, please refer to the application-specific configurations depending on the used dyes and markers and the existing instrument configuration. • Clicking on the Config button opens the Track Configurations window (Fig. 13) to load, store or delete track configurations. • For storing a new track configuration enter a desired name in the first line of the Configurations list box and click an Store. • For loading an existing configuration select it in the list box and click on Apply. Fig. 13 Track Configurations window • For deleting an existing configuration select it in the list box and click on Delete. Setting for multi track configuration in Channel Mode The Multi Track function permit several tracks to be defined as one configuration (Channel Mode Configuration) for the scan procedure, to be stored under any name, reloaded or deleted. The maximum of four tracks with up to 8 channels can be defined simultaneously and then scanned one after the other. Each track is a separate unit and can be configured independently of the other tracks with regard to channels, Acousto-Optical Tunable Filters (AOTF), emission filters and dichroic beam splitters. • Select Channel Mode if necessary (Fig. 14). Fig. 14 03/06 Configuration Control window for Multi Track 9 • Click on the Multi Track button in the Configuration Control window (Fig. 14). The following functions are available in the List of Tracks panel (Fig. 14). Add Track button An additional track is added to the configuration list. The maximum of four tracks can be added. One track each with basic configuration is added, i.e.: one ChL 1 channel is activated, all laser lines are switched off, emission filters and dichroic beam splitters are set in accordance with the configuration last used. Remove button The track previously marked in the List of Tracks panel in the Name column is deleted. Store/Apply Single Track button Opens the Track Configurations window. A selected track defined in a Recording Configuration can also be stored as a single track for single tracking applications. Also, it's possible to load a single track in a multitracking configuration A click on this arrow button will move the selected track (highlighted in blue) one position upwards in the list box. A click on this arrow button will move the selected track (highlighted in blue) one position downwards in the list box. • Configure each desired track for Multi Track function as described for Single Track. • For storing/applying or deleting a Channel Mode Configuration use the Config button. 10 03/06 Scanning an image • Click on the Scan button in the Acquire subordinate toolbar to open the Scan Control window. The Scan Control window appears (Fig. 15). Setting the parameters for scanning • Select Mode in the Scan Control window. • Select the Frame Size as predefined number of pixels or enter your own values (e.g. 300 x 600) in the Objective Lens, Image Size & Line Step Factor panel. The number of pixels influences the image resolution! Note: When using an Axioskop 2 FS MOT, indicate the objective that is in use in the Scan Control window. This ensures correct calculation of pinhole, Z stack optimization etc. Adjusting the exposure time • Use the Exposure Time slider in the Objective Lens, Image Size & Line Step Factor panel (Fig. 15) to adjust the exposure time. Fig. 15 Scan Control window, Mode settings Below the slider is shown the number Frames per Second. Start with 2 to 4 frames per second. Choosing the dynamic range • Select the dynamic range 8 or 12 Bit (per pixel) in the Pixel Depth, Scan Direction & Scan Average panel (Fig. 15). 8 Bit will give 256 gray levels, 12 Bit will give 4096 levels. Publication quality images should be acquired using 12 Bit data depth. 12 Bit is also recommended when doing quantitative measurements or when imaging low fluorescence intensities. Setting the scan averaging Averaging improves the image by increasing the signal : noise ratio. It can be achieved line by line or frame by frame. Frame averaging helps reduce photobleaching, but does not give quite such a smooth image. • Select the Line or Frame mode for averaging. • Select the desired scan average method Mean or Sum in the Method selection box. If you are using the Mean method, the image information is generated by adding up all scans pixel by pixel and then calculating the mean value. 03/06 11 In the Sum method, the pixel values of all scans are only added up, without a mean value being calculated. • Select the Number for averaging. Continuous averaging is possible in the Frame mode. In this case a Finish button for ending continuous averaging is displayed instead of the Cont. button. Adjusting the pinhole • Select Channels in the Scan Control window. • Set the Pinhole size to 1 (Airy unit) for best compromise between depth discrimination and efficiency. Pinhole adjustment changes the Optical Slice. When collecting multi channel images, adjust the pinholes so that each channel has the same Optical Size. This is important for colocalization studies. Image acquisition Fig. 16 Scan Control window, Channel settings Once you have set up your parameter as defined in the above section, you can acquire a frame image of your specimen. • Use one of the Find, Fast XY, Single or Cont. buttons to start the scanning procedure and acquire an image. Scanned images are shown in separate windows. • Click on the Stop button to stop the current scan procedure if necessary. Select Find for automatically preadjustment of detector sensitivity. Select Fast for continuous fast scanning – useful for finding and changing the focus. Select Single for recording a single image. Fig. 17 Image window Select Cont. for continuous scanning with the selected scan speed. Select Stop for stopping the current scan procedure. 12 03/06 Image optimization Choosing a lookup table • Select Palette in the Image window of the scanned image (Fig. 18). The Color Palette window appears. • In the Color Palette List panel, click on the Range Indicator item (Fig. 19). The scanned image appears in a false-color presentation (Fig. 18). If the image is too bright, it appears red on the screen. Red = saturation (maximum). If the image is not bright enough, it appears blue on the screen. Blue = zero (minimum). Fig. 18 Image window Fig. 19 Color Palette window Fig. 20 Scan Control window, Channel settings Adjusting the laser intensity • Set the Pinhole to 1 Airy Unit (Fig. 20). • Set the Detector Gain slider to a middle position. • When the image is saturated, reduce AOTF transmission in the Excitation panel (Fig. 20) using the slider to reduce the intensity of the laser light at the specimen. Adjusting gain and offset • Increase the Amplifier Offset until all blue pixels disappear, and then make it slightly positive (Fig. 20). • Reduce or increase the Detector Gain until the red pixels only just disappear. 03/06 13 Scanning a Z stack • Select Z Stack in the Scan Control window. • Select Frame if necessary. The Z Settings panel appears. • Select Mark First/Last on the Z Settings panel. • Click on the XY cont button. A continuous XY-scan of the set focus position will be performed. • Use the focusing drive of the microscope to focus on the upper position of the specimen area where the Z Stack is to start. • Click on the Mark First button to set the upper position of the Z Stack. • Then focus on the lower specimen area where the recording of the Z Stack is to end. • Click on the Mark Last button to set this lower position. • Click on X:Y:Z=1:1:1 button to set the Zinterval in such a way that the voxel has identical dimensions in the X-, Y- and Zdirections (cube). Fig. 21 14 Scan Control window, Z Stack settings • Click on the Start button to start the recording of the Z Stack. 03/06 Storing an image • Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the Main menu. The Save Image and Parameter As window appears. Fig. 22 Save Image and Parameter As window • Enter file name, description and notes in the appropriate text boxes. • Click on the OK button. Switching off the system • Click on the File button in the Main menu and then click on the Exit button to leave LSM 5 software program (Fig. 5). • Shut down the computer. • Put the LASER key switch to OFF position. Remove the key from the system rack. • Put the COMPONENTS switch and the System/PC switch to OFF position (Fig. 1). • Put the MAIN SWITCH on the system rack to OFF position (Fig. 1). • Switch off the HBO 100 mercury lamp (Fig. 2). 03/06 15
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