Slidebook user manual

Slidebook user manual
 SlideBook™ 5.0
Version 5.0.0.1 for Windows XP User Manual
Olympus America Inc. Reseller Edition Latest Version 4/1/09 End User License Agreement
This software interfaces with LibTiff version 3.6.1.1
Copyright (c) 1988-1997 Sam Leffler
Copyright (c) 1991-1997 Silicon Graphics, Inc.
Permission to use, copy, modify, distribute, and sell this software and
its documentation for any purpose is hereby granted without fee, provided that (i) the above
copyright notices and this permission notice appear in all copies of the software and related
documentation, and (ii) the names of Sam Leffler and Silicon Graphics may not be used in
any advertising or publicity relating to the software without the specific, prior written
permission of Sam Leffler and Silicon Graphics.
THE SOFTWARE IS PROVIDED "AS-IS" AND WITHOUT WARRANTY OF ANY KIND,
EXPRESS, IMPLIED OR OTHERWISE, INCLUDING WITHOUT LIMITATION, ANY
WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.
IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FOR ANY
SPECIAL, INCIDENTAL, INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND,
OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE, DATA OR
PROFITS, WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE, AND ON
ANY THEORY OF LIABILITY, ARISING OUT OF OR IN CONNECTION WITH THE USE
OR PERFORMANCE OF THIS SOFTWARE.
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SlideBook 5.0 User Manual
End-User License Agreement for 3i Software
IMPORTANT - READ CAREFULLY: This 3I End-User License Agreement (“EULA”) is a
legal agreement between you (either an individual or a single entity) and Intelligent
Imaging Innovations, Inc. (“3I”) for the 3I software product identified in the software
section of the system registration sheet, which includes computer software and a hardware
component (i.e., the “hardware key”) and may include associated media, printed materials,
and “online” or electronic documentation (“SOFTWARE PRODUCT”). The SOFTWARE
PRODUCT also includes any updates and supplements to the original SOFTWARE
PRODUCT provided to you by 3I. Any software provided along with the SOFTWARE
PRODUCT that is associated with a separate end-user license agreement is licensed to you
under the terms of that license agreement. By installing, copying, downloading, accessing
or otherwise using the SOFTWARE PRODUCT, you agree to be bound by the terms of this
EULA.
If you do not agree to the terms of this EULA, do not install or use the SOFTWARE
PRODUCT; you may, however, return it to your place of purchase for a full refund.
SOFTWARE PRODUCT LICENSE
The SOFTWARE PRODUCT is protected by copyright laws and international copyright treaties, as well as other intellectual property
laws and treaties. The SOFTWARE PRODUCT is licensed, not sold.
1. GRANT OF LICENSE. This EULA grants you the following rights:
 Applications Software. You may install, use, access, display, run, or otherwise interact with (“RUN”) one copy of the
SOFTWARE PRODUCT, or any prior version for the same operating system, on a single computer, workstation, terminal or other
digital electronic device (“COMPUTER”).
 Storage/Network Use. You may also store or install a copy of the SOFTWARE PRODUCT on a storage device, such as a
network server, used only to store the SOFTWARE PRODUCT. A license for the SOFTWARE PRODUCT may not be shared or used
concurrently on different COMPUTERS.
 Reservation of Rights. The SOFTWARE PRODUCT is licensed to You on a non-exclusive basis. All rights not expressly granted
are reserved by 3I.
2. DESCRIPTION OF OTHER RIGHTS AND LIMITATIONS.
 Not for Resale Software. If the SOFTWARE PRODUCT is labeled “Not For Resale” or “NFR,” then, notwithstanding other
sections of this EULA, your use of the SOFTWARE PRODUCT is limited to use for demonstration, test, or evaluation purposes and
you may not resell, or otherwise transfer for value, the SOFTWARE PRODUCT.
 Limitations on Reverse Engineering, Decompilation, and Disassembly. You may not reverse engineer, decompile, modify
or disassemble the SOFTWARE PRODUCT, except and only to the extent that such activity is expressly permitted by applicable law
notwithstanding this limitation.
 Separation of Components. The SOFTWARE PRODUCT is licensed as a single product. Its component parts may not be
separated for use on more than one COMPUTER.
 Trademarks. This EULA does not grant you any rights in connection with any trademarks or service marks of 3I.
 Rental. You may not rent, lease, or lend the SOFTWARE PRODUCT.
 Support Services. 3I may provide you with support services related to the SOFTWARE PRODUCT (“Support Services”). Use of
Support Services is governed by the 3I policies and programs described in the user manual, in “online” documentation, and/or in
other 3I-provided materials. Any supplemental software code provided to you as part of the Support Services shall be considered
part of the SOFTWARE PRODUCT and subject to the terms and conditions of this EULA. With respect to technical information you
provide to 3I as part of the Support Services, 3I may use such information for its business purposes, including for product support
and development. 3I will not utilize such technical information in a form that personally identifies you.
 Software Transfer. The initial licensee of the SOFTWARE PRODUCT may make a one-time permanent transfer of this EULA and
SOFTWARE PRODUCT only directly to an end user. This transfer must include all of the SOFTWARE PRODUCT (including all
component parts, the media and printed materials, any upgrades, this EULA). Such transfer may not be by way of consignment or
any other indirect transfer. The transferee of such one-time transfer must agree to comply with the terms of this EULA, including
the obligation not to further transfer this EULA and SOFTWARE PRODUCT.
 Termination. Without prejudice to any other rights, 3I may terminate this EULA if you fail to comply with the terms and
conditions of this EULA. In such event, you must destroy all copies of the SOFTWARE PRODUCT and all of its component parts.
3. UPGRADES. If the SOFTWARE PRODUCT is labeled as an upgrade, you must be properly licensed to use a product identified by
3I as being eligible for the upgrade in order to use the SOFTWARE PRODUCT. A SOFTWARE PRODUCT labeled as an upgrade
replaces and/or supplements (and may disable) the product that formed the basis for your eligibility for the upgrade. You may use
the resulting upgraded product only in accordance with the terms of this EULA. If the SOFTWARE PRODUCT is an upgrade of a
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End User License Agreement
component of a package of software programs that you licensed as a single product, the SOFTWARE PRODUCT may be used and
transferred only as part of that single product package and may not be separated for use on more than one COMPUTER.
4. PROPRIETARY RIGHTS. All proprietary rights in and to the SOFTWARE PRODUCT (including but not limited to patent,
copyright, trade secret, trademark, or other proprietary rights, any images, photographs, animations, video, audio, music, and text
incorporated into the SOFTWARE PRODUCT), the accompanying printed materials, and any copies of the SOFTWARE PRODUCT are
owned by 3I or its suppliers. All title and intellectual property rights in and to the content which may be accessed through use of
the SOFTWARE PRODUCT is the property of the respective content owner and may be protected by applicable copyright or other
intellectual property laws and treaties. This EULA grants you no rights to use such content. If this SOFTWARE PRODUCT contains
documentation which is provided only in electronic form, you may print one copy of such electronic documentation. You may not
copy the printed materials accompanying the SOFTWARE PRODUCT.
5. CONFIDENTIAL INFORMATION. You agree that the SOFTWARE PRODUCT contains proprietary information, including
trade secrets, know-how and confidential information, that is the exclusive property of 3I. During the period this Agreement is in
effect and at all times after its termination, you and any of your employees, agents, partners, associates, etc. shall maintain the
confidentiality of this information and not sell, license, publish, display, distribute, disclose or otherwise make available this
information to any third party nor use such proprietary information concerning the SOFTWARE PRODUCT, including flow charts,
logic diagrams, user manuals and screens, to any person(s) not an employee without the prior written consent of 3I. In addition,
You hereby agree to protect 3I’s proprietary information and take appropriate action against employees, independent contractors or
others who violate any of 3I’s proprietary rights.
6. DUAL-MEDIA SOFTWARE. You may receive the SOFTWARE PRODUCT in more than one medium. Regardless of the type or
size of medium you receive, you may use only one medium that is appropriate for your single COMPUTER. You may not RUN the
other medium on another COMPUTER. You may not loan, rent, lease, or otherwise transfer the other medium to another user,
except as part of the permanent transfer (as provided above) of the SOFTWARE PRODUCT.
7. BACKUP COPY. After installation of one copy of the SOFTWARE PRODUCT pursuant to this EULA, you may keep the original
media on which the SOFTWARE PRODUCT was provided by 3I solely for backup or archival purposes. If the original media is
required to use the SOFTWARE PRODUCT on the COMPUTER, you may make one copy of the SOFTWARE PRODUCT solely for
backup or archival purposes. Except as expressly provided in this EULA, you may not otherwise make copies of the SOFTWARE
PRODUCT or the printed material accompanying the SOFTWARE PRODUCT.
8. U.S. GOVERNMENT RESTRICTED RIGHTS. All SOFTWARE PRODUCT provided to the U.S. Government pursuant to
solicitations issued on or after December 1, 1995 is provided with the commercial rights and restrictions described elsewhere herein.
All SOFTWARE PRODUCT provided to the U.S. Government pursuant to solicitations issued prior to December 1, 1995 is provided
with RESTRICTED RIGHTS as provided for in FAR, 48 CFR 52.227-14 (JUNE 1987) or FAR, 48 CFR 252.227-7013 (OCT 1988), as
applicable.
9. EXPORT RESTRICTIONS. This SOFTWARE PRODUCT has been classified by the US Government as exportable under License
Exception TSU. Therefore the following terms apply: You agree that you will not export or re-export the SOFTWARE PRODUCT,
any part thereof, or any process or service that is the direct product of the SOFTWARE PRODUCT (the foregoing collectively
referred to as the “Restricted Components”), to any country, person or entity subject to U.S. export restrictions. You specifically
agree not to export or re-export any of the Restricted Components (i) to any country to which the U.S. has embargoed or restricted
the export of goods or services, which currently include, but are not necessarily limited to Cuba, Iran, Iraq, Libya, North Korea,
Sudan and Syria, or to any national of any such country, wherever located, who intends to transmit or transport the Restricted
Components back to such country; (ii) to any person or entity who you know or have reason to know will utilize the Restricted
Components in the design, development or production of nuclear, chemical or biological weapons; or (iii) to any person or entity
who has been prohibited from participating in U.S. export transactions by any federal agency of the U.S. government. You warrant
and represent that neither the BXA nor any other U.S. federal agency has suspended, revoked or denied your export privileges.
10. INDEMNIFICATION. You hereby agree to indemnify 3I and hold 3I harmless from and against, and shall defend against, any
and all claims and damages of every kind, including but not limited to fines, penalties, compensatory damages, consequential
damages, punitive damages, attorneys fees, or any other damages or fees arising out of or attributed directly or indirectly to your
negligent, reckless or intentional conduct, operations or performance of the SOFTWARE PRODUCT.
MISCELLANEOUS
If you acquired this SOFTWARE PRODUCT in the United States, this EULA is governed by the laws of the State of California.
If you acquired this SOFTWARE PRODUCT in Canada, unless expressly prohibited by local law, this EULA is governed by the laws in
force in the Province of Ontario, Canada; and, in respect of any dispute which may arise hereunder, you consent to the jurisdiction
of the federal and provincial courts sitting in Toronto, Ontario. If this SOFTWARE PRODUCT was acquired outside the United
States, then local law may apply.
Should you have any questions concerning this EULA, or if you desire to contact 3I for any reason, please contact 3I at:
Intelligent Imaging Innovations, Inc.
5124 Washington Street
Denver, CO 80216
http://www.intelligent-imaging.com
(303) 607-9429
(303) 607-9430 Facsimile
AUTHORITY TO BIND COMPANY/INSTITUTION: The individual signing this
Agreement hereby warrants, represents and covenant that they have the right,
power and capacity and are and will be duly authorized and empowered to enter
into, execute, deliver and perform this Agreement on behalf of the company,
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SlideBook 5.0 User Manual
organization, institution or other legal entity and that the execution, delivery
and/or performance by of this Agreement shall not, and will not, by the lapse of
time, the giving of notice or otherwise, constitute a violation of any applicable
law or a breach of any provision contained in the Articles of Incorporation, ByLaws, Articles of Partnership or similar document, or contained in any
agreement, instrument or document to which You are now or hereafter a party or
by which you may become bound.
SEVERABILITY: If any provision of this Agreement is held to be invalid or unenforceable, in whole or in part, such holding will not
affect the validity of the other provisions of this Agreement, unless the unenforceable provision is essential to this Agreement.
FAIR MEANING: The language in all parts of this Agreement shall in all cases be construed according to its fair meaning and not
strictly for or against either party. It is agreed that if any provision of this Agreement is capable of two constructions, one of which
would render the provision void and the other of which would render the provision valid, then the provision shall have the meaning
which renders it valid.
LIMITED WARRANTY
LIMITED WARRANTY FOR SOFTWARE PRODUCTS ACQUIRED OUTSIDE THE US AND CANADA. YOU HEREBY AGREE
THAT THE UNIFORM COMMERCIAL CODE (“UCC”) OF CALIFORNIA AND THIS AGREEMENT SHALL CONTROL ALL WARRANTIES
UNDER THIS LICENSE AGREEMENT AND EXPRESSLY WAIVE ANY RIGHTS YOU MAY HAVE UNDER INTERNATIONAL LAW OR
TREATY INCLUDING BUT NOT LIMITED TO THE INTERNATIONAL CONVENTION ON THE SALE OF GOODS.
LIMITED WARRANTY FOR SOFTWARE PRODUCTS ACQUIRED IN THE US AND CANADA. 3I warrants that (a) the
SOFTWARE PRODUCT will perform substantially in accordance with the accompanying written materials for a period of ninety (90)
days from the date of receipt, and (b) any Support Services provided by 3I shall be substantially as described in applicable written
materials provided to you by 3I, and 3I support engineers will make commercially reasonable efforts to solve any problem issues.
Some states and jurisdictions do not allow limitations on duration of an implied warranty, so the above limitation may not apply to
you. To the extent allowed by applicable law, implied warranties on the SOFTWARE PRODUCT, if any, are limited to ninety (90)
days.
CUSTOMER REMEDIES. 3I's and its suppliers' entire liability and your exclusive remedy shall be, at 3I's option, either (a) return
of the price paid, if any, or (b) repair or replacement of the SOFTWARE PRODUCT that does not meet 3I's Limited Warranty and
which is returned to 3I with a copy of your receipt. This Limited Warranty is void if failure of the SOFTWARE PRODUCT has
resulted from accident, abuse, or misapplication. Any replacement SOFTWARE PRODUCT will be warranted for the remainder of the
original warranty period or thirty (30) days, whichever is longer. Outside the United States, neither these remedies nor any product
support services offered by 3I are available without proof of purchase from an authorized international source.
NO OTHER WARRANTIES. To the maximum extent permitted by applicable law, 3I and its suppliers disclaim all
other warranties and conditions, either express or implied, including, but not limited to, implied warranties of
merchantability, fitness for a particular purpose, title, and non-infringement, with regard to the SOFTWARE
PRODUCT, and the provision of or failure to provide Support Services. This limited warranty gives you specific legal rights.
You may have others, which vary from state/jurisdiction to state/jurisdiction.
LIMITATION OF LIABILITY. To the maximum extent permitted by applicable law, in no event shall 3I or its
suppliers be liable for any special, incidental, indirect, exemplary or consequential damages whatsoever (including,
without limitation, damages for loss of business profits, business interruption, loss of business information, or any
other pecuniary loss) arising out of the use of or inability to use the SOFTWARE PRODUCT or the provision of or
failure to provide Support Services, even if 3I has been advised of the possibility of such damages. In any case, 3I's
entire liability under any provision of this EULA shall be limited to the greater of the amount actually paid by you for
the SOFTWARE PRODUCT or U.S.$5.00; provided, however, if you have entered into a 3I Support Services
Agreement, 3I's entire liability regarding Support Services shall be governed by the terms of that agreement.
Because some states and jurisdictions do not allow the exclusion or limitation of liability, the above limitation may not apply to you.
CONTRACTUAL STATUTE OF LIMITATIONS AND NOTICE. To the maximum extent permitted by applicable law, no
action or claim relating to this Agreement may be instituted more than one (1) year after the event giving rise to
such action or claim. In addition, you must notify 3I at least thirty (30) days prior to the instituting of any lawsuit
under this Agreement, with a full description of your claim and what you are seeking in damages.
DISPUTE RESOLUTION
Any controversy or claim arising out of or relating to this Agreement or the breach thereof, with the exception of injunctive relief
sought by 3I for any violation of the proprietary terms of this Agreement, shall be settled by arbitration in accordance with the rules
of the American Arbitration Association. Before entering into arbitration, You and 3I shall each appoint an arbitrator, and these two
arbitrators shall select a third arbitrator to be a member of the panel. Should the two arbitrators not be able to agree on a choice
of the third, then the American Arbitration Association shall make the appointment of a person who is neutral to the parties in
controversy. None of the arbitrators shall be officers or employees of the parties to this Agreement. Such arbitrators shall be
recognized experts in the computer software field. The cost of arbitration, including fees per arbitrator, shall be borne equally by
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the parties. The arbitration shall be held in the City of Los Angeles, in the County of Los Angeles, in the State of California in the
United States of America.
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SlideBook™ 5.0 User Manual
Table of Contents
END-USER LICENSE AGREEMENT FOR 3I SOFTWARE ...............................................................................II TABLE OF CONTENTS ......................................................................................................................................... VI MANUAL CONVENTIONS................................................................................................................................. XIV 1.1 1.2 TYPOGRAPHIC CONVENTIONS ................................................................................................................... XIV OF PIXELS AND VOXELS ........................................................................................................................... XIV USING THIS MANUAL ......................................................................................................................................... XV 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1 CHAPTER 1 - INSTALLING SLIDEBOOK™ ................................................................................................... XV CHAPTER 2 – QUICK TOUR......................................................................................................................... XV CHAPTER 3 – SLIDEBOOK™ ORGANIZATION ............................................................................................. XV CHAPTER 4 - CONFIGURING YOUR SYSTEM ............................................................................................... XV CHAPTER 5 - CONTROLLING THE CAMERA AND MICROSCOPE ................................................................... XV CHAPTER 6 - IMAGE CAPTURE AND IMPORT ............................................................................................... XV CHAPTER 7 – ADVANCED CAPTURE ........................................................................................................... XV CHAPTER 8 - IMAGE DISPLAY AND MANIPULATION .................................................................................. XVI CHAPTER 9 - PREPARING AN IMAGE FOR ANALYSIS OR EXPORT ............................................................... XVI CHAPTER 10 - IMAGE ANALYSIS ............................................................................................................... XVI INSTALLING SLIDEBOOK™....................................................................................................................... 17 1.1 COMPUTER SYSTEM REQUIREMENTS .......................................................................................................... 17 1.1.1 Operating System ................................................................................................................................... 17 1.1.2 Hardware ............................................................................................................................................... 17 1.1.2.1 1.1.2.2 1.1.2.3 1.2 1.3 1.4 1.5 1.6 1.7 2 Processors ..................................................................................................................................................... 17 Memory Requirements .................................................................................................................................. 17 Graphics Card ............................................................................................................................................... 17 CONNECTING THE HARDWARE KEY ............................................................................................................ 17 ABOUT SLIDEBOOK™ INSTALLERS ............................................................................................................ 18 COPYING THE SOFTWARE FROM CD-ROM ................................................................................................. 18 COPYING THE SOFTWARE FROM YOUR DOWNLOAD SITE ............................................................................ 18 INSTALLING SLIDEBOOK 5.0 ....................................................................................................................... 18 INSTALLING UPDATES ................................................................................................................................. 22 QUICK TOUR .................................................................................................................................................. 23 2.1 OPENING SLIDES AND IMAGES .................................................................................................................... 23 2.2 DISPLAYING A MAIN VIEW ......................................................................................................................... 23 2.3 USING THE INFO/TOOL BAR ........................................................................................................................ 25 2.3.1 Channel Menus and Data Values .......................................................................................................... 25 2.3.2 Up/Down Arrows, Scrolling through a 3D image .................................................................................27 2.3.3 Tool Menu .............................................................................................................................................. 27 2.3.3.1 2.3.3.2 2.3.3.3 The Marquee Tool......................................................................................................................................... 27 Zoom Tool .................................................................................................................................................... 28 Hand Tool ..................................................................................................................................................... 29 2.3.4 Axis Menu .............................................................................................................................................. 30 2.3.5 Renormalize Button ............................................................................................................................... 31 2.3.6 Thumbnail Button .................................................................................................................................. 32 2.4 GENERATING A THREE VIEW ...................................................................................................................... 33 2.5 SPAWNING A TILE VIEW ............................................................................................................................. 35 2.6 PERFORMING VOLUME RENDERING ............................................................................................................ 36 2.7 CREATING MASKS AND GENERATING STATISTICS ...................................................................................... 39 2.7.1 Creating a Mask using Threshold Techniques ....................................................................................... 39 2.7.2 Creating Masks Manually (Background Subtraction Example) ............................................................ 41 vi
Table of Contents
2.7.3 3 Defining Multiple Objects ...................................................................................................................... 45 SLIDEBOOK™ ORGANIZATION ............................................................................................................... 52 3.1 HARDWARE CONTROL AND IMAGE CAPTURE (CHAPTERS 5, 6 AND 7) ........................................................ 52 3.1.1 Focus Controls....................................................................................................................................... 52 3.1.2 Capture Dialog Box ............................................................................................................................... 52 3.1.3 Capture Controls ................................................................................................................................... 52 3.2 DATA STORAGE, DISPLAY, AND ANALYSIS (CHAPTERS 8, 9, AND 10)......................................................... 52 3.2.1 Images and channels.............................................................................................................................. 53 3.2.2 Views ...................................................................................................................................................... 53 3.2.3 Masks and Statistics ............................................................................................................................... 53 3.2.4 Slides...................................................................................................................................................... 53 4 CONFIGURING YOUR SYSTEM ................................................................................................................. 54 4.1 DESIGNATING USER LOGINS ....................................................................................................................... 54 4.2 CONFIGURING HARDWARE ......................................................................................................................... 56 4.2.1 General Hardware Configuration ......................................................................................................... 57 4.2.2 Configuring a Motorized XY Stage ........................................................................................................ 58 4.2.3 Configuring an Image Splitter ............................................................................................................... 60 4.2.4 Configuring Camera and Ocular Ports ................................................................................................. 61 4.3 DEFINING OBJECTIVES ................................................................................................................................ 62 4.3.1 Adding a New Objective ........................................................................................................................ 62 4.3.2 Modifying Information for an Existing Objective .................................................................................. 63 4.3.3 Removing an Objective .......................................................................................................................... 63 4.4 DEFINING FILTER CONFIGURATIONS ........................................................................................................... 64 4.4.1 Defining Filter Sets ................................................................................................................................ 64 4.4.2 Filter Configuration Parameters ........................................................................................................... 65 4.4.2.1 4.4.2.2 4.4.2.3 4.4.2.4 4.4.2.5 4.4.2.6 4.4.3 Light Source .................................................................................................................................................. 65 Filter Set........................................................................................................................................................ 65 Filter Positions .............................................................................................................................................. 66 Camera .......................................................................................................................................................... 66 Channel Types .............................................................................................................................................. 66 Default Color Display ................................................................................................................................... 66 Adding a New Filter Configuration ....................................................................................................... 68 4.4.3.1 4.4.3.2 4.4.3.3 Example Filter Configuration for Fluorescence Channels ............................................................................ 69 Configuring Channels for Color Cameras or Color LCD sliders .................................................................. 69 Defining Filters when using an Image Splitter .............................................................................................. 72 4.4.4 Modifying Information for an Existing Filter Configuration ................................................................. 73 4.4.5 Removing a Filter .................................................................................................................................. 73 4.5 DEFINING MAGNIFICATION CHANGERS ...................................................................................................... 73 4.5.1 Adding a New Magnification Changer .................................................................................................. 73 4.5.2 Removing or Modifying Magnification Changer Definitions ................................................................ 74 4.6 WORKING WITH SLIDEBOOK PREFERENCES AND HARDWARE PROPERTIES ................................................ 74 4.6.1 Backing Up SlideBook Configuration .................................................................................................... 74 4.6.2 Restoring SlideBook Configuration ....................................................................................................... 75 4.6.3 Exceptions .............................................................................................................................................. 75 4.7 DEFINING SYSTEM PARAMETERS ................................................................................................................ 76 5 CONTROLLING THE CAMERA AND MICROSCOPE HARDWARE (FOCUS WINDOW) ................ 77 5.1 FOCUS WINDOW FEATURES ........................................................................................................................ 77 5.1.1 Permanent Controls ............................................................................................................................... 78 5.1.1.1 5.1.1.2 5.1.1.3 5.1.1.4 5.1.1.5 5.1.1.6 Camera and Display Settings ........................................................................................................................ 78 Open Fluor, Open Bright, Open Alt .............................................................................................................. 79 Filter Controls ............................................................................................................................................... 79 XY Stage ....................................................................................................................................................... 79 Z Stage .......................................................................................................................................................... 80 Neutral Density ............................................................................................................................................. 80 vii
SlideBook™ 5.0 User Manual
5.1.2 Scope...................................................................................................................................................... 80 5.1.3 Z ............................................................................................................................................................. 81 5.1.4 5.1.5 XY .......................................................................................................................................................... 83 Camera .................................................................................................................................................. 84 5.1.2.1 5.1.2.2 5.1.2.3 5.1.2.4 5.1.2.5 5.1.2.6 5.1.3.1 5.1.3.2 5.1.3.3 5.1.5.1 5.1.5.2 5.1.5.3 5.1.5.4 5.1.5.5 5.1.5.6 5.1.5.7 Emission Selection ........................................................................................................................................ 80 Magnification Changer ................................................................................................................................. 80 Objectives ..................................................................................................................................................... 81 Stage Limits .................................................................................................................................................. 81 Lamp ............................................................................................................................................................. 81 Condenser ..................................................................................................................................................... 81 Set Top, Set Bottom, Set Reference, Center ................................................................................................. 82 Slider ............................................................................................................................................................. 83 Capture Information ...................................................................................................................................... 83 Histogram...................................................................................................................................................... 84 CCD Temp .................................................................................................................................................... 85 Speed............................................................................................................................................................. 85 Gain .............................................................................................................................................................. 85 Intensification ............................................................................................................................................... 85 Parameters..................................................................................................................................................... 85 Test Dual-View ............................................................................................................................................. 85 5.2 USING THE FOCUS WINDOW ....................................................................................................................... 85 5.2.1 Snapping an Image ................................................................................................................................ 86 5.2.2 Closing the Fluorescence Shutter Automatically when Closing the Focus Window.............................. 86 5.2.3 Labeling the Ocular Photo Prism Positions .......................................................................................... 87 5.2.4 How to Perform Streaming Capture ...................................................................................................... 87 6 IMAGE CAPTURE AND IMPORT ............................................................................................................... 89 6.1 SELECTING CHANNELS AND SETTING EXPOSURE TIMES ............................................................................. 89 6.2 SELECTING THE AREA TO BE IMAGED ......................................................................................................... 92 6.2.1 Using the Image Extent Menu ................................................................................................................ 92 6.2.2 Using the Test View ............................................................................................................................... 93 6.3 ENTERING IMAGE INFORMATION AND OPTICAL PARAMETERS BEFORE CAPTURE ....................................... 93 6.4 CAPTURING A SINGLE OR MULTI-CHANNEL 2D IMAGE .............................................................................. 93 6.5 PERFORMING AUTO-WHITE BALANCE FOR COLOR CAPTURE ..................................................................... 95 6.6 IMPORTING AN IMAGE ................................................................................................................................. 96 6.6.1 Importing SlideBook Spool Files ........................................................................................................... 97 6.6.2 Importing TIFF Files ............................................................................................................................. 98 6.7 GETTING AND EDITING IMAGE INFORMATION AFTER CAPTURE ............................................................... 101 6.7.1 Images that were captured in SlideBook.............................................................................................. 101 6.7.2 Imported Images .................................................................................................................................. 104 7 ADVANCED CAPTURE ............................................................................................................................... 105 7.1 3D CAPTURE ............................................................................................................................................. 105 7.1.1 Method 1 .............................................................................................................................................. 105 7.1.1.1 7.1.1.2 7.1.1.3 Setting Focus Window Parameters ............................................................................................................. 105 Setting Capture Window Parameters .......................................................................................................... 107 Setting Capture Preferences to Open and Close Shutter during Capture ..................................................... 108 7.1.2 Method 2 .............................................................................................................................................. 109 7.2 TIMELAPSE CAPTURE ................................................................................................................................ 112 7.2.1 Setting Capture Preferences ................................................................................................................ 112 7.2.1.1 7.2.1.2 7.2.1.3 7.2.1.4 7.2.1.5 7.2.1.6 7.2.2 7.2.3 viii
Performing Periodic Capture....................................................................................................................... 113 Opening and Closing Brightfield Shutter Between Exposures .................................................................... 113 Changing the Status Update Frequency ...................................................................................................... 114 Autofocus During Capture .......................................................................................................................... 115 Changing Capture Frequency ...................................................................................................................... 115 Saving Images to Disk ................................................................................................................................ 115 Setting Capture Window Parameters................................................................................................... 115 Creating Notes ..................................................................................................................................... 116 Table of Contents
7.2.4 7.2.5 Creating ROIs and Graphs to Monitor Regions of Interest ................................................................. 118 Initiating and Monitoring Timelapse Capture ..................................................................................... 119 7.2.5.1 7.2.5.2 7.2.5.3 Creating Graphs to Monitor Regions of Interest ......................................................................................... 120 Focusing During Capture ............................................................................................................................ 121 Viewing Previous Timepoints ..................................................................................................................... 122 7.3 4D CAPTURE ............................................................................................................................................. 124 7.3.1 Shifting the Volume During 4D Imaging ............................................................................................. 125 7.3.2 Mid-volume Capture During 4D Imaging ........................................................................................... 125 7.3.3 Multi-Channel Z-Series........................................................................................................................ 127 7.4 MULTIPOINT CAPTURE.............................................................................................................................. 127 7.4.1 Setting Capture Preferences ................................................................................................................ 127 7.4.2 Setting Focus Window Parameters ...................................................................................................... 128 7.4.2.1 7.4.2.2 7.4.3 Setting Points .............................................................................................................................................. 128 Saving and Loading a Multipoint List ......................................................................................................... 129 Setting Capture Window Parameters................................................................................................... 130 7.4.3.1 7.4.3.2 3D Multiple Location Capture .................................................................................................................... 130 Timelapse Multiple Location Capture......................................................................................................... 130 7.5 MONTAGE CAPTURE ................................................................................................................................. 131 7.5.1 Setting Focus Window Parameters ...................................................................................................... 131 7.5.2 Setting Capture Window Parameters................................................................................................... 132 7.6 MULTIWELL CAPTURE ............................................................................................................................. 133 7.6.1 Creating a Layout ................................................................................................................................ 134 7.6.2 Manipulating Hardware, Focusing on the Sample .............................................................................. 136 7.6.3 Calibrate the Stage Positions .............................................................................................................. 136 7.6.4 Save and Restore Calibration Positions .............................................................................................. 137 7.6.5 Selecting Wells for Capture ................................................................................................................. 137 7.6.6 Initiating and Monitoring Capture ...................................................................................................... 137 7.6.7 Exporting Point List ............................................................................................................................. 138 7.7 SIMULTANEOUS CAPTURE......................................................................................................................... 138 7.8 CONFIGURING YOUR HARDWARE FOR SPEED ........................................................................................... 138 7.8.1 Photometrics Cameras......................................................................................................................... 138 7.8.2 Sutter DG-4.......................................................................................................................................... 139 7.8.3 Z Stages................................................................................................................................................ 139 7.8.3.1 7.8.3.2 ASI/Mad City Piezoelectric Stage............................................................................................................... 140 Physik Instrumente PIFOC Piezoelectric Focusing Collar .......................................................................... 141 7.9 AUTOFOCUS .............................................................................................................................................. 144 7.9.1 Determining Auto Focus Parameters .................................................................................................. 144 7.9.2 Adding Auto Focus to an Image Capture Sequence............................................................................. 146 7.10 VARYING CAPTURE RATES DURING TIMELAPSE CAPTURE (SEQUENCES) ................................................. 149 7.10.1 Setting Capture Preferences ............................................................................................................ 149 7.10.2 Setting Capture Dialog Box Parameters ......................................................................................... 151 7.10.3 Examples for Variable Capture ....................................................................................................... 152 7.10.3.1 7.10.3.2 7.11 7.12 8 Example 1 ................................................................................................................................................... 152 Example 2 ................................................................................................................................................... 153 SAVING IMAGES TO DISK (SPOOLED CAPTURE) ........................................................................................ 153 SAVING CAPTURE PARAMETERS ............................................................................................................... 154 IMAGE DISPLAY AND MANIPULATION (VIEWS) .............................................................................. 156 8.1 INTRODUCTION TO SLIDE AND IMAGE DISPLAY IN SLIDEBOOK ................................................................ 156 8.1.1 Working with Slides ............................................................................................................................. 157 8.1.1.1 8.1.1.2 8.1.1.3 8.1.1.4 8.1.1.5 8.1.2 Saving Slides............................................................................................................................................... 157 Opening Slides ............................................................................................................................................ 157 Closing Slides ............................................................................................................................................. 157 Deleting Slides ............................................................................................................................................ 157 Changing the Slide View Display ............................................................................................................... 158 Working with Images ........................................................................................................................... 158 8.1.2.1 8.1.2.2 Selecting an Image ...................................................................................................................................... 158 Removing an Image from One Slide and Placing it in Another .................................................................. 158 ix
SlideBook™ 5.0 User Manual
8.1.2.3 8.1.2.4 Copying an Image ....................................................................................................................................... 159 Deleting an Image ....................................................................................................................................... 159 8.1.3 Image Display ...................................................................................................................................... 159 8.2 USING DATA VIEWS TO DISPLAY IMAGES................................................................................................. 160 8.2.1 Displaying a Main View ...................................................................................................................... 160 8.2.2 Displaying a Three View...................................................................................................................... 162 8.2.3 Spawning a Tile View .......................................................................................................................... 163 8.2.3.1 8.2.3.2 8.2.3.3 8.2.3.4 8.2.4 Displaying a 3D Tile View ......................................................................................................................... 163 Displaying a Timelapse Tile ....................................................................................................................... 164 Displaying a 3D or Timelapse Tile View of a Portion of the Image ........................................................... 165 Arranging your Tile View ........................................................................................................................... 166 Displaying a Multidimensional Channel View .................................................................................... 167 8.2.4.1 Multidimensional Channel View Settings ................................................................................................... 167 8.2.5 Displaying a Montage.......................................................................................................................... 169 8.3 MANIPULATING DATA VIEWS USING THE INFO/TOOL BAR ...................................................................... 169 8.3.1 Altering the Renormalization Parameters (Lookup Table) .................................................................. 171 8.3.1.1 8.3.1.2 8.3.1.3 Changing Renormalization Parameters for a Single View .......................................................................... 171 Changing Renormalization Parameters for a Group of Images in a Slide ................................................... 173 Applying Nonlinear Lookup Tables ............................................................................................................ 173 8.3.2 Changing the Display Colors .............................................................................................................. 174 8.3.3 Cropping an Image .............................................................................................................................. 177 8.3.4 8.3.5 8.3.6 8.3.7 8.3.8 Rotating an Image................................................................................................................................ 178 Changing the Invisible Axis ................................................................................................................. 179 Scrolling through the Invisible Axis in a 3D or Timelapse Image ....................................................... 180 Changing the Default Display ............................................................................................................. 180 Displaying Annotations........................................................................................................................ 180 8.3.2.1 8.3.2.2 8.3.2.3 8.3.2.4 8.3.2.5 8.3.3.1 8.3.8.1 8.3.8.2 8.3.8.3 Changing the Channels Displayed on an RGB Image ................................................................................. 174 Changing an Image Display to Monochrome .............................................................................................. 174 Changing an Image Display to Pseudocolor ............................................................................................... 175 Changing the Display to a User-Defined Color Palette ............................................................................... 175 Displaying a DIC image as Background of an RGB Image ........................................................................ 177 Extracting Timepoints from a Timelapse Image ......................................................................................... 178 Displaying Annotations............................................................................................................................... 181 Formatting Annotations .............................................................................................................................. 182 Setting Default Annotation Settings ............................................................................................................ 184 8.4 USING THE TOOL MENU ............................................................................................................................ 184 8.4.1 Making a 2D or 3D (x,y,z or x,y,t) Selection ....................................................................................... 186 8.4.1.1 8.4.1.2 8.4.1.3 8.4.1.4 8.4.2 8.4.3 Making a 2D Selection in any Data View ................................................................................................... 186 Making a 3D Selection in a Main, Three, and Channel View ..................................................................... 186 Making a 3D Selection in any Data View ................................................................................................... 186 Selecting an Entire Image ........................................................................................................................... 187 Scrolling through a Three View ........................................................................................................... 187 Making Distance or Velocity Measurements ....................................................................................... 187 8.4.3.1 8.4.3.2 8.4.3.3 8.4.3.4 Making a Measurement on a 2D Image ...................................................................................................... 187 Making a Measurement on a 3D or 2D Timelapse Image ........................................................................... 188 Making a Measurement on an Imported Image ........................................................................................... 188 Using the Ruler Tool to make a Scale Bar .................................................................................................. 188 8.5 USING DISPLAY VIEWS (CREATING RENDERINGS AND MOVIES) .............................................................. 189 8.5.1 Displaying a 3D or 4D Volume View (Volume Rendering) ................................................................. 189 8.5.1.1 8.5.1.2 8.5.2 8.5.3 8.5.4 Working with 3D Volume Views................................................................................................................ 190 Working with 4D Volume Views................................................................................................................ 193 Displaying a 3D Surface View ............................................................................................................. 193 Generating a Physically Proportional Rendering of 3D Data ............................................................ 195 Creating Series Movies ........................................................................................................................ 195 8.5.4.1 8.5.4.2 2D Timelapse Images.................................................................................................................................. 195 4D Images ................................................................................................................................................... 196 8.6 EXPORTING VIEWS .................................................................................................................................... 196 8.6.1 Exporting a Main View, Three View, Channel View, or Tile View as a TIFF ..................................... 197 8.6.2 Exporting a 3D or 2D Timelapse View as a TIFF or TIFF Series ...................................................... 197 x
Table of Contents
8.6.3 8.6.4 9 Exporting Default Views of All Images in a Slide ................................................................................ 197 Exporting Volume View and Surface View Movies .............................................................................. 198 PREPARING AN IMAGE FOR ANALYSIS OR EXPORT ...................................................................... 200 9.1 MANIPULATING INDIVIDUAL CHANNELS .................................................................................................. 200 9.1.1 Inserting an Image as a Channel ......................................................................................................... 200 9.1.2 Removing a Channel ............................................................................................................................ 201 9.1.3 Using Channel Math ............................................................................................................................ 202 9.1.4 Creating a Timelapse Composite Channel .......................................................................................... 204 9.2 CROPPING AN IMAGE ................................................................................................................................ 204 9.3 ALIGNING AN IMAGE................................................................................................................................. 204 9.4 MANIPULATING TIMELAPSE SERIES .......................................................................................................... 206 9.4.1 Removing Timepoints from a Timelapse Series ................................................................................... 206 9.4.2 Merging Timelapse Series ................................................................................................................... 207 9.5 PERFORMING FLAT FIELD CORRECTION.................................................................................................... 207 9.5.1 Collecting Flat Fields .......................................................................................................................... 207 9.5.1.1 9.5.1.2 9.5.1.3 9.5.2 Adding or Replacing a Flat Field ................................................................................................................ 208 When to Update the Flat Field Database ..................................................................................................... 209 Displaying Existing Flat Fields ................................................................................................................... 209 Applying Flat Fields ............................................................................................................................ 210 9.5.2.1 9.5.2.2 Applying Flat Field Correction During Image Capture ............................................................................... 210 Applying Flat Field Correction after Image Capture................................................................................... 211 9.6 PERFORMING PHOTOBLEACH CORRECTION .............................................................................................. 212 9.7 PERFORMING BACKGROUND SUBTRACTION ............................................................................................. 213 9.8 CREATING A PROJECTION IMAGE .............................................................................................................. 213 9.9 CREATING AN INTERPOLATED (ISOTROPIC) IMAGE ................................................................................... 215 9.10 APPLYING FILTERS ................................................................................................................................... 216 9.10.1 User-Defined Convolution Kernels ................................................................................................. 216 9.10.2 Gaussian, Hot Pixel, Mean, and Median Filters ............................................................................. 218 9.11 PERFORMING NO NEIGHBORS DECONVOLUTION ...................................................................................... 219 9.11.1 Running No Neighbors Deconvolution ............................................................................................ 219 9.11.2 Memory Requirements for Deconvolution ....................................................................................... 221 9.12 EXPORTING IMAGES .................................................................................................................................. 221 10 USING MASKS FOR IMAGE ANALYSIS ................................................................................................. 223 10.1 DISPLAYING IMAGE HISTOGRAM AND MEAN INTENSITY .......................................................................... 223 10.2 CREATING MASKS AND OBJECTS .............................................................................................................. 223 10.2.1 Creating a Mask using Threshold Techniques ................................................................................ 224 10.2.2 Creating Masks Manually ............................................................................................................... 226 10.2.2.1 10.2.2.2 10.2.2.3 10.2.2.4 Creating an Empty Mask............................................................................................................................. 226 Editing the Mask Manually ......................................................................................................................... 227 Moving the Mask ........................................................................................................................................ 228 Copying a Manually Created Mask to Other Planes or Other Images ......................................................... 229 10.2.3 10.2.4 Defining Objects .............................................................................................................................. 229 Splitting Objects .............................................................................................................................. 231 10.2.5 10.2.6 Removing Objects from Edge of Image ........................................................................................... 232 Tracking Objects (Particle Tracking).............................................................................................. 232 10.2.4.1 10.2.4.2 10.2.6.1 10.2.6.2 Manually Splitting Objects ......................................................................................................................... 231 Automatically Splitting Objects .................................................................................................................. 231 Automated Particle Tracking (Tutorial) ...................................................................................................... 232 Performing Manual Particle Tracking ......................................................................................................... 238 10.3 DISPLAYING OR DELETING A MASK .......................................................................................................... 239 10.4 BOOLEAN MASK OPERATIONS .................................................................................................................. 240 10.5 USING MASKS FOR SMOOTH CURVE ANALYSIS (KYMOGRAPH)................................................................ 241 10.6 GENERATING AND EXPORTING MASK AND OBJECT STATISTICS ............................................................... 243 10.6.1 Counting Objects in a Mask ............................................................................................................ 243 10.6.2 Generating Statistics for Masks and Objects................................................................................... 243 xi
SlideBook™ 5.0 User Manual
10.7 DISPLAYING GRAPHS ................................................................................................................................ 248 10.7.1 Viewing a Line Intensity Profile ...................................................................................................... 248 10.7.2 Viewing Timelapse Intensity Profiles .............................................................................................. 249 10.7.2.1 10.7.2.2 10.7.2.3 Method 1 – Using Masks ............................................................................................................................ 249 Method 2 – Point and Click ........................................................................................................................ 250 Method 3 – Using ROIs .............................................................................................................................. 251 APPENDIX A: SMOOTH CURVE ANALYSIS (KYMOGRAPH)................................................................... 253 xii
Technical Support
Technical Support
If you have any questions or experience any problems with SlideBook™, please contact
Olympus America ([email protected] or 1-800-446-5967 option 4).
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SlideBook™ 5.0 User Manual
Manual Conventions
1.1
Typographic Conventions
Menu commands are written in bold and follow the order of menu navigation. For instance,
choosing “Open” from the “File” menu is written as File > Open.
Dialog fields and other interface items are written in bold, as in Initial Offset.
References to other sections of the manual are underlined, as in Image Capture.
New terms are italicized.
NOTES will be written in bold and small capitals. Notes that you should pay particular
attention to are prefaced CAUTION.
1.2
Of Pixels and Voxels
In other contexts, image typically connotes a two-dimensional entity. However,
SlideBook™ was specifically designed to operate on three-dimensional data as easily and
intuitively as two-dimensional data. In light of this, a two-dimensional data set can be seen
as a special case of a three-dimensional set where data has been collected for only one value
of the z-axis. An image corresponds to all data collected during a particular capture.
To simplify this manual, we always refer to a single data element as a voxel and a region as
a volume, regardless of the depth of the image. These terms map directly to pixel and area,
respectively, for a two-dimensional set.
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Using this Manual
Using this Manual
The SlideBook ™ Manual describes all of the steps necessary for performing digital
microscopy, from installing the software to analyzing images. The chapters are outlined
below. If you are new to SlideBook™, you may wish to start by taking a step-by-step Quick
Tour as outlined in Chapter 2. This will familiarize you with basic elements and commands
in SlideBook™.
1.3
Chapter 1 - Installing SlideBook™
If your system was installed by 3i or a 3i reseller, then you will not need to install software
on your system. However, if it was not installed by 3i or a 3i reseller, or you need to
upgrade your software, you can learn how to do this in Chapter 1.
1.4
Chapter 2 – Quick Tour
This chapter will take you on a step-by-step guided tour of the basic elements of
SlideBook™. This is the perfect starting point for the beginning user.
1.5
Chapter 3 – SlideBook™ Organization
This chapter describes how hardware control, image capture, data storage, and data
analysis are organized into SlideBook™ menus, windows, and objects.
1.6
Chapter 4 - Configuring Your System
If your system was installed by 3i or a 3i reseller, then you will not need to configure your
system prior to use. However, if it was not installed by 3i or a 3i reseller, or you need to
make changes to your system configuration, you may do so by selecting Edit > Define
Optics or Edit > Hardware Configuration. Chapter 4 teaches you how to configure
hardware and define optics.
1.7
Chapter 5 - Controlling the Camera and Microscope
Next, you will learn how to set your microscope to the proper optics and bring your sample
into view and focus. Any motorized aspect of the microscope can be controlled through
SlideBook’s Focus Window. You may bring up this window by selecting Window > Focus
Window or clicking on the focus window button in the toolbar
.
1.8
Chapter 6 - Image Capture and Import
Parameters for image capture will be set in the Capture Window. Here you will set
camera parameters, such as exposure and binning, by performing test exposures. You may
bring up this window by selecting Image > Capture New Image or by clicking on the
capture button in the toolbar
. You may also import images that were captured using
other programs by selecting Image > Import.
1.9
Chapter 7 – Advanced Capture
In this chapter, you will learn to perform more advanced capture sequences, such as
timelapse, 3D, 4D, and montage capture.
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SlideBook™ 5.0 User Manual
1.10 Chapter 8 - Image Display and Manipulation
Once you have captured an image set, it may be displayed in various modes using the View
menu. Interactive data views allow you to display any combination of different channels
and masks (ROIs) as well as change lookup table parameters. You may also perform
operations such as volume rendering. Note that operations from this menu do not alter the
underlying intensity data contained in an image.
1.11 Chapter 9 - Preparing an Image for Analysis or Export
Before performing analysis on an image, you may want to perform additional operations on
an image. For example, you may wish to eliminate any constant artifacts from the image by
performing flat field correction. It may also be useful to deblur an image by performing
deconvolution. Other operations that may be necessary or useful prior to analysis are
cropping, aligning channels, or applying filters. Operations such as these are found under
the Image menu.
1.12 Chapter 10 - Image Analysis
Mathematical analysis can be performed on regions of the image that you select. You may
select these regions by creating a binary overlay called a mask. Masks are generated from
the Mask menu. Once you have made a mask, you may obtain information such as average
intensity and number of pixels in the region of interest by using the Statistics menu.
NOTE: Screenshots used in this manual were taken with SlideBook running in Windows
Vista™. Appearance may vary slightly with different operating systems.
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Chapter 2 – Quick Tour
1 Installing SlideBook™
1.1
Computer System Requirements
Your computer must meet the following system requirements in order to run SlideBook™
5.0.
1.1.1 Operating System
Windows XP Professional for computers driving hardware, also known as online systems.
Windows 32-bit or 64-bit Vista for computer running analysis only, also known as offline
systems.
1.1.2 Hardware
1.1.2.1 Processors
Recommend at least dual quad-core Xeon 3.2GHz processors
1.1.2.2 Memory Requirements
RAM: recommended 3 GB, minimum 2GB.
Hard drive: minimum 200 GB required for installation
1.1.2.3 Graphics Card
(for Interactive Volume Rendering only)
VRAM: minimum 256 MB, recommended 512 MB or more
Minimum models: NVIDIA GeForce or NVIDIA Quadro FX
Recommended models: NVIDIA GeForce 9800, NVIDIA Quadro FX 3540 or higher
Please contact Olympus America ([email protected] or 1-800-446-5967 option
4) with questions regarding hardware compatibility.
1.2
Connecting the Hardware Key
SlideBook™ comes with a hardware key, or dongle, that permits the application to run on
the machine that has the key attached. The key is a Sentinel series black (with purple
accent) USB device manufactured by SafeNet. Before you begin the software installation;
please connect the dongle to a USB port in the computer.
NOTE: Given the hardware key system, it is possible to install the SlideBook™ software on
several different machines. However, the software will only run on the machine that has
the key attached. Networked computers may run SlideBook™ if one of the computers on the
network has a network key.
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SlideBook™ 5.0 User Manual
1.3
About SlideBook™ Installers
SlideBook installers are available as “compact” or “complete”.
1.4

Compact – installs SlideBook program files, Sentinel dongle driver, user
manual, and QuickTime.

Complete – installs all SlideBook program files, user’s manual, QuickTime
program files, and Sentinel dongle drivers. Also allows the option to copy camera
driver files, Edgeport driver files, and QuickTour sample files to the
Intelligent Imaging Innovations, Inc directory.
Copying the Software from CD-ROM
1. Insert the SlideBook™ CD in your CD-ROM drive.
2. Navigate to the CD-ROM and copy the SlideBook™ setup program,
InstallSlideBook.exe to your desktop.
3. Double-click on the icon to begin installation and proceed to the section
“Installing SlideBook 5.0” (below).
1.5
Copying the Software from your Download Site
Every SlideBook™ user may access a private download site in order to obtain current
installers and updaters. Please contact Olympus America ([email protected]
or 1-800-446-5967 option 4) to obtain your user name and password.
To access your download site:
1. Go to www.intelligent-imaging.com
2. Go to Downloads, and then Customer Login, and click on link “Click Here to
Login.”
3. Enter your username/password (case-sensitive).
4. Click on the desired link to download the software. When prompted, select Save
and then navigate to the desired destination for the file.
5. Click OK to save the file.
1.6
Installing SlideBook 5.0
The SlideBook installer allows you to install the program, drivers, adjunct programs, the
manual, and tutorials. If you are installing SlideBook for the first time, continue with this
section. If you already have SlideBook installed on your machine, please see Installing
Updates.
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Chapter 2 – Quick Tour
1. Double-click on the InstallSlideBook icon to start the setup utility. The following
dialog box will be displayed.
2. Select Next and you will be prompted to review and accept the terms of the
license agreement.
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SlideBook™ 5.0 User Manual
3. Select Next and the following window will appear:
4. If the default destination directory is acceptable, select Next. If you would like to
install to a different directory, click Change and navigate to the desired location
and then select Next to continue installation. The following dialog box will
appear:
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Chapter 2 – Quick Tour
5. Select Complete then Next. The following dialog box will appear:
6. Select Install and the setup utility will display status of the installation:
7. When the installation is complete, the following message will confirm
installation:
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SlideBook™ 5.0 User Manual
8. Click Finish to complete the setup.
You may now launch SlideBook™ from either the Start Menu > Programs or
from the Shortcut to SlideBook.exe.
NOTE: When you start SlideBook™ for the first time, it will notice that a preferences file,
which is stored along with the application, does not exist. SlideBook™ will create a default
preferences file. As you configure your hardware and define optics (discussed in Chapter 4,
Configuring Your System), SlideBook™ will update this preferences file with specific
information about your setup.
An electronic copy of this manual, called SlideBook 5.0 Users Manual.pdf, is in
Adobe Acrobat format, and can be opened by an Acrobat reader. Feel free to distribute the
manual to all users of SlideBook™.
1.7
Installing Updates
You may download software updates at www.intelligent-imaging.com as described above in
Copying Software from your Download Site. Before installing an update, be sure to back up
your configuration files. To backup your files, go to Edit > Setup Guides > Backup
Preferences. You will be prompted for a location. If you use the defaults settings, a folder
called Global Preferences will be created in the SlideBook 5.0 directory. After
downloading is complete, double-click on the file that you have just downloaded and follow
the installation directions. Install the update in Program Files\Intelligent Imaging
Innovations, Inc\SlideBook. If SlideBook™ is not located in the above path; refer to
section 4.6 Working with SlideBook Preferences and Hardware Properties on page 74.
22
Chapter 2 – Quick Tour
2 Quick Tour
This Quick Tour will introduce you to some basic elements of SlideBook. By the end of this
tour, you will know how to do the following:





Open slides and images
Display a Main View, Three View, and Tile View
Use basic tools in the Info/Tool Bar to Maneuver within a view
Perform volume rendering
Create a mask and obtain statistics
NOTE: Menu operations, such as choosing “Open Slide” from the “File” menu are written as
File > Open Slide.
In order to run the Quick Tour, first make sure that the SlideBook program and Quick Tour
slides have been installed. If not, please go to Chapter 1, Installing SlideBook™ and follow
the procedures for installing the software and the Quick Tour slides.
2.1
Opening Slides and Images
To begin the tour, we will first open a slide file, SlideBook’s native file type (*.sld). A slide
file corresponds to a single file on the disk and can be thought of as SlideBook’s document
type. It can contain many multi-plane, multi-channel images in any combination.
1. Launch SlideBook by pointing to Programs > SlideBook from the Start menu.
An empty slide will appear in the window under the SlideBook menus.
2. Close the new slide by choosing File > Close Slide or by clicking on the
upper right corner.
in
3. Choose File > Open Slide and navigate to the Documentation folder in the
SlideBook 5.0 directory. Double click on QuickTourWIN1.sld.
The following slide window appears that shows a thumbnail of one image. This
slide contains a single 3D image of a dividing B cell.
2.2
Displaying a Main View
SlideBook can display any image using a variety of views. Each data set can have as many
views open at one time as you desire. SlideBook has seven kinds of views that display
individual images:
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SlideBook™ 5.0 User Manual







Main View
Three View
Tile View
Channel View
Multidimensional Channel View
3D Volume View
3D Surface View
NOTE: Views are only used to display and alter the look of an image. The underlying data
are not affected.
Now, we will open a Main View.
1. Double-click anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail. A new Main View window will open that displays the
image.
2. Create another Main View of the Dividing B Cell image by first selecting the
image with a single click, then selecting View > New Main View.
At this point you should have three open windows: the Slide Window and two
Main View windows. Both Main Views show the dividing B cell in three colors,
where each color corresponds to a different channel, or fluorophore. DNA appears
in blue (AMCA channel), surface immunoglobulin appears in green (FITC
channel), and tubulin appears in red (Cy3 channel).
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Chapter 2 – Quick Tour
2.3
Using the Info/Tool Bar
The Main View window contains an Info/Tool Bar placed above the image of the data. The
Info/Tool Bar displays a variety of useful information and gives you quick access to tools
you will use to explore and manipulate the view. The Info/Tool Bar is present in all data
views, namely the Main View, Three View, Tile View, and Channel View.
X, Y, Z, (T) Position
Mask Menu
Axis Menu
Set Default
View
Zoom Controls
Plane Selection
Tool Menu
Adjust Renormalization
Show Graphs
View Settings
Pixel Intensity Values
Red Menu
Green Menu
Blue Menu
Background Menu
2.3.1 Channel Menus and Data Values
On the far right of the Info/Tool Bar are three pop-up menus with colored red, green, and
blue text next to them. These menus allow you to select the fluorescence channel that is
displayed for each color. The Main View in this data set has the red menu set to the CY3
channel, the green menu set to the FITC channel, and the blue menu set to the AMCA
channel.
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SlideBook™ 5.0 User Manual
1. Click on one of the Main View images to select it as the active window.
2. Click on the CY3 bar (red menu) and select FITC from the pop-up menu. Notice
that the FITC label in the view now appears yellow from the combination of red
and green pixels.
3. Click on the red menu and select None. Note that there are no data displayed in
red.
4. Next, set all color menus to the same channel, FITC, and observe that the image
is now displayed in black and white.
Try changing the settings for each menu, noting how the display changes.
5. Reset the red menu to CY3, the green menu to FITC, and the blue menu to
AMCA.
To the left of the channel menus are colored numbers. These numbers correspond
to the intensity of the voxel that the cursor is positioned over for each channel.
6. Move the cursor over the image displayed in the Main View. Note how the
intensity values increase as you move over brighter regions and decrease as you
move to darker regions.
7. Visualize just the DNA by setting the red, green, and blue menus to DAPI. As
you move the cursor over the image, note that the red, green, and blue values are
equal.
26
Chapter 2 – Quick Tour
8. Close this Main View window by clicking on the
in upper right corner. One
Slide View and one Main View window should now be open.
2.3.2 Up/Down Arrows, Scrolling through a 3D image
The “Dividing B-Cell” image is actually a 3D data set containing 50 planes captured at 0.2
micron spacing. You may navigate through the stack in a variety of ways.
1. Click on the remaining Main View. Scroll through the z-axis using the small
arrow buttons
on the far left of the info/tool bar.
Note that the coordinates in the upper left corner give you the x, y, z position of
the mouse pointer. As you scroll through the image stack, the z position will
update to your current location.
2. Select View > Go To Plane. Enter “2” in the data entry field and select OK. The
value of the z coordinate is now 2, which corresponds to the third plane in the
image, as the count starts at 0.
3. Next, select View > Previous Plane and View > Next Plane commands,
observing the value of the z coordinate in the upper left corner of the info/tool
bar.
4. If your mouse is equipped with a scroll wheel it can also be used to move through
the planes of the image.
2.3.3 Tool Menu
The tool menu lets you choose the function the mouse cursor performs.
2.3.3.1
The Marquee Tool
The marquee tool is the default tool of the tool menu and allows you to select a two- or
three-dimensional region of the image.
1. Click and drag the mouse over a region in the Main View. Note that the cursor
appears as cross-hairs. Release the mouse button.
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SlideBook™ 5.0 User Manual
2. You have just selected a 2D region using the marquee tool. In the visible axes (x
and y), the marquee tool works as in any drawing program, but because
SlideBook is designed around the 3D images, a marquee must be manipulated in
the invisible plane as well. To illustrate this point move to different z positions
in the image using one of the methods outlined above. Notice that the marquee
selection only appears in the plane in which it was originally drawn.
3. With the marquee region still selected, go to View > Select Rect in All Planes.
Again, scroll through the different z planes in the image. Notice that now the
marquee selection exists in all planes of the image.
4. Click once on the image to remove the marquee from the view.
NOTE: There are other ways to select 3D regions that will be demonstrated later
in the tour.
2.3.3.2
Zoom Tool
The zoom tool is used to expand or shrink the size of the window (and the corresponding
zoom factor of the displayed image).
1. Click on the tool menu and select the zoom tool.
2. Click on the Main View image two times, zooming in on the image. Note that
the percent magnification is displayed in the upper left hand corner.
3. To zoom out on the image simply hold the shift button down while the zoom tool
is selected and click on the Main View image.
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4. Alternately, you may click on the “-“ and “+” buttons or utilize the drop down
menu in the upper left hand corner of the view window as illustrated below.
2.3.3.3
Hand Tool
The hand tool lets you move the displayed portion of an image that is in a window smaller
than the extent of the displayed axes.
1. Zoom in on the Main View image until it is larger than the confines of the
window as illustrated on the next page.
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SlideBook™ 5.0 User Manual
2. Select the hand tool button from the tool menu.
3. Click and drag the hand tool in the window, moving the portion of the image that
is displayed.
Notice that the hand tool performs the same function as the scroll bars on the
right and bottom sides of the Main View.
4. Return to a 100% zoom factor by any of the methods discussed previously.
2.3.4
Axis Menu
SlideBook is capable of visualizing data along all three axes.
1. Click on the axis menu on the tool bar and select y. Enlarge the image by using
the zoom tool.
2. Use the up and down arrows to move through the planes.
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Chapter 2 – Quick Tour
The y-axis is now the invisible axis. You are observing the xz-plane and scrolling
through the y-axis. You may view all three Cartesian planes simultaneously
using the Three View, which will be discussed later in the tour. The axis menu
options are unavailable in the Three View because all three planes are already
displayed.
3. Select the z axis from the axis menu to return the view to the xy-plane.
2.3.5
Renormalize Button
The renormalize button brings up the Renormalize Image dialog box. This tool allows you to
change the range of intensities that are displayed for any channel that you choose.
1. Click on the renormalize button. The following dialog box will appear:
A histogram is displayed showing the relative number of pixels on the y axis and
intensity values on the x axis for the specified channel. In this image, the CY3
channel intensities range from 0 to 14281.
2. Click on the drop-down channel menu and select FITC.
3. Click on the red and green bars in the histogram window and drag them to the
left or right, observing the change in the display of the green data.
The red and green bars allow you to select the minimum and maximum
intensities that correspond to the absence (black) or full saturation of color in the
display, respectively. Again, the underlying values in the image are not affected.
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SlideBook™ 5.0 User Manual
4. Now enter the numbers 300 and 800 in the Low and High data entry fields.
Note that the image does not automatically update as it does when moving the
red and green bars.
5. Click Apply to register the change and update the image.
6. Enter 400 and 1000 in the Low and High data entry fields and click on Apply to
register the change.
7. Choose OK to exit the Renormalize Image dialog box.
NOTE: You may also bring up the Renormalize Image dialog box by selecting View >
Renormalize.
2.3.6
Thumbnail Button
The thumbnail button lets you set the default view settings for a particular image: color
assignment of particular channels, renormalization information, and the z position of a
newly opened image. If you close the Main View that is currently open without setting a
default, the changes that you just made using the renormalize button will be lost.
1. Select View > Go to plane... and enter “10” in the edit box. (Note that plane
number 10 is actually the eleventh plane, as plane numbering starts at zero.)
2. Select OK.
3. Click on the thumbnail button to select your current display as the default.
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Chapter 2 – Quick Tour
Now all subsequent views of the image will default to the same display. You have
also changed the “thumbnail” image that is displayed in the slide window.
4. Open another Main View of the image by double-clicking on the thumbnail.
Note that the new settings that you entered previously apply to this new Main
View. Also, notice that the eleventh plane is displayed.
5. Close the Main View that you just created.
2.4
Generating a Three View
SlideBook allows you to view all three axes simultaneously using a Three View.
1. Select View > Three View to generate a new Three View.
A new window will open with an xz pane on the top, a yz pane on the left and an
xy pane in the middle. The most useful tool for exploring this view is the point
selection tool.
2. Choose the point selection tool icon in the tool menu
.
3. Click on the xy pane (center pane) of the Three View with this tool. Notice that
you update the xz and yz panes.
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SlideBook™ 5.0 User Manual
4. Hold the mouse down and drag it around the center pane to get a cutting plane
effect.
5. Click and drag the mouse along the x axis of the center pane, and note that the
yz plane is rapidly changing, while the xz pane is relatively still.
6. Next, click and drag the mouse on the yz pane to the left, and observe that the
other two planes are being updated.
Now we will make 3D selections in the Three View using the marquee tool.
7. Select View > Go To Plane. Enter plane 20 and select OK.
8. Next, choose the marquee tool from the tool menu in the info/tool bar.
9. Select the chromosomes by clicking and dragging a rectangle around them.
10. Then go to View > Define Selection Cube. The following dialog box will
appear:
11. The Define Selection Cube dialog box allows you to manually enter coordinates
for the selection box. Change the Beginning Z value to 10 and the End Z value
to 30. Notice that Extent Z has updated to indicate 20 planes are selected. Leave
the x and y coordinates unchanged and press OK.
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Chapter 2 – Quick Tour
Notice that the z extent of the marquee selection only spans the 20 planes that were
selected.
Next, we will spawn a Tile View that contains the data defined by the selection cube.
2.5
Spawning a Tile View
SlideBook can generate a plane-by-plane mosaic called a Tile View from any Main View or
3D selection. Now we will spawn a Tile View of the selection cube that we just created.
1. Select View > Tile View. A large a mosaic of the chromosomes is displayed with
each panel representing one of the 20 different z planes that was contained
within the marquee selection.
2. Select the hand tool
from the tool menu in the info/tool bar.
3. Click and drag around a single pane of the mosaic. This adjusts the position of
the display cube within the image, and allows all of the tiles to be readjusted
simultaneously.
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SlideBook™ 5.0 User Manual
4. Close the Tile View.
5. Open a Tile View from the Main View by first clicking on the Main View to
remove the marquee selection and then selecting View > Tile View. Notice that
the entire image space is now displayed.
NOTE: In order to display a Tile View of a portion of the image from the Main
View, you must first define a 3D selection.
6. Close the Tile View.
7. You may close the slide by either clicking on the
corner, or by selecting File > Close Slide.
2.6
in the upper right hand
Performing Volume Rendering
SlideBook can perform interactive volume renderings using the 3D Volume View. The
following rendering types are available. When you go to View > 3D Volume View, two
options will be available, High Speed and High Quality. High Speed will render the
image at the highest speed possible. High Quality will render the image with a more
rigorous algorithm that may improve the quality of the image with a small decrease in the
speed performance. These setting apply to all modalities of volume rendering but are most
noticeable when using Dynamic Lighting mode.

Dynamic Lighting – allows you to illuminate your object as you wish by
allowing control of the lighting angle. Useful for determining surface
characteristics.

Fixed Lighting – uses a fixed lighting pattern for shading, approximates
surface characteristics.

MIP – Displays the pixel of maximum intensity along the axis perpendicular to
the viewing plane. This type of rendering does not require an advanced graphics
card; however, performance of certain functions will be improved with one.

X Ray – Displays the summed intensity along the axis perpendicular to the
viewing plane.
1. Choose File > Open Slide and navigate to the Documentation folder in the
SlideBook directory. Double click on QuickTourWIN3.sld.
The following slide window appears that shows a thumbnail of one image. This
slide contains one 3D image of a mouse mammary gland section.
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Chapter 2 – Quick Tour
2. Click anywhere in the area of the image in the list and select View > 3D
Volume View > High Speed or High Quality, depending on the desired mode.
NOTE: If the image contains a marquee selection, SlideBook will only render the
portion of the image that is selected.
1. To change the channels that are displayed, use the channel menus as you would for
the Main View, Three View, or Tile View.
2. To alter the shading, grab the lighting tool by clicking on the icon in the upper right
hand corner
and rotating it to achieve the desired lighting.
3. To zoom into the view, click on the zoom tool
down in your view to zoom in and out.
and then click and drag up and
4. To rotate the image, click on the rotate tool
and then click and drag in the
direction that you wish to move the image. You may click and drag either directly
on the image or on the coordinate image in the lower right hand corner.
5. To move the image in the field of view, click on the hand tool icon
and move the image by clicking and dragging on it.
and then grab
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6. To instantly reorient your image, you may choose to display a specific face of the
volume by choosing the axis tool
Front, or Back.
and selecting Top, Bottom, Left, Right,
7. To select a different rendering mode, or to alter the colors displayed, select the
renormalization icon
. The Volume View Settings window will appear.
NOTE: The Volume View Settings window automatically opens when you open a 3D
Volume View.
Several features are offered in addition to the ability to renormalize the image
(change the lookup tables).
8. Alter the background color using the Background slider and change the brightness
of the gridlines using the Gridlines slider.
9. You may alter the opacity of an individual channel using the Opacity slider control
that corresponds to each color.
10. Once you have explored this view, you may close the slide by selecting the
upper right hand corner, or by choosing File > Close Slide.
in the
For further information about volume rendering, please see Displaying a 3D or 4D Volume
View (Volume Rendering) on page 189.
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Chapter 2 – Quick Tour
2.7
Creating Masks and Generating Statistics
A mask is a two- or three-dimensional set of binary values that has the same extent as the
image to which it is assigned. Masks are often termed regions of interest (ROIs) in other
imaging programs. Masks are used for performing advanced selection and analysis of image
data and can either be created and edited manually or generated automatically through
threshold segmentation and other techniques. A single image can contain several different
masks. We will now learn how to create masks using a variety of techniques.
2.7.1 Creating a Mask using Threshold Techniques
1. Select File > Open Slide and navigate to QuickTourWIN2.sld in the
Documentation folder within the SlideBook directory. A slide window will
open that contains two images.
2. Double click on the “Fibroblast” thumbnail to open a Main View of the image.
3. Create a threshold mask by clicking on the mask menu icon
and selecting
Create Segment Mask. Alternately, you can reach this dialog by selecting
Mask > Segment. The following dialog box will appear:
4. Select Apply. The Generating Mask dialog box will appear and report the
segmentation progress by plane.
Once segmentation is complete, your entire image will have a blue overlay.
SlideBook has selected, or masked, any pixel that has intensity values that lie
between the red and green bars in each of the three channels.
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SlideBook™ 5.0 User Manual
5. Select Cy3 from the drop-down menu.
6. Move the red bar to the right by clicking and dragging it. Note that when you
release the mouse button, the image will be segmented again with the new
parameters and the mask will be updated.
7. Set the low threshold edit box to “550” and click on Apply.
Now SlideBook is masking any pixel with a Cy3 channel intensity value between
550 and 2715.
8. Scroll through the image using the scroll wheel or the up and down arrows in the
info/tool bar. Note that the mask persists throughout the entire image.
9. Close the Segment Image dialog box by pressing the OK button.
10. Change the red channel from Cy3 to None. Observe the mask that is displayed.
11. Click on the mask menu button
in the tool bar and select None. The mask
has been removed from the display.
12. Select Mask 1 from the mask menu. The mask is now displayed.
13. To delete the mask, select Mask > Delete. The following dialog box will appear.
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Chapter 2 – Quick Tour
14. Click OK to delete the mask from the image.
2.7.2 Creating Masks Manually (Background Subtraction Example)
SlideBook also offers a variety of tools that can be used to manually select regions of
interest. Note that this example illustrates multiple features of SlideBook.
1. Click on the Main View of the “Fibroblast” image.
2. Go to the middle of the focus range by selecting View > Go to Plane, entering
“7” in the edit box, and selecting OK.
3. Next, select Create Empty Mask from the mask menu or go to Mask > Create.
The following dialog box will appear.
4. Click OK to create a new mask for the current image.
Often when you perform analysis, you would like to specify a region to be used
for background subtraction. This can easily be done using the marquee tool.
5. Click and drag over a region that would be suitable for calculating the
background intensity.
6. Select Mask > Mark Selection, and notice that the region is highlighted in
blue.
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SlideBook™ 5.0 User Manual
7. This mask only exists in the current plane. To copy this region to multiple
planes go to Mask > Copy This Plane. The following dialog box will appear:
8. Leave the all planes radio button selected and click OK.
9. Scroll through the image and note that now the same masked region is selected
in all planes.
10. You can use SlideBook to get statistics from masked regions. Go to Statistics >
Mask Statistics. The following dialog box will appear:
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Chapter 2 – Quick Tour
11. Check the Entire Mask radio button in the Mask Scope section and then expand
the Intensity menu in the Features section of the Mask Statistics dialog box by
clicking on the + to the left of the checkbox. Then check Mean Intensity, and
then click Display. A table will appear that resembles the following.
These statistics can be exported as a tab-delimited file by selecting Export.
12. To perform background subtraction, choose Image > Channel Operations >
Channel Math. The following dialog box will appear:
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SlideBook™ 5.0 User Manual
13. Enter the mean background intensity value for the given channel (DAPI) to the
offset data field for the first channel, then select (no op). Enter a name, such as
“DAPI-Background”, for the new channel that will be generated. The dialog box
will look like this:
14. Select OK to create the background subtracted channel. This new channel will
now be present in the channel pull down menu within the Main View:
15. You may repeat steps 12-14 for each channel by selecting the appropriate
channel in the Channel Math dialog box and subtracting the relevant
background intensity value. When you are finished, close the Main View of the
Fibroblast image.
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Chapter 2 – Quick Tour
2.7.3 Defining Multiple Objects
Often you may have several distinct objects that will have intensities that are very similar,
and thus will segment together. A mask that contains several distinct objects may be
separated into objects, as we will now demonstrate.
1. Open a Main View of the “HeLa” image within the QuickTourWIN2.sld by double
clicking on the thumbnail.
2. Create a new threshold mask by selecting Create Segment Mask from the
mask menu. The following dialog box will appear:
3. Drag the red bar to the right until the low threshold value is approximately 550.
As you drag the bar, note the masked areas that appear on the image. Also, note
that the data that is being selected is shown in white on the histogram. Pixels
with intensity levels that lie in the gray area are not included in the mask.
Alternatively, type “550” in the Low data entry field and select Apply to set the
low threshold value. Your dialog box will appear as follows:
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SlideBook™ 5.0 User Manual
4. Select OK to create the mask.
5. Next, select Masks > Define Objects in Mask. The following dialog box will
appear:
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Chapter 2 – Quick Tour
Here you may choose to exclude objects based on minimum and maximum size
gates. Leave the box unchecked for now.
6. Select OK. A dialog box will appear that gives the status of the object generation.
When object generation is complete, the following box will appear, noting the
number of objects generated.
7. Click on OK, and now position the cursor over one of the objects in the Main
View. Note that the object number appears to the right of the cursor location
coordinates in the info/tool bar.
8. SlideBook can generate individual statistics for each object. Select Statistics >
Mask Statistics. The following dialog box will appear:
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SlideBook™ 5.0 User Manual
9. Expand the Morphometry and Intensity statistics selections by clicking on the
+ signs to the left of the checkboxes.
10. Select Area (pixels) and Mean Intensity, then select Display to generate
statistics. The following table will appear.
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Chapter 2 – Quick Tour
11. Expand the column headers by clicking and dragging the edge of the header to
the right. Click on any object number in the table. Notice that the corresponding
object in the image is highlighted in pink.
Notice that some of the objects are of very small size. If you would like to exclude
those regions when making objects, you may gate the objects by size. This can be
done either by going back to the Define Objects dialog box or through the
Statistics View itself.
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SlideBook™ 5.0 User Manual
12. To gate objects by size using the Define Objects window, first close the statistics
window. Go to Mask > Update Object Definitions (Note the menu item is
now named “Update Object Definitions” as opposed to “Define Objects” since
objects already exist for this image). The following dialog box will appear:
For the purposes of this demonstration, select the Gate Objects by Size
checkbox and enter a minimum size gate of 100 voxels. This will exclude any
objects from the mask that are less than 100 voxels.
13. Select OK to update the object definitions. Notice that SlideBook has generated
fewer objects than when this operation was performed without a size gate.
14. Statistics can now be generated on these new objects as described above.
15. To gate objects by size using the Statistics window select Statistics > Mask
Statistics. The following dialog box will appear:
16. Now click the Remove objects smaller than: check box located under the
Primary Mask drop down menu and enter a minimum size gate of 100 in the
active edit box.
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Chapter 2 – Quick Tour
17. Select the statistics you would like to have computed and click Display.
SlideBook will automatically remove objects smaller than the minimum size gate
during statistics computation.
18. The statistics table may be exported for use in other programs by clicking the
Export button. A dialog box will appear that will allow you to choose the file
destination. The file will be saved as a tab-delineated text file (*.txt), which
can be opened by various programs, such as Excel.
19. Close the slide by clicking on the
by selecting File > Close Slide.
in the upper right corner of the slide view or
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3 SlideBook™ Organization
SlideBook was designed to seamlessly integrate microscope control and image operations
such as capture, display, and analysis. In this chapter you will learn about the organization
of SlideBook elements. The following topics will be covered:


3.1
Hardware Control and Image Capture
Data Storage, Display, and Analysis
Hardware Control and Image Capture (Chapters 5, 6 and 7)
In order to capture an image, two distinct processes are performed. First, a sample is
brought into view and focus using the Focus Controls. Second, image capture parameters
are set and capture is initiated in the Capture dialog box. Once the capture begins, it can be
monitored in the Capture Controls.
3.1.1
Focus Controls
In this window, the microscope parameters are selected and moved into position, if
motorized. A semi-live stream of images are captured and displayed in the Focus Controls.
Additionally, parameters for the XY stage, such as points of interest, and for the Z stage,
such as the depth for a 3D capture, can be set in this window. Once the hardware is set as
desired and the sample is in focus, the user will move to the Capture dialog box. See
Chapter 5 for further explanation of the Focus Window.
3.1.2
Capture Dialog Box
Camera and capture parameters are set in the Capture dialog box. The appropriate
exposure time and capture sequence are determined in this window. For instance, the user
will decide whether the capture will be two-dimensional, three-dimensional, twodimensional over time or three-dimensional over time. Parameters set in the Focus
Controls, such as multiple points in the xy plane and z distances, can be imported into the
Capture dialog box and utilized during the capture sequence. See Chapters 6 and 7 for
further explanation of the Capture dialog box.
3.1.3 Capture Controls
The Capture Controls window shows the progress of a capture sequence. The features in
this window are most useful when performing timelapse acquisition and are discussed in
Chapter 7.
3.2
Data Storage, Display, and Analysis (Chapters 8, 9, and 10)
SlideBook’s image visualization and analysis tools are designed around five principal
objects: image, channel, view, mask, statistics, and slide. This organization is somewhat
different than other image processing programs. For instance, with this organization,
operations that change data are kept separate from those that only affect its display or
analysis.
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Chapter 3 – SlideBook™ Organization
3.2.1 Images and channels
An image is a two- or three-dimensional set of data collected by a digital microscope or
imported from another application. It consists of one or more channels, where each channel
corresponds to a different fluorophore. For instance, one channel may represent a FITC
labeled protein while another represents a CY3 labeled protein.
An image (and its constituent channels) represents the underlying data. Operations that
can alter the underlying data (such as deconvolution, flat field correction, and cropping) are
found under the Image menu.
3.2.2 Views
Unlike images, views represent a visual interpretation of the image’s data. SlideBook
currently has two classes of views: data views and display views. It is within a view that
data can be renormalized (altering the mapping from image intensity levels to red, green, or
blue intensities on the display) and colors can be assigned to different channels. You can
have multiple views open on a given image at one time. Commands used to create views are
found under the View menu.
3.2.3 Masks and Statistics
A mask is a two-, three-, or four-dimensional set of binary values that has the same extent
as the image to which it is assigned. Masks are often termed regions of interest (ROIs) in
other imaging programs. Masks are used for performing advanced selection and analysis of
image data and can either be created and edited manually or generated automatically
through threshold segmentation and other techniques. A single image can contain several
different masks. Commands used to create and manipulate masks are found under the
Mask menu. Statistics can be viewed and exported for both masks and image tools from
the Statistics menu.
3.2.4 Slides
Images in SlideBook are organized into slides. Typically, a slide corresponds to a number of
images captured from a single sample, and it is the native file type saved by SlideBook. By
collecting related images into a single slide, SlideBook makes managing files easier. Slides
appear on your file system with a .sld extension.
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4 Configuring Your System
In order for SlideBook to control any motorized aspects of your microscope system and to
perform deconvolution and analysis of images, it must know the optical and system
parameters of the microscope used to collect the image. By the end of this chapter, you will
know how to:







Designate User Logins
Configure Hardware
Define Objectives
Define Filters
Define Magnification Changers
Work with SlideBook Preferences and Hardware Properties
Define System Parameters
Once the hardware is configured and the optical parameters are defined, they can be
utilized in several different ways. If the optics are motorized, they may be selected and
brought into the light train using the focus window. Once an image is captured using the
selected motorized optics, the capture information is automatically stored with the image. If
the optics are not motorized, they may be selected in the capture window and then
automatically stored with the image information. Also, optics information can be added or
edited post-capture, which is especially useful for imported images.
4.1
Designating User Logins
You may define multiple user logins to separate user preferences. The user login will allow
individual users to access their capture preferences (see Saving Capture Parameters on
page 154). Hardware, filter, and objective configurations are shared among all users. To
configure user logins:
1. Go to Edit > Hardware Properties and scroll down until User Login
Properties is visible.
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Chapter 4 – Configuring Your System
2. Click on the + next to User Login Properties to expand the section, and then
click on Enable User Login.
3. Click Yes and then Apply Change.
4. Restart SlideBook. Upon startup, you will see the following screen:
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5. To add a user, click Add User. The following dialog will appear:
6. Enter the desired user name in the User Name in the edit field and click OK.
7. Upon restart, choose your user name from the drop-down list and then click
Login. Your capture preferences will be available in the Capture dialog.
4.2
Configuring Hardware
SlideBook has the ability to control a range of hardware, including the following:







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CCD cameras
Automated microscope components
 Objective turret
 Magnification changer turret
 Ocular/Photo switch
Fluorescence lightpath hardware
 Filter wheels
 Filter turret
 Shutter
Automated xy and z stages
Image Splitters
Transmitted (brightfield) lightpath hardware
 Shutter
 Lamp
 LCD filter
TTL controllable hardware
Chapter 4 – Configuring Your System
When you start SlideBook for the first time, it will notice that a preferences file, which is
stored along with the application, does not exist. SlideBook will create a default
preferences file. As you configure your hardware and define optics, SlideBook will update
this preferences file with specific information about your setup.
4.2.1 General Hardware Configuration
To configure your hardware:
1. Start SlideBook by selecting the program from the Start menu.
2. Select Edit > Hardware Configuration. The following dialog box will appear:
3. Name the system in the System name edit field. This should be descriptive and
include the laboratory name so that the preferences file can be identified by 3i
technical support.
4. Select the components that are present from the drop-down menus (set to None
in the dialog box above).
5. For each component that is controlled through a COM port (or USB port mapped
to a COM port), select the COM port to which it is connected in the drop-down
menus to the right of each component. If a component is not present, or you
would not like it to communicate with SlideBook, be sure the COM port is set to
Off.
NOTE: A typical PC has two serial COM ports, port 1 and port 2, located at the rear of the
machine. Additional COM ports are often added (e.g., through use of an Edgeport adapter
or PCI card). These additional ports are then typically termed port 3, port 4, etc. instead of
following the labels on the adapter.
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6. Select OK.
7. Restart SlideBook to register the changes and begin hardware communication.
8. If you plan to control your hardware via TTL control, please consult the
SlideBook TTL Module Manual.
NOTE: There are instances when you will need to select your objective and filter in the
Focus Window, even if these components are not motorized. For example, if you would like
to perform auto focus you will need to select the objective and filter you are using. In these
instances, you will define Manual Filter Turret or Manual Objective Turret (COM port set
to OFF). Make sure that you specify the position of your objectives in the Objective
Definition section (see page 62).
4.2.2 Configuring a Motorized XY Stage
In order for your motorized xy stage to work properly when collecting montage images, you
will need to specify the relationship between the stage and camera.
1. To do so, you will first need to bring a sample into view and focus in the focus
window (see Using the Focus Window on page 85).
2. Using the joystick, move the stage so that the image on the screen moves to the
left (in other words, pan and move the image field to the right). When panning to
the right, notice whether the x coordinate position in the XY Stage section of the
Focus Controls window increases or decreases.
3. Repeat for the y axis by panning down so that the image appears to move up.
Note the change in the y axis coordinate position (increasing or decreasing) when
panning.
4. Select Edit > Hardware Properties. The following Edit Hardware Properties
dialog box will appear.
5. Click on the + sign next to XY Stage to expand the list.
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Chapter 4 – Configuring Your System
6. Click on the item in the list titled MoveFieldRightSign. The left side of the
dialog box will be updated.
7. Enter the appropriate value, 1 or -1 based on the change in x position values
when panning right, in the edit field and select Apply Change.
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8. Repeat steps 6 and 7 for the y axis by clicking on the MoveFieldDownSign.
9. Close the dialog box using the Close button.
4.2.3 Configuring an Image Splitter
SlideBook supports the use of image splitter devices. An image splitter allows you to
capture two separate channels for the right and left halves of the camera. To configure an
image splitter:
1. Select Edit > Hardware Properties and click on the + sign to expand the
Simultaneous Capture section.
2. Click on Split Capture. The left side of the dialog box will change.
3. Select Yes, Apply Change, and then Close.
4. Restart SlideBook to register the changes.
To use the image splitter, you must define filters appropriately (see Defining Filters when
using an Image Splitter on page 72). To align your image splitter, use the Test Dual-View
function in the Focus Window as described on page 85. To capture images using your image
splitter, use the Simultaneous Capture checkbox as described on page 133.
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4.2.4 Configuring Camera and Ocular Ports
If your system has motorized ocular photo and CCD/Video prisms as well as multiple
cameras, you may define the positions of the prisms that correspond to each camera. To do
so:
1. Select Edit > Hardware Properties and expand the section titled Camera 1.
2. Click on Prism1Pos. The right side of the dialog will be updated.
3. Enter the value of the ocular photo prism (Prism 1) position that corresponds to
the position of Camera 1 in the edit field.
4. Select Apply Change.
5. Repeat steps 4 and 5 for Prism2Pos (CCD/Video prism).
6. Repeat steps 2 through 5 for any other cameras and the ocular lightpath.
Once you have defined the prism positions, you can click on any of your cameras in the
Camera tab and the prisms will move to direct light to that camera. Additionally, if you
have defined filters for specific cameras, the prism positions will move to the appropriate
camera when you select a filter in the Focus Controls.
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4.3
Defining Objectives
Once an objective is defined, it can be controlled (if the objective turret is motorized) from
the focus window. If the objective turret is not motorized, it may be selected in the capture
window and the objective definition will automatically be stored with the image
information. If the objective was used to capture an image that has been imported, the
objective information can be added to the image information.
4.3.1 Adding a New Objective
In order to define a new objective for your system, do the following:
1. Select Edit > Define Optics > Objectives. The following dialog box will
appear:
2. To add a new objective definition, click on the Add button.
3. Enter the objective name, magnification, numerical aperture, working distance,
and m/pixel. You must give the lens a name and fill out all parameter fields,
except those in the Parfocality and Parcentricity and Transmitted Light
Configuration groups.
NOTE: For optimal results, objective pixel size should be measured by
traditional means using a stage micrometer. If this is not possible, a close
estimate can be calculated by dividing the camera pixel size by the magnification
of the objective. Please note that this will not take into account any optics that
alter magnification such as the Spherical Aberration Correction device by 3i.
4. Select the location of objectives from the Position drop-down menu. If you have
a motorized objective turret, Position should reflect the position of the objective
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on the microscope; if you are using SlideBook offline or with a non-motorized
turret, you should select Unmounted. If you are using a non-motorized turret,
but have selected Manual Turret in your hardware configuration, select the
appropriate turret position.
5. Select the UV checkbox for UV objectives and select the appropriate medium
from the radio button menu.
6. Select the appropriate configurations for the desired transmitted light condenser
turrets using the drop-down menus on the right. Once you have defined the
condenser positions for a given objective, you may define filters that utilize the
appropriate condenser positions. Filter definitions are discussed in the next
section.
7. Repeat steps 1 through 6 for any additional objectives that you wish to add.
8. Select OK when you are finished.
9. Restart SlideBook to register the changes that you have made.
4.3.2 Modifying Information for an Existing Objective
You can modify existing settings as follows:
1. Select Edit > Define Optics > Objectives to bring up the Objective Parameters
dialog box.
2. Select the appropriate lens using the Objective Lens dropdown list.
3. Change the appropriate fields as desired.
4. Select OK to save the changes.
5. Restart SlideBook to register the changes that you have made.
4.3.3 Removing an Objective
1. Select Edit > Define Optics > Objectives to bring up the Objective Parameters
dialog box.
2. Select the appropriate lens using the Objective Lens dropdown list.
3. Choose Remove then OK to save the changes.
4. Restart SlideBook to register the changes that you have made.
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4.4
Defining Filter Configurations
You can add, modify, and remove filter configurations using the Filter Configuration
Parameters dialog box. The components of this dialog box and instructions for adding,
modifying and removing filter configurations are explained below.
4.4.1 Defining Filter Sets
SlideBook allows you to assign a particular filter to one of six groupings, or filter sets. These
sets can be chosen in the focus window and capture window. The default sets are Fixed,
Live, User 1, User 2, User 3, and User 4. Sets allow you to work with many different
filter definitions in an orderly fashion. To change these default group names:
1. Select Edit > Hardware Properties. The Filter Configuration Parameters
dialog box will appear.
2. Scroll down to find the property Filter Set Information and click on the + sign
to expand as shown below:
3. Click on the Filter Set Label that you wish to change.
4. Enter the text for your filter set. Examples may be a specific user name or a type
of experiment.
5. Click Apply Change and then Close to register your changes.
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4.4.2 Filter Configuration Parameters
The Filter Configuration Parameters dialog box is found by selecting Edit > Define Optics
> Filter Configurations.
4.4.2.1 Light Source
Here you may choose which light source will be used. If your microscope has an automated
brightfield lamp, the lamp will be switched on and off (or shuttered) for each brightfield
image that is acquired. Also, if you have a motorized condenser turret, you may select
whether a transmitted light channel is brightfield, darkfield, DIC (Nomarski), or phase. If
you check the Prompt if mode is not available for current objective checkbox,
SlideBook will automatically warn you if objective and filter definitions are inconsistent.
For fluorescent channels, input the emission wavelength in microns in the edit field. You
should also indicate whether the fluor requires a UV objective. SlideBook will automatically
check for consistency between objective type and fluorescent channel type and prompt you if
there is a problem.
NOTE: You MUST have condenser turret positions defined for objectives if you would like
the condenser turret position to correspond to a specific transmitted light channel (see
Adding a New Objective).
4.4.2.2 Filter Set
SlideBook allows you to assign a particular filter to one of six groupings, or sets. The
default sets are Widefield, Confocal, Transmitted, TIRF, Life Time, and FRAP. You
may also edit these set names as shown on page 64. These sets can be chosen in the Focus
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Controls and the Capture Controls. Sets allow you to work with many different filter
definitions in an orderly fashion.
4.4.2.3 Filter Positions
SlideBook permits filters to be defined in up to five positions within the optic path. For
systems equipped with filter wheels or a rapid filter switcher (such as the Sutter DG-4 or
TILL Polychrome V), use the Excitation Wheel Position and Emission Wheel Position
fields. If your system has a motorized filter cube turret, you must specify the Internal
Turret Position for the filter configuration’s cube. If your system does not have a
motorized filter cube turret, you should still enter the position of the cube. Then, when you
select the filter from the Focus Controls, SlideBook will prompt you to move the turret to
the appropriate position. Some examples of how to use filter positions are described in the
section Adding a New Filter Configuration on page 68.
NOTE: If you have a manual filter turret, SlideBook will prompt you to move the beam
splitter when performing multi-channel capture. In cases where you do not want a prompt
for a different beam splitter (or in the motorized case, you do not want the internal turret to
move), you can simply set Internal Turret Position to Unmounted. If you believe you
are seeing a prompt in error, you may wish to review your filter configuration to make sure
that your positions are defined correctly and also check your hardware configuration to
ensure that any motorized elements are defined properly.
Finally, if you have an LCD color filter as part of your system, the LCD/BF Filter
Position field can be set to select positions for individual colors.
4.4.2.4 Camera
SlideBook supports use of multiple cameras. If you have more than one camera, you may
select the camera that will be used for capturing the given filter configuration.
4.4.2.5 Channel Types
All fluorescent filter configurations that are not used for ratio imaging should be set to
Independent. Configurations for ratio and FRET channels are discussed in the Ratio/
FRET Module manual. Brightfield channels that use a color camera should be set to RGB
Color. Brightfield channels that use an LCD color filter, or three separate color filter
cubes, should be set to Virtual RGB (red), (green), or (blue).
4.4.2.6 Default Color Display
You can always change the colors (or color display type) of data captured with different
filter configurations. The Default Color Display, however, is a convenient way to choose
how data from different filter configurations is displayed in the capture status dialog box as
well as the initial view created for an image. Channels may be set to standard RGB,
pseudocolor, monochrome, user-defined, or set to the emission wavelength.
4.4.2.6.1 Pseudocolor
Pseudocolor (Color) and Pseudocolor (Intensity) together determine the look of a
pseudocolored image. A filter configuration that has Pseudocolor (Color) as its default
will set the hue value of the display according to the normalized (i.e., scaled from minimum
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to maximum) value of each voxel. A filter configuration that has Pseudocolor (Intensity)
as its default will gate the intensity of the pseudocolored image according to the normalized
value of each voxel. If a captured image contains a channel with a filter configuration set to
Pseudocolor (Color) but not Pseudocolor (Intensity), the corresponding view will show
a range of hues at maximum intensity (i.e., ranging from bright blue to bright red). If the
image also contains a channel with a filter configuration set to Pseudocolor (Intensity),
the corresponding view will show a range from dark blue (black) to bright red. If the image
only contains a channel with a filter configuration set to Pseudocolor (Intensity) and no
Pseudocolor (Color) counterpart, the default setting will be treated as if it were set to
None.
Pseudocolor color types cannot be displayed along with red, green, or blue channels.
Monochrome settings may only be used for a background channel when displayed with RGB
channels. In multiple channel images, the pseudocolor setting will override any red, green,
or blue settings. Red, green, or blue settings will in turn override a monochrome setting.
Again, this doesn’t mean you can’t easily get back to viewing any other channels, but simply
determines what will be visible initially.
4.4.2.6.2 User-Defined Color
You may elect to set the default color to one of your choosing. To do so, select the UserDefined radio button, and then click on the color block to the right. A dialog box similar to
the following will appear.
Select one of the basic colors by clicking on the color block in the upper left hand color, or
design a custom color using the color spectrum on the right and choose Add to Custom
Colors.
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4.4.3 Adding a New Filter Configuration
1. Select Edit > Define Optics > Filter Configurations. The Filter
Configuration Parameters dialog box will appear.
2. To add a new filter configuration definition, click on the Add button.
3. Enter the filter name in the data entry field.
4. Select the light source, and enter the emission wavelength in m (for fluorescent
filters). Also, if the filter requires a UV objective, select the checkbox under the
emission wavelength data field. The Light Source field permits sequential
brightfield (or Nomarski) and fluorescence imaging on systems equipped with
both a fluorescence shutter and either a brightfield shutter or electronic voltage
control.
NOTE: You may configure multiple transmitted light channels if you have a
motorized condenser turret. For instance, you may define a DIC channel by
selecting Transmitted light source and DIC from the Mode dropdown menu.
You could also define a phase channel by creating a new channel with Phase
selected from the Mode drop-down menu. Then, if you performed capture with
either of these channels, the condenser turret would move to the appropriate
position automatically. You must define the condenser turret positions when
defining objectives for this feature to work properly (see section Adding a New
Objective).
5. Select the desired Filter Set from the dropdown menu.
6. If the exciter is located in an external excitation wheel, select the position of the
filter from the Excitation Wheel Position dropdown menu. If it is mounted in
a cube in the internal turret or if it is not currently mounted on the system,
select Unmounted.
7. Select the internal turret position of the dichroic mirror from the Internal
Turret Position dropdown menu. If the dichroic mirror is not currently
mounted, you should select Unmounted.
8. If the emitter is located in an external emission wheel, select the position of the
filter from the Emission Wheel Position dropdown menu. If it is mounted in a
cube in the internal turret or if it is not currently mounted on the system, select
Unmounted.
9. Repeat steps 1 through 8 for any additional filter configurations that you wish to
add.
10. Select OK when you are finished.
11. Restart SlideBook to register the changes that you have made.
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4.4.3.1 Example Filter Configuration for Fluorescence Channels
Example – Quad Pass and Dual Pass sets on a system with filter wheels and motorized
turret
Some systems with filter wheels will need different beam splitters to be in position for
different sets of fluorophores. For instance, a system may have excitation and emission
filter wheels loaded with filters for DAPI, CFP, FITC, YFP, CY3, and CY5. The DAPI,
FITC, CY3, and CY5 filters require a quad pass beam splitter designed for that set of
fluorophores, while CFP and YFP require a dual pass beam splitter which is designed
specifically for those two fluorophores.
In a system with filter wheels and a motorized turret, Internal Turret Position should be
set to the appropriate beam splitter (or dichroic) position for each filter configuration. In a
system with filter wheels but no motorized turret, the Internal Turret Position field
should be set differently for each set of fluors that require a different beam splitter if you
want SlideBook to prompt you to move to the necessary beam splitter. Given the example
above, even if the system does not have a motorized turret, the filter configurations for
DAPI, FITC, CY3, and CY5 should have Internal Filter Position set to say, 1, while CFP
and YFP have Internal Filter Position set to 2.
4.4.3.2 Configuring Channels for Color Cameras or Color LCD sliders
If you have a color camera, you will need one filter definition that appears as follows (you
must enter the appropriate location of an empty turret and emission wheel position if
applicable):
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If you have a LCD slider, such as that made by CRI, you will need to configure three
separate channels; however, these three channels will appear as one channel in the capture
dialog. You must define one channel each for red, green, and blue. For the color LCD slider,
the filter definitions must appear as follows. Create the definitions in the following order:
red, blue, green. This is because you will most often perform autofocusing with the green
channel, and autofocusing will be performed with the last channel captured. For the red
channel, you may also choose “Red” as the Default Color Display. RGB Settings allows
you to manually enter white balance values. You may find that using the white balance
function in the capture dialog is sufficient (please see Performing Auto-White Balance for
Color Capture on page 95).
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4.4.3.3 Defining Filters when using an Image Splitter
If you have configured an image splitter, such as the Dual-View from Optical Insights, you
should define two filters that have identical excitation wheel positions (if applicable) and
identical internal turret positions. Then, depending on the position of the image splitter,
select one channel Camera position as Camera 1, and the second channel as Camera 2.
For instance, if you are performing CFP/YFP FRET, you may wish to define four filter
configurations:
1. YFP excitation, YFP emission,
2. YFP excitation, CFP emission (this is a dummy filter that must be configured,
but will not contain information),
3. CFP excitation, YFP emission,
4. CFP excitation, CFP emission.
In this example, filters 1 and 3 will use Camera 1, while filters 2 and 4 will use Camera 2.
Also, filters 1 and 2 will use the same excitation position, as will filters 3 and 4. You must
define the filters in order so that the channels within each pair that share an exciter are
adjacent. In the example above, channels would be defined in order of 1, 2, 3, 4.
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4.4.4 Modifying Information for an Existing Filter Configuration
1. Select Edit > Define Optics > Filter Configurations. The Filter
Configurations Parameters dialog box will appear.
2. Select the appropriate filter using the Filter Configuration dropdown list.
3. Modify the appropriate fields as desired.
4. Select OK to save the changes.
5. Restart SlideBook to register the changes that you have made.
4.4.5 Removing a Filter
1. Select Edit > Define Optics > Filter Configurations. The Filter
Configurations Parameters dialog box will appear.
2. Select the appropriate filter using the Filter Configuration dropdown list.
3. Choose Remove, then OK to save the changes.
4. Restart SlideBook to register the changes that you have made.
4.5
Defining Magnification Changers
A magnification changer, or optovar, is a component which your microscope may or may not
have. It acts to increase the effective magnification of an objective. If you do not have a
magnification changer, you should use the predefined “1.0 x” definition.
4.5.1 Adding a New Magnification Changer
1. Selecting Edit > Define Optics > Mag. Changers. The Mag. Changer
Parameters dialog box will appear:
2. Click on the Add button.
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3. Enter a Name in the data entry field, and the Magnification value.
4. Click OK to save the definition.
5. Restart SlideBook to register the changes that you have made.
4.5.2 Removing or Modifying Magnification Changer Definitions
You may remove and alter magnification changer definitions as you would for objectives
and filter configurations.
Note that if you switch between several camera adapters with different relay lens
magnifications, you can also use magnification changer definitions to specify which relay
lens is in place.
4.6
Working with SlideBook Preferences and Hardware Properties
SlideBook stores two different types of configuration files, User Preferences and System
Preferences. User Preferences refers to settings that are user-specific such as saved capture
settings or saved particle tracking protocols. System Preferences refers to system wide
settings such as hardware configuration and hardware properties. There are several tools
built into the software to help you manage and backup preference files as discussed below.
4.6.1 Backing Up SlideBook Configuration
1. To back up your SlideBook configuration, go to Edit > Setup Guides > Backup
Preferences, the default behavior is to save the backup files file in C:\Program
Files\Intelligent Imaging Innovations, Inc\SlideBook 5.0.
2. Select where you would like the backup files to be located. If desired, create and
name a new folder.
3. Click OK.
4. Three folders containing system configuration will be placed in the directory
designated. The folders are as follows:
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
Exceptions – This folder contains backup data from interrupted sessions
and will be discussed later in this section.

Global Preferences – This folder contains the system configuration files
such as SlideBookHardwareProperties.dat, SlideBookPrefs.dat,
PSFs.dat, and FlatFields.dat.

Users – This folder will contain files specific to individual users or the
default user. This is where saved capture settings, point lists, and
analysis protocols are saved.
Chapter 4 – Configuring Your System
4.6.2 Restoring SlideBook Configuration
1. In order to restore your system preferences, you must first open the backup
configuration folder. This file may be located at C:\Program
Files\Intelligent Imaging Innovations, Inc\SlideBook 5.0 or a
user-designated location as specified in Step 2 above.
2. Double-click on the Global Preferences folder and select all of the files and
folders. Copy the data by right-clicking and selecting Copy from the menu.
3.
Now, return to SlideBook and go to Edit > Setup Guides > Open System
Preferences Folder.
4. Click inside of the Windows Explorer folder view that opens. Right-click and
select Paste from the menu.
5. You will be informed that there is already a file in this location with the same
name. Select the option to overwrite all files or folders and click the box to apply
to all files and continue.
6. You have now replaced the active system configuration files with the backup
copies. Now you will restore the user preferences.
7. Go back to the backup configuration from Step 1.
8. Double-click on the Users folder and select all of the files and folders. Copy the
data by right-clicking and selecting Copy from the menu.
9. Now, return to SlideBook and go to Edit > Setup Guides > Open User
Preferences Folder.
10. Click inside of the Windows Explorer folder view that opens. Right-click and
select Paste from the menu.
11. You will be informed that there is already a file in this location with the same
name. Select the option to overwrite all files or folders and click the box to apply
to all files and continue.
12. Close all folder views and restart SlideBook.
4.6.3 Exceptions
Exceptions is a folder that is backed up with your system and user configurations. This
folder may contain a number of folders that begin with “ExceptDump”. These files are
created when a process is interrupted in SlideBook. If your SlideBook session is
interrupted and your data was not saved, you can recover the data by looking in the
Exceptions folder. It may be useful to monitor this directory and delete unnecessary files.
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4.7
Defining System Parameters
You can adjust the z-axis increment size, the default interplane spacing, the index of
refraction of the oil you are using, and the disk drive on which to put temporary files using
Edit > Define Optics > System Parameters:
For microscope automation to work correctly, you need to enter the size of a single z-axis
increment of the stage (in microns) in the Microscope Step Size field. When you import
files from other applications, SlideBook will assume a default interplane spacing (in
microns) that is defined in the Default Import Spacing field.
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5 Controlling the Camera and Microscope Hardware (Focus Window)
SlideBook provides a Focus Window for automated hardware control and semi-live camera
readout. The Focus Window will allow you to choose the appropriate optics and bring your
sample into focus. It will also allow you to set parameters for advanced capture, including
top and bottom z limits for 3D imaging, xy coordinates for multipoint acquisition, and
boundaries for a generating a montage. This chapter covers the following topics:


Focus Window Features
Using the Focus Window
The use of the Focus Window for advanced capture is discussed in detail in Chapter 7.
5.1
Focus Window Features
The Focus Window can be opened by either selecting Window > Focus Window or
clicking on the focus window button in the toolbar
. The Focus Window consists of a
Focus Controls dialog box and the Live Window, which operates in a live mode when the
Focus Window is selected. The Live Window is also used for capture as discussed in
Chapters 6 and 7.
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The Focus Controls dialog box always has at least two tabs: Scope, and Camera that
control and report the status of automated microscope components and cameras. Typically
systems also have either a Z tab or an XY tab or both. Additionally, there are a number of
permanent controls in the lower half of the dialog box that are displayed no matter which
tab is selected.
5.1.1 Permanent Controls
The lower half of the Focus Controls dialog is present regardless of the tab selected.
5.1.1.1 Camera and Display Settings
The Exposure time slide bar allows you to select the duration of a single camera exposure
(between 1 and 500ms). This exposure is repeated continuously to form the semi-live image
display. You can also select between four bin factors, all of which are based on square pixel
groupings (i.e., 1x1, 2x2, etc). Binning increases camera sensitivity and readout speed. This
is useful in the focus window because it allow shorter exposure times and faster image
refresh rates, ultimately making it easier to find a sample and bring it into focus.
Importantly, the focus window exposure time and bin factor adjustments are used only for
optimizing image display in the focus window. Adjusting the camera settings for capturing
an image is covered in Chapter 6 on page 89.
On systems with multiple cameras and motorized camera port selection, the Camera dropdown menu lets you direct the light to the desired camera and display the resulting image.
On systems with two cameras running simultaneously from a single port, these buttons let
you select which camera’s output will be displayed in the Focus Window.
The Start/Stop button starts (or stops) continuous camera capture. This can be
particularly useful if you have a mechanically shuttered camera since the focus window can
be left open without taking unnecessary camera exposures. You may also press Snap to
grab a single image that will be stored in an open slide.
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The
and
buttons in the bottom left corner are used to zoom in and out on the image
display. Zooming in on the display can be particularly useful for focusing on small objects.
Selecting the Guides check box will bring up yellow guides that mark a 256 × 256, 512 ×
512, and 1024 × 1024 pixel region in the image. The Color check box is used to display a
color brightfield image in the focus window. Use of this feature requires specific hardware
for capturing color images, such as an LCD color filter changer or color camera.
5.1.1.2 Open Fluor, Open Bright, Open Alt
The Open Fluor button is used to toggle the fluorescence light source if there is a
computer-controlled shutter or dark slider in the reflected light path.
The Open Bright button is used to toggle the brightfield light source if there is either a
computer-controlled shutter in the transmitted light path or the microscope has computer
control over the transmitted light source voltage.
The Open Alt button is used to toggle an alternate source if it is present. For example, if
you have a NEOS AOTF laser system, or a shuttered TIRF laser, Open Alt will toggle the
laser light on and off.
5.1.1.3 Filter Controls
You may select a filter set using the filter set drop-down menu. Each button corresponds to
a filter configuration that you have defined for the system. (For more information on
creating new filter configurations, please refer to the section Defining Filter Configurations
on page 64). If filter configurations have been assigned to a particular filter set, they can be
accessed using the drop-down menu. Depending on the configuration of your microscope
system, clicking on a filter configuration tab will change one or more of the following:




excitation filter wheel position
emission filter wheel position
internal filter turret (or slider) position
camera that is used for display (when the system has multiple cameras)
The filter configuration that is currently selected will be highlighted and displayed in bold
letters, while the rest of the configurations will have a normal button appearance.
5.1.1.4 XY Stage
On systems equipped with a motorized xy stage, you can use the directional buttons to
move the stage in increments specified by the value in the adjacent edit field. This value is
always given in microns (or fractions thereof). You can change this increment distance at
any time. The defaults are 100, 10, 1 and FOV (Field of View is an integer value defined by
the pixel array of the CCD) which can be selected from the drop-down menu.
TIP: If you double-click on an object in the image display, the xy stage will move so that the
object is centered.
The current x and y positions read from the stage are also displayed in this section.
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5.1.1.5 Z Stage
On systems equipped with motorized z focusing, you can move the focus up and down using
the directional buttons. There are three sets of buttons with predefined stage movement
increments (10, 1, and 0.1 microns). In addition, an user-defined stage movement increment
value can be entered in the edit field below the last set of buttons on the right. The default
value of the edit field will be set to the theoretical z resolution for the selected objective.
With few exceptions, the up and down buttons correspond to the direction of mechanical
movement. For instance, on an upright microscope with either an integrated or external
stepper motor, the direction corresponds to stage movement, so “up” means focusing further
past the cover slip and towards the slide since the stage is moving up. On an inverted
microscope, “up” corresponds to moving the nosepiece up, and therefore also means focusing
further past the cover slip.
The Auto focus Button is used to define and test parameters for auto focusing during
capture. Use of this feature is covered in Chapter 7.
5.1.1.6 Neutral Density
If you have defined neutral density positions in your hardware configuration, you may
select the position from the Neutral Density drop-down menu.
5.1.2 Scope
The Scope tab lets you observe and control other encoded and automated features of your
system.
5.1.2.1 Emission Selection
On systems with a motorized emission light path, these radio buttons let you choose
between letting the light go 100% to the camera, 100% to the oculars (eyepieces), or to 50%
each (if available).
5.1.2.2 Magnification Changer
On systems with an encoded magnification changer, this field reports its current position.
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5.1.2.3 Objectives
On systems with a motorized objective turret, you can move to a different objective by
selecting the corresponding button. (For information on adding objectives to those defined
for your system, please refer to the section Defining Objectives on page 62).
If you try to move between objectives that use differing immersion media, such as an oil
objective to an air objective, SlideBook will bring up a dialog box warning about the pending
move.
CAUTION: On most systems with motorized objective turrets, you can safely move between
objectives of similar immersion media (e.g., air, oil). However, there are situations where
you will definitely not want to move between objectives automatically. For instance, if you
are using a water immersion objective on an upright microscope, where the objective is
dipped into the chamber, trying to move the objective could break the chamber or damage
the motorized turret.
5.1.2.4 Stage Limits
On certain microscopes with motorized z focus drives, stage limits are used to control the
maximum and minimum position of the stage. These limits prevent the microscope from
being focused above or below certain z positions. The Stage Limits radio buttons are used
to enable or disable this feature.
5.1.2.5 Lamp
On microscopes with a computer controlled lamp voltage adjustment, the intensity of the
brightfield illumination can be controlled using this slider. Moving the slider to the far left
corresponds to turning the lamp off, while moving the button to the far right corresponds to
turning the lamp to maximum intensity.
5.1.2.6 Condenser
On microscopes with an automated condenser turret, you may select condenser positions
from the drop-down menu.
5.1.3 Z
There are several possible ways to set up 3D capture using the Focus Window. These
methods are explained in further detail in Chapter 7, 3D Capture on page 105. You must
first choose the basis for your capture. You may define your capture based on the current z
position, a selected reference position, or by defining the top and bottom of capture.
Examples of using the Focus Window for 3D capture are as follows:

Current z position - Use the Focus Window to find a plane of interest (for example
the focal plane), and then choose to range your capture around that position in the
Capture dialog.

Define a reference position in the Focus Window, and then define a total range in
microns or number of planes to capture in the Capture dialog.
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
Define a top and bottom position in the Focus Window, and choose to use those
positions in the Capture dialog. You may define the step size in either the Focus
Window or the Capture dialog.
The Z tab contains controls that let you make an interactive selection of a z axis range that
can be imported into the Capture dialog when performing 3D acquisition. It is available on
all systems that have motorized z focusing.
NOTE: You can also select a z axis range directly in the Capture dialog box (see 3D Capture
on page 105). The Z tab allows you to interactively define the 3D capture range and is
generally used under circumstances where the precise z extent of the sample is unknown.
5.1.3.1 Set Top, Set Bottom, Set Reference, Center
The Set Top and Set Bottom buttons let you specify the end points of a 3D capture range.
You can select these in either order. For instance, suppose you have found a cell that you
want to capture and are in the middle of the focus range. To set the boundaries of capture
in the z direction, first move the stage (or nosepiece) up until the focal plane is at the top of
the volume that you wish to capture. This can be done either by repeatedly clicking on one
of the “up” buttons or by adjusting the fine focus of the microscope itself. After you are
finished, click on the Set Top button. A number will appear above the Set Top button
indicating that the top z position has been set. Next, focus in the opposite direction until the
focal plane is at the bottom of the volume of interest. Click on the Set Bottom button.
Again, a number will appear below the Set Bottom button indicating that the bottom z
position has been set. Once you have set a top and bottom position, you may drag the z
position slider to return to either of those positions. In addition, SlideBook will calculate the
Center position based on your top and bottom positions. You may select Go to return to the
center position.
You may also set a reference, or home, position. This reference position can be the point of
best focus, or some other position that is of importance for your experiment. Once you set
this reference position, it may be used for 3D capture. To set the reference position, press
Set. This position will be saved until you reset the position or close SlideBook.
NOTE: If you do not wish to continuously expose your sample while performing z focusing,
you may set these ranges using single images generated in the Capture dialog box. Please
see 3D Capture on page 105.
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5.1.3.2 Slider
Once top and bottom z positions have been selected, the slider to the left of the buttons
provides the ability to move the z focus of the microscope to any position along the specified
range. This is useful for confirming that the specified region covers all of (and not
significantly more than) the volume you wish to capture and provides a means of returning
the z focus to the brightest part of the sample without altering the 3D capture range. When
you later wish to find optimal exposure times that won’t saturate the camera, it is best to be
focused on the z position with the highest peak intensities.
5.1.3.3 Capture Information
Choosing top and bottom positions in the Focus Window to set the extent of z travel is only
one part of setting up a 3D capture. You also need to specify how many planes (discrete z
focus positions) should be collected. Typically, the most important consideration is the step
size, as this governs the ultimate z axis resolution of the acquired image. The step size (in
microns) is specified in the corresponding edit box. SlideBook suggests a z step size based
on the theoretical resolution of the lens. The theoretical resolution is a function of
wavelength of the chosen filter and numerical aperture. You may adjust this step size by
entering a value in the Step Size edit field. Once top and bottom positions are defined,
SlideBook will use the step size to compute how many planes must be acquired to cover the
specified range. At any point a different step size can be entered and the computed number
of planes will be updated. Note that you can also specify the direction of travel of the stage
(or nosepiece). The stage and objective should always travel toward each other. Thus, the
direction of the arrow should normally point upwards.
You may also choose to set either number of planes or the step size in the Capture dialog,
as shown in 3D Capture on page 105.
5.1.4 XY
If your system is equipped with a motorized xy (and z) stage, the XY tab lets you specify,
store and visit multiple xy (or xyz) locations. These points can be used for visiting multiple
locations of interest in a sample and returning to them later as well as for specifying a
sequence of locations to repeatedly visit during timelapse acquisition.

Set Point - adds the current xyz location to the list

Clear Point - removes the currently selected xyz location from the list
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
Visit Point - moves the stage (and z focus) to the selected xyz location

Clear All - removes all locations from the list

Reset All Z – updates the z positions in all points to the current z position

New Point Z - updates the z position of the selected point to the current z
position

Montage Extent - sets the boundaries that will be used to create a montage
image.

Load – allows you to load a saved x,y,z point list

Save – allows you to save an x,y,z point list

Home – allows you to calibrate your x,y stage before loading a point list.
Using these features in the context of preparing to capture an image is also discussed in
Chapter 7-Advanced Capture.
NOTE: If your system has a motorized xy stage, but not motorized z focus, then the XY tab
works the same way except with xy rather than xyz locations.
5.1.5 Camera
The Camera Tab lets you control various settings on the CCD camera as well as view a
histogram of the pixel intensities for the image in the focus window.
5.1.5.1 Histogram
The histogram shows a plot of pixel intensities values for the focus window image. Pixel
intensity values are plotted on the x axis, while numbers of pixels at a given intensity are
plotted on the y axis. In addition, the minimum, maximum, and mean pixel intensity
values are reported beneath the histogram. This data is continuously updated when the
camera is actively acquiring images. The histogram can be operated in two modes as shown
in the Scale Image Display region of the window, Manual and Auto. In manual mode,
you may drag the red and green bars to set the min and max values that are displayed in
the live image. You may also enter numbers in the Low and High edit fields. In auto mode,
the live image will use the minimum and maximum intensity values of the live image to set
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the contrast in the image. Thus, if you increase the exposure time, the live image will
update and automatically reset the contrast of the image to match the range of intensities
in the live image. You also have the option to display the full dynamic range of the camera
using the Show Full Dynamic Range checkbox. When this box is checked, the histogram
will not show the minimum and maximum intensities of the image, but rather the
minimum and maximum possible intensities based on your camera’s readout.
5.1.5.2 CCD Temp
On systems with a camera that has electronic temperature readout, this field reports the
current sensor temperature in degrees Celsius.
5.1.5.3 Speed
On cameras that support this adjustment, the CCD read-out speed can be adjusted using
this dropdown menu.
5.1.5.4 Gain
On cameras that support this adjustment, the gain can be set using this dropdown menu.
5.1.5.5 Intensification
On cameras that support this adjustment, the intensification factor can be adjusted using
the slider and edit field located below the speed menu.
5.1.5.6 Parameters
This dropdown menu allows for support of additional features on certain cameras. For
instance, on Hamamatsu cameras, the parameters menu allows you to select the camera
offset.
5.1.5.7 Test Dual-View
The Test Dual-View check box is used with systems capable of performing dual camera
simultaneous capture. It is also available when SlideBook is configured with an image
splitter such as the Optical Insights Dual-View (see Defining Filters when using an Image
Splitter on page 72 and Configuring an Image Splitter on page 60) This option is only
available when multiple cameras or an image splitter have been configured in SlideBook.
When this box is checked, the focus window display will rapidly alternate between images
from camera 1 and camera 2.
5.2
Using the Focus Window
The following general procedure describes how to use the focus window to focus on a
sample. If the microscope that you are using is not automated, you will need to move the
optics manually. Procedures for using more advanced features of the focus window are
detailed in Chapter 7 –Advanced Capture.
1. Open the Focus Window by either selecting Window > Focus Window or
clicking on the focus window button in the toolbar
. The Focus/Capture
Window will begin updating as soon as the Focus Window is opened.
2. Choose the desired objective and magnification changer from the Scope tab.
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3. Choose the desired filter configuration from the Focus Controls below the
Scope tab.
4. Place the sample on the stage.
5. Click on Open Fluor or Open Bright to open the desired lightpath.
Next, you will need to bring the sample into focus. This may be accomplished by
either viewing the sample through the eyepieces or viewing the sample in the
focus window. If your microscope is equipped with a motorized emission light
path, the image can be directed to the eyepieces or the camera using the Scope
tab. Moving the xy stage and changing the z focus can be accomplished by either
using the manual controls on the microscope or the computer controls (see
above).
6. Using the slider, set the exposure time so that the image is not overexposed.
7. Using the controls in the Focus Controls window (or joystick or manual
controls), adjust the x and y position of the stage and z position of the stage or
nosepiece to find and focus on an area of interest.
TIP: If you double-click on an object in the image display, the xy stage will move
so that the object is centered.
8. Once the sample focus is satisfactory, you may wish to close the lightpath for
light sensitive samples, and then close the focus window by pressing the X
button in upper right corner. If desired, the Focus Window and Capture dialog
box may be open simultaneously.
5.2.1 Snapping an Image
You may find that you want to capture a single image as you are using the Focus Window.
To do so, simply press Snap once you are happy with the image in the live view. The image
will be stored into the open slide file.
5.2.2 Closing the Fluorescence Shutter Automatically when Closing the Focus
Window
If you would like your fluorescence shutter to close automatically when you close the focus
window, select Edit > Hardware Properties, then expand the Focus Window section.
Click on ShutterUponClose.
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Set the value to Yes and then click on Apply Change. Click Close and then restart
SlideBook.
5.2.3 Labeling the Ocular Photo Prism Positions
You may also provide custom labels for the emission selection positions (ocular photo prism
positions) that appear in the Scope tab of the focus window. To do so, select Edit >
Hardware Properties, then expand the Focus Window section. Click on the Ocular
Photo Prism Pos you wish to edit and enter the label in the edit field.
Click Apply Change, Close, and restart SlideBook.
5.2.4 How to Perform Streaming Capture
The streaming capture function allows you to run your Hamamatsu or Photometrics camera
at the fastest possible speed. There are limitations with Streaming Capture. When a
camera is recording in this mode, you may only alter other hardware via the Focus Window.
Streaming Capture does not work using the Capture dialog box, so you may not perform
automated multi-channel, 3D, 4D, multipoint or montage capture. This capture mode is
ideal for rapid 2D timelapse capture. To run your camera in streaming capture mode:
1. Open the Focus Window and select the Stream tab.
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2. Select the Start button. A new image capture window will open with a counter
for number of images captured.
3. Your camera will continuously generate images with the exposure time that you
have selected in the Focus Window. If desired, select the Number of frames to
average from the drop-down list.
4. Name the file in the Image Name field.
5. Press Record to save the captures to disk. Once you have pressed record, images
will be saved to disk and the Record button will convert to Pause. To end the
recording, press Stop. This will also stop the streaming update. To resume the
streaming update, press Start. You will not be able to change camera settings
while the camera is streaming. You must press Stop and make changes to
camera settings, then press Start again.
CAUTION: Recording at this speed will rapidly use computer disk space.
Spool files will be opened automatically once you press Stop. They will also be
placed in the open slide with the name designated in Step 4.
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6 Image Capture and Import
Image capture parameters are set in SlideBook’s Capture window. In order to capture an
image, you must first set your optics and bring your sample into view and focus using the
Focus Window (please see Chapter 5 - Controlling the Camera and Microscope Hardware
(Focus Window) on page 77). Next, you must set camera parameters such as binning,
exposure time, and image extent, as described in this chapter. Once all of these parameters
have been set, you may initiate capture. You may also choose to import an image created
using another program. This chapter covers the following topics:







Selecting Channels and Setting Exposure Times
Selecting the Area to be Imaged
Entering Image Information and Optical Parameters before Capture
Capturing a 2D Image
Performing Auto-White Balance
Importing an Image
Getting and Editing Image Information after Capture
Advanced topics such as 3D, timelapse, 4D, montage, and multipoint imaging are covered
in Chapter 7 – Advanced Capture.
6.1
Selecting Channels and Setting Exposure Times
Exposure times are set by generating a series of test images for each channel that will be
exposed. If you are performing a transmitted light capture with a color camera or color
LCD filter, you may wish to first perform an auto-whitebalance (see below).
1. Once you have a sample in focus, open the Capture dialog by selecting Image >
Capture or by selecting the Capture icon in the toolbar
have a slide file open in order to open the Capture dialog.
. Note that you must
The dialog box that appears is divided into eight parts starting from the upper
left corner: capture settings, image extent and binning factor, multiplane capture,
multipoint capture, exposure settings by channel, image histogram, optical
parameters, and image info. The Capture dialog uses the same Live View as the
Focus Window.
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2. First select the binning factor in the upper left drop-down menu.
Binning lets you group several pixels together to increase sensitivity (at the cost
of resolution). For instance, selecting 2 × 2 binning effectively gives you a
camera with half the horizontal and vertical resolution, but four times the
sensitivity. Note that as you increase binning, you will need to reduce the
exposure time.
3. Select the desired Filter Set from the dropdown menu.
4. Click on the checkbox of the channel that you would like to expose. The channel
that you select will be highlighted in blue.
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5. Enter an exposure time in the Expose field, if a different one is desired, and
press Test.
Pressing the Test button for a particular filter configuration will give you a
sample single-plane image at the specified exposure time for that channel. You
may resize the image by dragging and dropping the corners or edges of the Live
View. An intensity histogram and the minimum and maximum data values will
be displayed in the Capture dialog in order to show you where in the dynamic
range of the CCD your exposure lies.
Making a test image for each channel gives you the opportunity to choose the
best exposure times (and of course make sure that the light is directed towards
the CCD camera and not the eyepieces).
6. Press the Once button in the Adjust Exposure section of the dialog and notice
that the exposure time has been adjusted to take advantage of the CCD camera’s
full dynamic range.
7. Press the Test button again to update the image in the test view.
8. Repeat the test and adjust exposure process until the histogram shows an
adequate intensity distribution (see note below).
For multi-channel capture, simply repeat this process for each channel that you
wish to expose. For instance, a two channel capture using both the DAPI and
the FITC filter configurations will appear as follows:
NOTE: The maximum value for a pixel depends on the bit depth of the camera. For
instance, on 12-bit cameras, the pixel values can range from 0 to 4095. In order to
take advantage of the full dynamic range of the camera, it is important to try to get
the maximum pixel value well into the four digit figures. Pressing the Once button
will automatically adjust the exposure time to try to achieve a maximum pixel value
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of about 3000. In extremely dim samples, this may not be practical, but it is a good
target value. Conversely, it is important for deconvolution that no pixel values are at
the camera’s maximum value, as that means that the image is overexposed and past
the linear range of the CCD detector. Pressing the Once button will reduce exposure
times if necessary to try to reach a lower maximum value.
6.2
Selecting the Area to be Imaged
Typical cameras contain either a 1392 × 1040 or a 512 × 512 pixel chip, but often you will
want to capture from a smaller portion of the chip in order to reduce the memory required
for capture and/or increase the camera frame rate. You may select the area to be imaged in
two ways.
6.2.1 Using the Image Extent Menu
The image area can be selected using the features in the Image Extent and Binning section
of the Capture window.
1. Select the x- and y-extent of the image from the Width and Height drop down
menus. You may also manually enter the desired values in the edit field.
You may select to add yellow guides in the test view that correspond to 256 ×
256, 512 × 512, and 1024 × 1024 pixels. By default, SlideBook reads from an area
centered on the chip with the provided dimensions. To add the guides, you must
select the Guides checkbox in the Focus Controls.
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2. If you want to move the region that is read to some other location on the chip,
enter offset values for both the x- and the y-axes in the X Offset and Y Offset
fields.
3. Select Test to update the Live View so that it has the desired size.
6.2.2 Using the Test View
Alternatively, you may choose a region of the chip to be imaged by directly selecting it from
the Image Window.
1. Be sure that the
button at the top of the window is depressed.
2. Click and drag the mouse over the desired portion of the test view.
3. Select Update to register the new image size.
4. Select Test to update the Live View so show the new image size.
Clicking on the Full Chip button will automatically select the maximum extent of the
camera.
NOTE: After you have selected the area to be imaged, you may wish to adjust the exposure
times using the Test and Once buttons to take advantage of the full dynamic range of the
camera.
6.3
Entering Image Information and Optical Parameters before Capture
You may specify the following image information and optical parameters in the Capture
dialog box.


Image Name and Comments – can be entered in edit fields
Objective and Mag. Changer - can be selected (or will automatically be selected if
they are either encoded or automated) using the dropdown lists
The channels to be captured and their exposure times are automatically stored with the
image. Although these settings don’t immediately affect the microscope settings or capture,
they are stored with the image and are important for correct deconvolution and
measurement.
You can always go back and add or alter this information by selecting Image > Get Info,
which is described in the section Getting and Editing Image Information After Capture on
page 101.
6.4
Capturing a Single or Multi-Channel 2D Image
The default mode of the capture window is for a single 2D image. The 2D image may be
either single or multi-channel depending on the number of channels that you have checked.
If you wish to capture 3D, timelapse, 4D, multipoint, or montage images, please see
Chapter 7 –Advanced Capture.
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1. Once you have set all of the parameters (channels, exposure times, binning, etc.)
in the Capture dialog box, select OK to begin capture.
If your system does not have automated filter control, you will be prompted to
move the filters into the correct position before the image is captured.
NOTE: If you see these prompts in error, you need to make sure that you do not have
positions defined for hardware components that are not motorized. For example, if
you have a filter cube (reflector) turret that is not motorized, make sure that your
filter definitions are designated Unmounted in the filter turret position.
2. Move the hardware into position and click OK.
The Live View will show the image in color as it is being acquired. If you have
more than one channel selected for capture, it will show a combined image. Thus
if you have designated your DAPI and FITC filters to display as blue and green,
respectively, the Image Window will first display the DAPI image in blue and
then overlay the FITC image in green. In addition to the Image Window updates,
the Capture Controls will become active and report the progress of your capture.
The default Status tab of the Capture Controls displays the current channel in
monochrome. The features of the Capture Controls are best utilized when
performing timelapse imaging and are discussed in Timelapse Capture on page
112.
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6.5
Performing Auto-White Balance for Color Capture
If you are performing a color capture with an LCD filter, you may first wish to perform a
white balance procedure (you must first define your filters as discussed on page 69). This
will yield the truest color rendering. To do so:
1. In the focus window, find a clear portion of your slide. Alternatively, use a piece
of white lens paper.
2. Make sure that the halogen lamp is set to 3200K and that you microscope is set
for Köhler illumination.
3. Select the green filter and check that your exposure time is at least 25ms (this
yields the best white balance results). If you are saturating the camera at 25ms,
you should use neutral density filters.
4. Once you have achieved an adequate exposure time, go to Image > Capture or
select the image capture icon
.
5. Select the filter configuration checkbox corresponding to the color channel and
then Test.
6. Select the area to be imaged and any binning desired.
7. Adjust the exposure time as described above.
8. Select the area of the test view that you would like to use for performing white
balance by clicking and dragging over the region.
9. Select White Balance from the radio buttons and then click Update.
10. A white balance operation is performed and a dialog box similar to the following
will appear.
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NOTE: If you are getting inconsistent color maximums when performing capture
and the RGB factors are varying significantly each time you perform a white
balance, then you should adjust your illumination. Bright, dim or inconsistent
lighting may cause this phenomenon.
11. Select OK to close the dialog box.
12. You may now capture an image by selecting OK in the Capture dialog box.
The white balance settings will remain in the system memory until the slide file
is closed.
13. If you would like to manually enter white balance settings, go to Edit > Define
Optics > Filter Configuration and select the red filter configuration for an
LCD filter, or to the color filter configuration for a color camera.
14. In the default Color Display section, select the Settings button. The following
dialog box will be displayed:
15. Enter the white balance gain factors. In general, the green factor will be the
largest.
16. Click OK to save the settings.
6.6
Importing an Image
You may also use SlideBook to display and analyze images captured using other programs.
The following file types are supported by SlideBook:
 SlideBook Spool (*.spl)
 TIFF
 TIFF Sequence
SlideBook Spool is the file type used when capturing images to disk. Please see Saving
Images to Disk (Spooled Capture) on page 153.
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6.6.1 Importing SlideBook Spool Files
To import a SlideBook Spool file:
1. Select the destination for import by either opening a new slide or opening an
existing slide file. You may do this by selecting File > New Slide or File >
Open Slide. If the slide is already open, make it the active window by clicking
on it.
2. To import an image, select Image > Import > SlideBook Spool. The following
dialog will appear:
3. Navigate to the desired spool file and select Open. The Load Spool File dialog
will appear.
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4. Select the timepoints that you wish to load. For large data sets, it can sometimes
be easier to load a subset of data.
5. Select if you would like to subsample the data. This may speed up processing for
large data sets but will decrease the image resolution.
6. If applicable, select pre-processing and Dual-View parameters.
7. Name the image and add comments.
8. Click Load. The image will be added to the active slide.
6.6.2 Importing TIFF Files
To import individual TIFF files or TIFF sequences:
1. Select the destination for import by either opening a new slide or opening an
existing slide file. You may do this by selecting File > New Slide or File >
Open Slide. If the slide is already open, make it the active window by clicking
on it.
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2. To import an image, select Image > Import and select the file type that you
wish to import.
If there are already images in your slide file, and one is selected, the following
dialog box will appear for TIFF files (but not TIFF Sequence):
Select whether you would like to import the file as a new image or as a new
channel in the current image, and click OK.
3. The following dialog box will appear:
4. Navigate to the file and select Open. The following dialog box will appear if you
have selected TIFF. (If you have selected TIFF sequence, proceed to step 6.)
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5. Select the planes you would like to import as well as the channels to be used for
each color in the image and select OK.
For example, if you have an image that displays FITC as green data, you will
designate the green data as FITC in the dropdown menu.
6. The following dialog box will appear:
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7. Enter image information as outlined in the following section.
8. Click OK after you have entered the information. The imported image will
appear as a Main View. You may always go back and edit the image
information by selecting Image > Get Info.
6.7
Getting and Editing Image Information After Capture
You can get information about an image after capture or import. The Image Info dialog box
gives you the name of the image, comments associated with the image, and the time and
date of capture if the image was collected using a digital microscopy workstation (as
opposed to imported). It also gives you the optical settings as well as which filter
configurations were used to collect each channel along with the exposure time. If you would
like to add optical information to an imported image, you must first define the objective,
magnification changer, and filter configurations as described in Chapter 4 - Configuring
Your System.
You may view and edit information as described below.
6.7.1 Images that were captured in SlideBook
1. Open a Main View of the desired image by either double-clicking on the
thumbnail in the slide file, or by clicking once on the thumbnail and then
selecting View > New Main View.
2. Select Image > Get Info. The following dialog box will appear. View and edit
the Image Name and Comments fields as desired.
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3. Press the Edit Info button if you wish to change any of the optical configuration
or collection information. This brings up the following dialog box:
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NOTE: If you open a SlideBook file that was created on another computer or with a
different SlideBook preferences version, the filter and objective configurations will
be displayed as Other. To prevent this, make sure that the SlideBook preferences
match those that were used for capture.
4. Select the Objective and Mag. Changer used during capture from the drop
down menus.
NOTE: If you have imported an image from another program, you must first enter
the objective and magnification changer definitions as discussed in Chapter 4. Then
you may select the proper objective and magnification changer from the drop down
menus.
5. Enter the Interplane Spacing (µm) for 3D images.
6. Select the filter configurations from the dropdown menus, and enter the
exposure times for each channel.
7. Select OK when you are finished. The data that you entered is saved with the
image.
NOTE: Even if you are working with 2D images, it is a good idea to provide a value for
Interplane Spacing as this will be used in the no neighbors deconvolution algorithm for
the theoretical spacing value. That way, you will not have to change any values that appear
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in the deconvolution dialog box. As a guideline, you should use the approximate z resolution
for the objective used.
6.7.2 Imported Images
You may also add information to images that are imported into SlideBook from other
programs.
1. Import the image as discussed above in Importing an Image on page 96.
2. Make sure that the objective definitions exist by selecting Edit > Define Optics
> Objectives and looking at the available objectives in the dropdown list. If the
objective is not available, add the objective definition as described on page 62.
3. If a magnification changer (optovar) or relay lens was used, make sure that its
definition exists by selecting Edit > Define Optics > Mag. Changers and
looking at the available magnification changers in the dropdown list. If the
magnification changer is not available, add the definition as described on page
73.
4. Make sure that the filter definitions exist by selecting Edit > Define Optics >
Filter Configurations and looking at the available filters in the dropdown list.
If the filters are not available, add the filter definitions as described on page 68.
5. Open a Main View of the imported image by either double-clicking on the
thumbnail in the slide file, or by clicking once on the thumbnail and then
selecting View > New Main View.
6. Follow steps 2-7 above for getting and editing info for images that were captured
in SlideBook. You should also select the Capture Type for your imported
image and make sure that the image information is accurate.
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7 Advanced Capture
SlideBook can automate many different types of capture. This chapter covers the following
topics:












7.1
3D Capture
Timelapse Capture
4D Capture
Multipoint Capture
Montage Capture
Multiwell Capture
Simultaneous Capture
Configuring Your Hardware for Speed
Auto Focus
Varying Capture Rates for Timelapse Capture (Sequence Feature)
Saving Images to Disk (Spooled Capture)
Saving Capture Parameters
3D Capture
Similarly to 2D capture, 3D capture is accomplished by setting microscope parameters in
the Focus Window and then setting capture parameters in the Capture dialog box. In order
to perform 3D capture, your system must have motorized z control.
There are two procedures for performing 3D capture. In Method 1, you will use the focus
window both to bring the sample into view and to interactively set the capture extent in the
z dimension. These z parameters will then be imported into the capture dialog box. In
Method 2, you will use the focus window to bring the sample into view and focus, but then
set the z parameters in the capture dialog box. You may wish to use Method 2 if you already
know the z range of capture. For example, if you are imaging an object that you know to be
30 m in height, you may bring the cell into focus and then capture 20 m above and below
the plane of focus. However, if you are unsure of the extent of the object of interest in the z
dimension, you should use Method 1 since it allows the range of capture to be interactively
defined. Both methods are outlined below.
7.1.1 Method 1
7.1.1.1 Setting Focus Window Parameters
1. Open the Focus Window by either selecting Window > Focus Window or
clicking on the focus window button in the toolbar
. The current camera image
will begin updating as soon as the Focus Window is opened.
2. Select your objective, magnification changer, and filter configuration, and bring
your sample into view and focus as discussed on page 85 in Chapter 5 Controlling the Camera and Microscope Hardware (Focus Window).
3. Click on the Z tab. Now, you will need to set the z limits for capture.
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4. Starting at the middle of the focus range, move the stage (or nosepiece) up until
you are at the top of the volume that you wish to capture. This can be
accomplished either by repeatedly clicking on one of the “up” arrow buttons or by
manually adjusting the fine focus of the microscope.
NOTE: When moving the z position by computer control you may either use one
of the fixed increment buttons (10, 1 or .1 micron steps) or manually enter a step
size in the adjacent edit box.
5. Click on the Set Top button. SlideBook will save the current z position and
display it to the left of the Set Top button.
6. Focus in the opposite direction until you are at the bottom of the volume of
interest.
7. Click on the Set Bottom button. SlideBook will save the current z position and
display it to the left of the Set Bottom button.
8. Return to the point of best focus (the brightest plane) using the slider, Go
button, or microscope controls. Returning to the brightest point in the sample is
necessary so that the correct exposure times can later be defined in the capture
window.
9. Close the lightpath for light sensitive samples by choosing Close Fluor.
10. Enter the step size that you would like to use for capture in the Step Size (µm)
edit field of the Capture Information group. The number of planes that will be
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required to capture the entire z range will be calculated and displayed above the
step size.
Note: You may wish to alter the capture step size so that the size of the captured
image is not excessive. Alternately, you may decrease the total distance traveled
by resetting the top and bottom parameters.
11. Close the focus window by pressing the Close button.
7.1.1.2 Setting Capture Window Parameters
1. Once you have set the z parameters in the Focus Window, open the Capture
dialog box by selecting Image > Capture or by selecting the capture icon in the
tool bar
.
2. Select channels and set exposure times as described in the section Selecting
Channels and Setting Exposure Times in Chapter 6 (page 89).
NOTE: It is important that z parameter is set at the middle of the focus range
(brightest) when setting exposure times so that the brightest planes are not
overexposed.
3. Select the area to be imaged as described in the section Selecting the Area to be
Imaged in Chapter 6 (page 92).
4. Enter image information and settings as described in the section Entering Image
Information and Optical Parameters before Capture in Chapter 6 on page 93.
5. Click on the 3D checkbox in the Capture Type section. The radio buttons and
edit fields in the 3D Capture section will become active. Note that default value
of the # Planes field is one, signifying a single plane capture.
6. Import the z parameters that you set in the focus window by selecting the Use
top and bottom positions radio button. The # Planes field will display the
number of planes to be captured (see dialog box below). The Spacing field
determines how many microns the stage needs to move between planes. The
Offset field is not used with this method of setting 3D capture parameters.
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7. If you wish for your z focus position to return to the center of the volume once
capture is completed, select the Return to center of volume after capture
checkbox.
8. Click OK to begin capture. The Capture Controls window will appear and
display each plane as it is captured. You may choose to Pause, Cancel, or Stop
your capture any time after it has started.



Pause – pauses the capture until you press Continue
Cancel – cancels capture and does not save any images
Stop – stops capture and saves a truncated sequence
7.1.1.3 Setting Capture Preferences to Open and Close Shutter during Capture
If you are performing single channel 3D capture, the default fluorescence shutter action in
SlideBook is to leave the shutter open during capture. If you would like the shutter to open
and close after every capture, select the Advanced button in the Capture Settings
section of the Capture dialog box. Select the 2D/3D capture checkbox labeled Open and
close shutter on single channel or simultaneous 3D capture in the General tab.
The default action for multiple channel capture is to open and close the shutter after every
capture.
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7.1.2 Method 2
Use this method if you already know the distance above and below the plane of focus or
other reference position that you would like to capture.
1. Bring your sample into view and focus by following steps 1-2 as described above
in Method 1: Setting Focus Window Parameters. If desired, you may set another
position of interest by pressing the Set button next to Reference.
2. Set capture preferences as described above in Method 1: Setting Capture
Preferences.
3. Open the Capture dialog box by selecting Image > Capture or by selecting the
capture icon in the SlideBook toolbar
.
4. Follow steps 2 through 5 as described above in Method 1: Setting Capture
Window Parameters. If you have set a reference position, the dialog will appear
as follows:
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5. The edit fields that appear are described below.

Range (m) – Total distance traveled in microns.

# (Number of) Planes - Total number of z planes acquired during capture.

Step Size (m) – Distance the stage (upright) or objective (inverted or fixedstage) must move between z planes. A positive number typically corresponds
to the stage or objective moving up during capture, while a negative number
corresponds to the stage or objective moving down during capture.

Offset (m) – The distance above or below the current stage location that the
stage or objective will move before starting image collection. For example, on
an inverted microscope, a positive number corresponds to the stage moving
up while a negative number corresponds with the stage moving down. Thus,
on an inverted microscope, you will need to use a positive spacing and
negative initial offset.
If your sample is already in focus, you may select the Use current position
radio button and choose the Range around current checkbox and simply enter
in the total range of travel (Range) and either the number of planes or the step
size. The remaining capture parameters will be calculated for you. If you have
set a reference position, you may choose to use the reference position as the basis
for capture.
6. If you would like your z focus to return to the current position or reference
position after capture check the Return to current position or Return to
reference position checkbox as appropriate.
7. Click OK to begin capture. The Capture Controls window will appear and
display each plane as it is captured. You may choose to pause, cancel, or stop
your capture any time after it has started, as described above in Method 1.
A comparison of these capture situations are shown in the figures below.
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Top
Center
(calculated from top
and bottom positions)
Bottom
Fig. 7.1 Method 1 is accomplished by setting top and bottom positions in the Focus Window
and then setting the capture window parameters as shown.
Current
position
Current
position
Fig. 7.2 Method 2 can be accomplished using either the current position or a chosen
reference position. This figure shows how capture is performed when using the current
position of the z stage with the given capture window parameters.
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Reference
position
Reference
position
Reference
position
Fig. 7.3 This figure shows how Method 2 is performed when a reference position is selected
in the Focus Window. Note that the offset is different for the two situations on the right.
7.2
Timelapse Capture
In order to perform timelapse capture, first bring a sample into view and focus as described
in the Using the Focus Window section of Chapter 5 on page 85. Then, proceed as follows.
7.2.1 Setting Capture Preferences
Before beginning a timelapse capture, you may alter your capture preferences to do the
following:






Change capture frequency for a single channel in a multi-channel timelapse
capture
Open and close shutter between exposures for single channel capture
Change the rate of live updates (to increase capture speed)
Autofocus during capture
Change capture frequency
Save images to disk
All of these options can be accessed in the Capture Preferences dialog. This dialog box is
displayed when you select the Advanced button in the Capture Settings portion of the
Capture dialog.
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7.2.1.1 Performing Periodic Capture
You may choose to capture a single channel at a slower rate than other channels when
performing a multi-channel 2D timelapse capture. You may do so by bringing up the
Capture Preferences dialog and selecting the Periodic tab. Select the check box, the
channel that you would like to capture at a slower rate, and the frequency at which you
would like to capture that channel. For example, a frequency of 5 indicates that you would
like to capture a channel every 5th image. If you would like to copy the image of the
selected channel to timepoints where you are not collecting the channel, select the check
box to Fill uncaptured timepoints with data from last captured timepoint.
7.2.1.2 Opening and Closing Brightfield Shutter Between Exposures
For fluorescence shuttering, please see the above section Setting Capture Preferences to
Open and Close Shutter during Capture on page 108. If you are performing 2D timelapse
capture, the default fluorescence shutter action in SlideBook is to open and close the
shutter if your timelapse interval is non-zero. If you have a 0ms timelapse interval, the
shutter will remain open to allow you to capture as quickly as possible. This default
behavior is designed to protect the sample at non-zero timelapse intervals, yet maximize
acquisition speed at 0ms timelapse intervals. Thus, the Open and close shutter on
single channel or simultaneous 3D capture checkbox is only applicable when you wish
to shutter for 2D timelapse images with a timelapse interval of 0ms.
If you would like your brightfield shutter to close between transmitted light images:
1. Select Edit > Hardware Properties and expand the Advanced Capture
Settings list.
2. Click on ShutterBrightFieldTimelapse. The right side of the dialog box will
update.
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3. Select Yes and then Apply Change.
4. Select Close and then re-launch SlideBook to register the changes.
7.2.1.3 Changing the Status Update Frequency
You may change the frequency with which the elements in the Capture Status window
are updated by choosing Advanced in the Capture dialog box to bring up the Capture
Preferences dialog and then entering the desired update frequency in the edit boxes in the
General tab. An update frequency of “3” would update the given element at the conclusion
of every 3rd capture. Reducing the update frequency is primarily useful for increasing
capture rate with single processor computers.
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You may suppress all updates for optimal speed performance. To do so, select the
Suppressed radio button. If When Possible is selected, updates will be provided when
the computer processor allows. Some updates will be dropped in the interest of speed. If
Always is selected, each frame requested will be displayed (at the expense of speed).
7.2.1.4 Autofocus During Capture
Please see the section Autofocus on page 144.
7.2.1.5 Changing Capture Frequency
Please see the section Varying Capture Rates During Timelapse Capture on page 149.
7.2.1.6 Saving Images to Disk
Please see the Saving Images to Disk (Spooled Capture) section on page 153.
7.2.2 Setting Capture Window Parameters
1. Open the Capture dialog box by selecting Image > Capture or by selecting the
capture icon in the SlideBook toolbar
.
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2. Follow steps 2-4 in the above section 3D Capture: Setting Capture Window
Parameters.
3. Create any annotations that you would like to record during capture (see
Creating Notes below).
4. Check the Timelapse checkbox in the Capture Type section of the Capture
dialog box. You now have the option to define a variety of timelapse parameters.
Enter the desired values in the following fields:

# of Timepoints – Number of timepoints that will be captured.

Duration – Total length of time for the experiment. Units of time, in
milliseconds (ms), seconds (s), minutes (m) or hours (h), can be selected from
the dropdown menu. Due to the inability to predict time required for
hardware movement, this option is only available when the interval for
capture is over 30 seconds.

Interval – Delay between the beginning of one timepoint and the beginning
of the next timepoint. The interval unit can be selected from the dropdown
menu. If the capture sequence at one timepoint takes longer than the
interval, SlideBook will capture the next timepoint immediately following the
preceding timepoint.
NOTE: As you type in two of these values, the third field will be calculated
automatically.

Renormalize to First Timepoint – When checked, the minimum and
maximum pixel intensity values for the first timepoint in a timelapse capture
series will be used to determine the renormalization values of all subsequent
timepoints. This option only affects the image display during capture and
will not affect the captured image.
7.2.3 Creating Notes
SlideBook allows you to record the exact time of experimentally relevant actions. To do this,
you may either create notes prior to capture or during capture. These notes will then
appear as buttons in the Capture Status Window during capture. If you do not wish to
create notes, proceed to the next section, Initiating and Monitoring Timelapse Capture.
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1. To create annotations, first bring up the Capture Preferences dialog from the
Capture dialog by clicking on the Advanced button.
2. Select the Notes tab. The following dialog box will appear.
You may specify up to eight notes, which will appear in Capture Controls and the
exported data.
3. Click OK once you have entered the desired annotations.
Once capture is initialized (by clicking OK in the Capture dialog box), the notes
will appear in the Capture Status Window (see below).
4. To record an event, simply click on the desired annotation button during capture.
You can access notes from the Notes tab of the Capture Controls. You may also
add new notes at this point. You need not specify the notes before capture.
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NOTE: You can make multiple clicks during capture (even of the same event), but
currently only one event can be recorded with any single timepoint. After
capture, elapsed time and timelapse annotations can be displayed as a text
overlay in the image by selecting Annotations > Timestamps. In addition,
these events are displayed next to the annotated timepoint when data is
exported using Statistics > Ratio/Timelapse Data.
7.2.4 Creating ROIs and Graphs to Monitor Regions of Interest
Before beginning your capture, you may wish to create regions of interest in order to
monitor intensities during. You may also do this during capture. To draw regions before
capture, perform the following:
1. Make sure that the Live View is displaying the specimen. You can do this by
pressing Test in the Capture dialog box.
2. Choose one of the ROI tools, which are located on the SlideBook toolbar at the
top of the application window.
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
- Rectangle Tool, click and drag to draw a rectangle

– Ellipse Tool, click and drag to draw an ellipse
Chapter 7 – Advanced Capture

– Polygon Tool, click to draw a series of straight lines, double-click to
close the shape

– Free Hand Tool, click and drag to draw freehand, release mouse button
to close the shape

– ROI Selection Tool, use to select an ROI. Holding shift and clicking on
multiple regions will allow you to select more than one region. Clicking and
dragging on a selected region will move the selected ROIs. If the ROI
number is displayed in white, this indicates that the ROI is selected.
3. Continue to draw regions as desired. The following operations can be performed
by selecting the ROI with the ROI selection tool, and then right-clicking to bring
up the ROI menu.

Set as background – uses the selected ROI for background subtraction
during graphing

Duplicate – creates a copy of the selected ROI

Delete – removes the selected ROI (s)
4. Once you have created the desired regions of interest and begun capture, you can
display a graph by selecting the desired channel in the Graph Channels section
at the bottom left of the Capture Controls and then clicking on the Show button.
The regions of interest that you have created can also be displayed after capture as
discussed in Displaying Annotations on page 181. To recreate the graph post-capture,
please see page 251.
7.2.5 Initiating and Monitoring Timelapse Capture
In the Capture dialog box, click Start to begin capture. The Capture Controls will appear
and display the current single channel image in the Status tab. The Live View will show a
larger, resizable version of the current capture which can combine multiple channels.
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7.2.5.1 Creating Graphs to Monitor Regions of Interest
If you have created ROIs, you may choose to display intensity graphs. To do so:
1. Select the channel that you wish to graph from the drop-down in Graph
Channels section and click Show. You may choose to display the graphs for
multiple channels individually or by type.
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Open Data Table in Excel
Save Graph as Tiff
Save Data Table as .txt
Channel Menus
Expand Time
Range
Zoom In/Out
X-axis
Expand
Intensity
Range
Zoom In/Out
Y-axis
2. Use the zoom in and zoom out tools to explore your data.
3. Once your capture is finished, your graph will remain open for viewing.
4. To export your graph, simply select the icon.
The regions of interest that you have created can be displayed as discussed in Displaying
Annotations on page 181. To recreate the graph post-capture, please see page 251.
7.2.5.2 Focusing During Capture
You may adjust the focus of your image during capture. To do so, go to the Live tab. The
Live View works similarly with the Capture Controls as it did with the Focus Controls.
NOTE: Before using the Live tab, you must first select Pause. Once you are finished
focusing, you must select Continue to resume capture.
The window has the following features:
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Z-Stage
Snap
Filter
Start/Stop
Shutter Buttons

Start / Stop – begins (or ends) the semi-live camera readout

Snap – snaps a single image

Shutter Buttons – toggle fluorescence, brightfield, or alternate source shutters
open and closed, boxes to the left of the buttons indicate status

Filter – moves motorized filters into position when you select Go

Z Stage – allows you to refocus your sample using precise movements
To use this window, press Start, open the desired shutter, select the appropriate filter, and
focus your sample. When you are finished, close the shutter, press Stop, and then
Continue. Alternately, you can press Snap to check a single focal plane then adjust the zstage, then Snap again to update the view. The later approach may minimize exposure to
the sample.
7.2.5.3 Viewing Previous Timepoints
You may choose to view previously captured images while your 2D timelapse or 4D
experiment is in progress. To do so:
1. Set up your capture as you would normally in the Capture dialog box. Make sure
that you are saving images to memory, or to memory and disk (see page 149).
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You can do this by selecting Advanced to bring up the Capture Preferences
dialog. Then, select Spool and be sure that one of the first two radio buttons is
selected.
2. Once you have set up your capture, click OK. The Capture Controls will appear.
3. Select the Live tab.
4. Select the Show button next to Captured Images and the following window
will appear:
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5. Use the slider to scroll through previously captured images or enter a specific
timepoint into the edit field and select Go. You may also use the + and – buttons
to move one timepoint at a time.
6. If you would like to view a single channel, select the Single Channel radio
button and the desired channel from the dropdown list.
7. The default mode is “Single Frame.” If you are capturing a 3D stack over time,
you may also choose MIP to view a maximum intensity projection of each z stack
at any timepoint.
8. Once you are finished viewing images, close the dialog box by clicking on the X in
the upper right hand corner of the dialog box or selecting Hide from the Live
tab.
7.3
4D Capture
SlideBook allows you to capture 3D images over time. To set up for 4D capture simply set
up for 3D capture and timelapse capture as described in the above sections, then make sure
that both the 3D and Timelapse checkboxes are selected in the Capture dialog box and the
appropriate parameters are entered in the edit fields before selecting OK. The resulting
slide file will contain a single 4D image that includes all of the z sections and timepoints.
NOTE: If you have specified a timelapse interval that is shorter than the time required to
capture a single 3D stack, SlideBook will default to capturing 3D stacks as quickly as
possible.
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In addition to all of the options available for 2D timelapse imaging, SlideBook also allows
you to perform mid-volume capture and sequential multi-channel z series, as discussed
below.
7.3.1 Shifting the Volume During 4D Imaging
You may find that your sample exhibits focus drift during image acquisition. You may
correct for this by selecting the Live tab during capture. To move your volume:
1. Click on the Live tab and press Pause to pause the capture.
2. Click Start to go into “live” mode in the Live View. Refocus your sample either
manually or by using the Z Stage controls and select Recenter Z. You may also
shift the whole 3D volume by checking the Shift 3D Volume box.
7.3.2 Mid-volume Capture During 4D Imaging
SlideBook offers the ability to capture up to three 2D mid-volume images during each
timepoint of a 4D capture. This feature is useful for acquiring channels that often do not
need to be imaged in three-dimensions (such as DIC or Fura-2) during a 4D capture. To
setup a 4D mid-volume capture:
1. Go to Capture Preferences dialog box by pressing the Advanced button in the
Capture dialog box.
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2. Select the 4D tab from the capture preferences dialog box. The following window
will appear:
3. Select the Capture midvolume plane checkbox. This will activate the edit box
to the right allowing you to specify the plane at which a midvolume image will be
acquired. The midvolume plane must fall within the plane limits of the 4D
capture as specified in the Capture dialog box.
4. SlideBook defaults to capturing the midvolume plane during each 3D acquisition.
For instance, consider the case of a 4D capture with a 15 plane 3D image being
acquired at every timepoint. If the midvolume capture plane is set to 10,
SlideBook will acquire 10 planes of the 3D image, acquire the midplane channel
and then acquire the remaining 5 planes of the 3D image. To have SlideBook
capture the midvolume image at the end of the 3D capture select the Capture
midvolume plane at end of 3D acquisition checkbox. When this option is
selected, SlideBook will capture the entire 3D volume, return to the specified
midvolume plane and then capture the selected channels.
5. Select the midvolume channels that will be captured at each timepoint during
the 4D capture. In the edit box to the right of each channel specify the exposure
time that will be used to capture the channel. Importantly, channels that will be
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captured at midvolume planes should not be selected for exposure in the Capture
dialog box when setting up the 4D capture parameters.
6. Click OK and then proceed to the Capture dialog box to set up for 4D capture as
described above.
7.3.3 Multi-Channel Z-Series
In order to speed up capture, you may wish to capture multi-channel z stacks one channel
at a time.
1. Go to Capture Preferences dialog box by pressing the Advanced button in the
Capture dialog box.
2. Select the 4D tab from the capture preferences dialog box. The lower half of the
dialog box appears as follows:
3. Select the desired capture order. If you would like to reduce the number of filter
switches that occur during capture, select Capture one channel at a time for
all z positions.
4. Click OK and then proceed to the Capture dialog box to set up for 4D capture as
described above.
7.4
Multipoint Capture
If your stage is equipped with a motorized xy stage, SlideBook allows you to select multiple
locations to visit throughout the course of capture. You may perform timelapse and/or 3D
capture at each location. As with the other types of capture, you will set parameters in the
focus window and in the capture window.
7.4.1 Setting Capture Preferences
Depending on the type of capture you plan to perform (2D timelapse, 3D or 4D), you may
wish to select capture preferences such as periodic capture, midvolume capture, multichannel z options, autofocus and spooled capture as described in the sections above and
below (see pages 113, 115, 125, 127, and 146).
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7.4.2 Setting Focus Window Parameters
You may choose to select new points, or you may load points that you have saved
previously.
7.4.2.1 Setting Points
1. Open the Focus Window by either selecting Window > Focus Window or
clicking on the focus window icon in the SlideBook toolbar
. The Live View
will open and begin updating.
2. Select your objective, magnification changer, and filter configuration, and bring
your sample into view and focus as discussed in Chapter 5 - Controlling the
Camera and Microscope Hardware (Focus Window) on page 77.
3. To select a field to be visited during capture, first click on the XY tab.
4. Choose Set Point to add a field of view to be visited during capture. The xy
location coordinates will be added to the window of the XY tab.
NOTE: If your system also has encoded z focusing, you may specify xyz
coordinates.
Once you have added a location to the list, you may remove, visit, or add
locations. To perform these operations, first highlight the location by clicking on
the coordinates in the list, then click on the appropriate button to perform the
following actions:
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
Visit Point – moves the stage (and z focus) to the selected xyz location.

New Point Z – allows you to alter the z position of a given xyz location.
This allows you to update the focus position of a certain sample if it has
drifted.

Reset All Z – resets the z location on all points to the current z location.

Clear Point – removes the selected location from the list.

Clear All – clears all locations from the list.
Chapter 7 – Advanced Capture
5. Once you are satisfied with your list of locations, close the lightpath for light
sensitive samples by choosing Close Fluor.
6. Return to the brightest field that you are planning to capture by selecting the
location from the list in the XY tab and choosing Visit Point. It is important
that the brightest field is in view when setting exposure times in the Capture
dialog box.
7.4.2.2 Saving and Loading a Multipoint List
This feature allows you to save the xyz multipoint list so that you may visit the same points
in future experiments. To do so:
1. Before placing your sample on the stage, open the Focus Controls and go to the
XY tab.
2. Before setting points, you will need to home the stage. Some stages do not
maintain an absolute coordinate system, but rather reset to zero at whatever
position the stage is in when it is turned on. If it is your first time performing the
home operation with the stage, make sure that your objectives are in the “load”
position, as far from the stage as possible. Once you have all possible
obstructions cleared, select Home. If Home is grayed out, you must manually
home the stage by taking the stage to its limit at any of the four corners, then
resetting your stage to zero (for instance, on an ASI stage, you would take the
stage to the limit, then turn the stage off and on to zero the stage). You must
perform this procedure each time you plan to save or load a multipoint list.
3. Once the stage is homed, place your sample on the stage and proceed with
setting your point list. Once the list is complete, select Save. You will be
prompted to enter a description for your point list.
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4. Enter a description and then select OK.
Now that you have saved XY stage positions, you can load them for subsequent
experiments. To load them, simply home the stage before loading your sample, then
select Load to populate the point list.
7.4.3 Setting Capture Window Parameters
1. Once you have set the appropriate parameters in the Focus Window, open the
Capture dialog box by selecting Image > Capture or by selecting the capture
icon in the SlideBook toolbar
.
2. Follow steps 2 through 4 in the above section 3D Capture: Setting Capture
Window Parameters in order to select channels to be captured and set
parameters for image extent and binning.
3. Click on the Entire List/Montage radio button in the Multiple XY Location
Capture section. Alternately, if you would only like to capture the current point,
you may choose the Current Location radio button.
If you wish to take a single image at each location, proceed to step 8.
7.4.3.1 3D Multiple Location Capture
4. If you would like to capture 3D stacks at each location, set up for 3D capture as
discussed in 3D Capture on page 105.
5. Click on the 3D checkbox in the Capture Type section.
7.4.3.2 Timelapse Multiple Location Capture
6. If you would like the points to be visited repeatedly, click on the Timelapse
checkbox in the Capture Type section of the Capture dialog box.
7. Enter the number of timepoints to be collected and the interval between capture
in the appropriate edit fields.
NOTE: In multiple location timelapse capture, the timelapse interval corresponds
to the time that elapses between the completion of capture for the last point in
the list and the beginning of the capture for the first point in the list.
8. Click OK to begin capture. The Capture Status Window will appear and display
each plane as it is captured. You may choose to pause, cancel, or stop your
capture any time after it has started.
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7.5
Montage Capture
Often, you may wish to capture an image that is larger than a single field of view.
SlideBook can stitch multiple fields together into what is called a montage image. It does
this by taking successive camera fields and moving the motorized xy stage a computed
amount. The procedure for capturing a montage image is described below.
NOTE: Before attempting to perform montage capture, make sure your xy stage is properly
configured as discussed in Configuring a Motorized XY Stage on page 58.
7.5.1 Setting Focus Window Parameters
1. Open the Focus Window by either selecting Window > Focus Window or
clicking on the focus window icon in the SlideBook toolbar
. The Live View
will begin updating.
2. Select your objective, magnification changer, and filter configuration, and bring
your sample into view and focus as discussed in Chapter 5 - Controlling the
Camera and Microscope Hardware (Focus Window) on page 77.
3. The XY tab of the focus window has a panel of buttons that assist in selecting the
range of the montage:
4. These buttons are used to define the boundaries of the entire region to be
included in the montage image. SlideBook needs enough coordinate information
to generate an imaginary “box” around the region to be imaged. Thus, the extent
of the montage image can be defined by selecting one of the following sets of
coordinates: upper left/lower right, upper right/lower left, top/bottom/left/right.
In the following example the upper left and lower right buttons are used to
define the montage extent.
5. Navigate to the upper right corner of the region that you want to image by either
moving the stage controller’s joystick or using the up, down, left, and right
arrows that appear in the Focus Controls.
6. Click on the
button. The x, y, and z coordinates of that position will appear
in the XY tab’s list of points with a UR designation next to it.
7. Now, search for the lower left corner of the selection and click on the
button.
The x, y, and z coordinates of that position will appear in the XY tab’s list of
points with a LL designation next to it.
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Selecting these two corners lets SlideBook automatically compute how many
camera fields need to be captured in order to stitch together a montage image
that will cover the entire region. When a montage extent has been successfully
defined, the number of vertical and horizontal camera fields needed to cover the
region will be displayed (in this case a 2 × 3 grid of 6 images). If X axis? or Y
axis? is displayed, SlideBook cannot compute a montage extent because the
selected coordinates are not correctly orientated with respect to one another. For
instance, Y axis? would be displayed if the selected lower right position is
actually located above the selected upper left position. If you continue to see
these errors, please check that your xy stage is configured properly (page 58).
If you want to change the montage extent, you can simply go to a different
location and click on one of the buttons again, as appropriate. The previous
coordinates will remain on the list of points, but the LL or UR designation will
move to the new point.
Once you have added the locations to the list, you may visit or adjust them. To
perform these operations, first highlight the location by clicking on it and then
click on the appropriate button to perform the following actions:

Visit Point – moves the stage (and z focus) to the selected location

Clear All – clears all locations from the list

Reset All Z – resets the z coordinate of all locations to the current z position

New Point Z – resets the z coordinate of the selected location to the current
z position
8. Once you have selected two corner boundaries, close the lightpath for light
sensitive samples by choosing Close Fluor.
7.5.2 Setting Capture Window Parameters
1. Once you have set the boundaries of the montage image in the Focus Window,
open the Capture dialog box by selecting Image > Capture or by selecting the
capture icon in the SlideBook toolbar
.
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2. Select channels and set exposure times as described in the section Selecting
Channels and Setting Exposure Times in Chapter 6 on page 89.
3. Enter image information and settings as described in the section Entering Image
Information and Optical Parameters before Capture in Chapter 6 on page 93.
NOTE: It is critical to verify that the Objective and Mag. Changer fields under
Optical Parameters are set correctly. SlideBook uses its knowledge of the size
of the camera field at the current magnification to compute the number of images
needed to span the montage extent. If your microscope has an automated
objective nosepiece, SlideBook can read its position and the Objective field
should already be set correctly. If it has an automated magnification changer
turret (or only a single magnification changer setting) then the Mag. Changer
field should also be set correctly. However, if your microscope has manual
selection of objectives or more than one manually selected magnification changer,
you must make sure the Objective and Mag. Changer fields are correct before
proceeding.
4. Click on the Entire List/Montage radio button in the Multiple XY Location
Capture section of the Capture dialog. The image extent is adjusted to reflect
the montage extent in pixels.
NOTE: If the Entire List/Montage button is not enabled, a montage extent was
not properly defined in the Focus Window.
5. Click on the OK button to start the capture.
SlideBook will return the xy stage to the upper left corner and begin its
acquisition sequence. The Capture Status Window will appear and display each
plane as it is captured. You may choose to pause, cancel, or stop your capture any
time after it has started. Once the capture is complete, the current slide will
contain images of all of the fields that contributed to the montage. To generate a
montage image please see page 169, Displaying a Montage.
7.6
MultiWell Capture
Multi-well capture uses a different interface than other forms of capture. In this section,
you will learn how to:





Create a Layout
Manipulate Hardware and Focus on the Sample
Calibrate the Stage Position
Select Wells for Capture
Initiate and Monitor Capture
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7.6.1 Creating a Layout
In order to perform multi-well capture, you must first create a layout, or template, that
defines capture parameters. These layouts may be saved and applied for future captures. To
create a layout:
1. Go to Image > Advanced Operations > Capture Multi-well Plate. The
Multi Well Capture dialog will appear:
2. Go to the upper left corner and select New to generate a new layout. The
following dialog will appear:
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3. Enter the Layout Name in the edit field. This layout will be saved so you may
use it again for future experiments.
4. Select the Microscope Components from the Objective and Mag Changer
dropdown menus.
5. Enter Plate Setup parameters by either entering numbers or using the up and
down arrows.

X spacing, Y spacing – center-to-center distance in x and y directions in
microns (can be determined by contacting the plate manufacturer; values
show in above dialog box are typical for a standard 96-well plate)

Well Diameter – can also be determined by contacting the plate
manufacturer

Calibration Row – row used for setting top of upper left and upper right
wells (see below). You can generally specify this to be 1 (e.g. row A) unless
you cannot move your stage to focus on the top edge of the top row and
must use row 2.

Calibration Column – column used for setting right side of lower right
well (see below). You can generally specify this to be the rightmost column
of the plate. For a 96 well plate often you cannot reach the right edge of
the wells in the 12th column and must set this value to the 11th column.
6. Select the channels that you wish to capture by clicking in the appropriate
checkboxes. You may choose to perform an autoexposure or you may set the exact
exposure time.
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7. Select the type of capture that you wish to perform for each well as shown in the
Capture Parameters section of the dialog. You have the following choices:

Images at the center of the well - you may choose the size of the grid,
and you may generate a montage of the grid

Single center frame

Image pattern - for the predefined grid pattern, you may enter a
separation value, which will allow you to sample at various points in the
well
8. Select autofocus parameters. You may wish to determine these parameters first
using the Focus Window (see Autofocus on page 144.)
9. Click Save to exit the Multiwell Setup dialog box.
7.6.2 Manipulating Hardware, Focusing on the Sample
Once you have determined your layout, you can interface with hardware in the Multi Well
Capture dialog. This works very similarly to the Focus Window. You can select filters, move
x,y, and z positions, and open the fluorescence or transmitted light shutters. Before
proceeding to the next section, make sure that your sample is in view and focus.
7.6.3 Calibrate the Stage Positions
1. Go to a column towards the left of the plate and find the top of a well in the
calibration row (as defined by Calibration Row in your layout, generally row
A).
2. Focus on the upper middle edge of the meniscus of the well and center it in the
camera image (see picture below), and click Set UL.
3. Go to a column towards the right of the plate and find the top of a well in the
calibration row (as defined in your layout, generally row A).
4. Focus on the upper middle edge of the meniscus of the well and center it in the
camera image (see picture below), and click Set UR.
5. Go to a row towards the bottom of the plate in and find the right of a well in the
calibration column (as defined by Calibration Column in your layout, column
11 by default).
6. Focus on the right middle edge of the meniscus of the well and center it in the
camera image (see picture below), click Set next to Lower Right.
7. Test calibration by clicking on a well (it will turn white), and selecting Goto.
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U
UR
LR
7.6.4 Save and Restore Calibration Positions
If the currently selected layout has been calibrated before, then you can use the Restore
button to load the previously set Upper Left, Upper Right and Lower Right positions.
Caution: Your xy stage MUST be Home’d before you set the positions or they will not be
saved (see Setting Points on page 128). After you restore calibration positions you should
use the corresponding Visit buttons to view and then the Reset button to adjust for
changes in the plate placement.
7.6.5 Selecting Wells for Capture
1. Select the wells you wish to image by clicking on the well and then clicking
Select. If you wish to select multiple wells, first select a single well (it will be
gold). Starting from the selected well, click and drag over the wells you want to
image. The selected wells will turn gold.
2. To unselect wells, click on the well that you don’t want to image and press
Unselect. To unselect multiple wells, first unselect a single well, then click and
drag over the wells you wish to unselect. The unselected wells will turn gray.
7.6.6 Initiating and Monitoring Capture
To begin imaging, you will first make sure that the first well is in focus.
1. Go to the first well that you will capture (by clicking on the first well and using
the Goto command).
2. Open the desired light path by clicking Open Fluor or Open Bright.
3. Select the desired filter from the Filter Configuration section.
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4. Obtain focus on your sample in the camera view.
5. If you would like to view images as they are captured, select the Live Picture
during Capture checkbox.
6. Click Start to begin capture.
7.6.7 Exporting Point List
To capture 3D images or to take advantage of other advanced capture options you must use
the Export Points button to transfer the list of positions for each selected well to the XY
tab of the Focus Controls window. Once transferred, you can perform a multipoint
capture using the exported points (see Multipoint Capture on page 127). The well name
and position index within the well will be saved as the image name and spool file name for
each image captured.
7.7
Simultaneous Capture
SlideBook allows for simultaneous dual camera or image splitter operation for systems so
equipped (see Configuring an Image Splitter on page 60 and Defining Filters when using an
Image Splitter on page 72). Simply set up for the type of capture that you would like to
perform as described in the sections above. Before initiating capture, make sure that the
Simultaneous Capture checkbox is selected in the Multiple XY Location Capture
section of the Capture dialog box.
7.8
Configuring Your Hardware for Speed
The following sections describe how to get the best speed performance when performing
timelapse imaging. In addition, to get best speed performance, you may wish to change the
frequency of image and histogram updates during capture (see page 114).
7.8.1 Photometrics Cameras
If you feel that your camera is not running at appropriate speeds, go to the Camera tab in
the Focus Window and make sure that the drop-down Speed menu is set to 0. Also, you
may choose to set the clear cycle on the camera so that charge is not cleared from the chip
before each image is captured. Charge accumulates between image captures, and the
default action of SlideBook is to clear the accumulated charge before capturing an image. If
you choose not to perform the clear action, the camera will run at a faster rate. However,
the accuracy of the intensities measured will be reduced.
To change the clear cycle setting, do the following:
1. Select Edit > Hardware Properties.
2. Click on the + sign to expand the PVCAM section.
3. Click on Clear Cycles and the right hand side of the dialog box will update.
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4. Enter “0” in the edit field and then Apply Change.
5. Click Close and then restart SlideBook to register the change.
6. Before performing an experiment, make sure to capture a test image
immediately before beginning the experiment. This will help reduce the effect of
charge build up in the first frame of the image capture.
7.8.2 Sutter DG-4
You may increase the switch time on your Sutter DG-4 by employing TTL control. You must
purchase the SlideBook TTL Synchronization Module to enable TTL control (please contact
Olympus America ([email protected] or 1-800-446-5967 option 4) if you wish
to purchase this module). Note that in order to use TTL control, the exposure times of the
various channels to be captured must be equal. Please see the TTL module documentation
for details on configuring a system for TTL control. The TTL module for SlideBook includes
software plus a National Instruments multifunction DAQ board and breakout box.
7.8.3 Z Stages
There are delays that may be reduced in order to increase the speed performance of the
ASI/Mad City piezoelectric stage and the Physik Instrumente PIFOC piezoelectric focusing
collar. The following adjustments allow the user to fine-tune the amount of time the
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software waits for a Z movement to complete. The appropriate delay depends on system
configuration and experimental conditions. The following parameters must be set to give
you the minimum possible delay to achieve maximum system performance. Real-world
conditions may make it necessary to increase the delays to achieve scientifically acceptable
results. Should you experience issues with image quality we recommend you reset these
parameters to the default value.
7.8.3.1 ASI/Mad City Piezoelectric Stage
To increase the speed of this device, perform the following steps:
1. Select Edit > Hardware Properties.
2. Click on the + sign to expand the section next to Advanced Z Stage
Parameters.
3. Click on StaticSerialMoveDelay and enter “0” in the edit field on the right side
of the dialog box.
4. Click on the + next to ASI Z Stage to expand the section.
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5. Click on Mad City Static Delay and the right hand side of the dialog box will
update.
6. Enter the number “1” in the edit field and then select Apply Change.
7. Select Close and then restart SlideBook to register the change.
You may find that you need to increase the delay above 0ms in order to achieve
scientifically acceptable results.
7.8.3.2 Physik Instrumente PIFOC Piezoelectric Focusing Collar
The PIFOC collar can be operated in TTL mode (see TTL module documentation) or in
serial mode. In order to operate in TTL mode, you must purchase the TTL module for
SlideBook.
You should perform the following procedure to increase speed in serial mode whether you
are configured for TLL or serial mode. There may be instances where the TTL mode will not
be activated, so you will need to increase the performance in serial mode. To increase the
speed of this device in serial mode, perform the following steps:
1. Select Edit > Hardware Properties.
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2. Click on the + sign to expand the section next to Advanced Z Stage
Parameters.
3. Click on StaticSerialMoveDelay and enter “0” in the edit field on the right side
of the dialog box.
4. Click on the + next to Polytech PI to expand the section.
5. Click on ShortMoveSerialDelay
6. Enter “0” in the edit field and then select Apply Change.
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7. Click on EnableLongDelay, the right side of the dialog will update.
8. Select Yes and then Apply Change.
9. Select Close and then restart SlideBook to register the change.
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You may find that you need to increase the delay above 0ms in order to achieve
scientifically acceptable results
7.9
Autofocus
SlideBook offers the ability to perform an automatic focus routine during a 2D timelapse, a
2D multipoint timelapse or a montage capture. As with the other types of capture, you will
set parameters in the focus window and in the capture window. The basic procedure for
adding an auto focus routine to a multiplane capture is given below:
7.9.1 Determining Auto Focus Parameters
1. Open the Focus Window by either selecting Window > Focus Window or
clicking on the focus window button in the SlideBook toolbar
.
2. Select your objective and filter configuration then bring your sample into view as
discussed in Chapter 5 - Controlling the Camera and Microscope Hardware
(Focus Window) on page 77.
NOTE: It is critical that the Objective information is set correctly under Edit >
Optical Parameters > Objectives. SlideBook uses its knowledge of the
objective to compute the z spacing that it will use during auto focus. If your
microscope has an automated objective nosepiece, SlideBook can read its
position; however, if your microscope has a manual objective turret you must
make sure the proper Objective selected. To view objectives in the focus window
on a manual microscope, go to Edit > Hardware Configuration and select
Manual Objective Turret from the Objective Turret dropdown menu.
3. Select the Auto Focus button from the Focus Window.
4. The Auto Focus dialog box is used to determine the optimal auto focus
parameters for your particular sample.
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5. Enter parameters into the edit boxes according to the following descriptions:

Method – The computational method used to evaluate and compare focal
planes. Changing the method can affect the success of the auto focus
routine. The default method of Spectral 2D Template will work best in
almost all cases. The other methods are experimental. You should consult
3i if you are not having success with the default method.

Total Search Range – The total distance in the z dimension that will be
searched during the auto focus routine starting from half the specified
distance below the current z position. SlideBook will automatically
compute the number of planes to image based on the total search range
and the calculated depth of field of the microscope objective. This
information is displayed below the Total Search Range edit box. For
instance, in the above example in order to span the 8 micron search
distance with an objective that has a focal distance (FD) of 0.27 microns, a
total of 29 planes will be imaged.

Post Focus Offset – A positive or negative offset distance that the z
stage will be moved from the optimal focus position. This feature is useful
for always focusing a constant distance above or below the automatically
determined focal plane.

Peak Delta Threshold – Used to determine when no optimal plane of
focus should be computed from a particular image position. When
performing a montage capture, for example, some fields might not contain
tissue and should not be used to change the current z position. This value
can range between 0.0 and 1.0, but should not be decreased below 0.1 in
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most cases. For captures where you know that each image in the sequence
will have sufficient detail to focus, this threshold can be raised. If the
value is set to 1.0 then the threshold will be ignored.

X-Y Threshold – Not currently used.

Exposure – The exposure time used for image capture during the auto
focus routine. This edit box will have the same value as the exposure
time used in the focus window.

Save TIFFs – An advanced debugging feature that will generate a series
of TIFF files in a numbered directory created in SlideBook program
directory.

Save Logfile – An advanced debugging feature that creates an autofocus
log file in the SlideBook program directory.
6. Press the Autofocus button to initiate a single auto focus scan. When finished,
SlideBook will report the optimal focal z position in the Best Z information box
and automatically move the z stage to this location. In addition, timing statistics
for the autofocus routine are given in the Total Time and Image Time
information boxes.
NOTE: If SlideBook is unable to determine the optimal focal plane in the sample
it will return one of two messages in the Best Z information box. The No
Gradient message indicates that SlideBook was unable to find a focal gradient
within the specified search range. In this case, the peak delta threshold used for
autofocus computation may need to be increased or the search range may need to
be increased. The At Edge message indicates that the optimal focal plane
appears to lie above or below the specified search range. Since SlideBook will
automatically move the z stage to the best focal plane within the specified search
range, simply running the auto focus routine again (starting from this new z
position) will often find the optimal focal plane.
7. Press the OK button in the Auto Focus dialog box.
8. Use the focus window to evaluate the success of the autofocus routine. Using the
zoom button
to enlarge the image often aids in determining the best plane of
focus (see page 78). Once an acceptable set of auto focus parameters has been
determined, an auto focus routine can be added to the capture sequence.
7.9.2 Adding Auto Focus to an Image Capture Sequence
1. To include an auto focus routine in an image capture sequence go to Capture
Preferences dialog box (via the Advanced button in the Capture dialog box)
and select the Focus tab. The following window will appear:
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2. Select the Auto focus during timelapse/multipoint captures checkbox. This
will enable specification of several auto focus parameters.
3. Enter the autofocus frequency for the image capture series in the Update focus
every __ image capture(s) edit box. The autofocus routine will always be
initiated before the first image of a capture series. Specifying the autofocus
frequency will determine at which subsequent timepoints the autofocus will
occur. For instance, setting a frequency of 2 would cause an auto focus to occur
every other timepoint.
NOTE: SlideBook keeps track of the optimal auto focus plane during the image
capture series. Thus, after an auto focus routine has determined an optimal focal
plane, all subsequent images will be captured at this z position until another
auto focus routine is initiated. The z position of this new auto focus routine will
be determined by the results of the previous auto focus.
4. Select the channel that will be used for auto focus. This should be the same
channel that was used to test the auto focus parameters in the focus window.
5. The Total Search Range, Post Focus Offset, and Peak Delta Threshold
values will be automatically entered based on the auto focus parameters
determined in the focus window. If needed, these values can be further modified.
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6. If the autofocus channel will be a captured channel during the image series, click
the OK button and proceed with setting up the timelapse image capture
parameters as described on page 112. Make sure to expose the autofocus channel
in the image capture window and set an appropriate exposure time. SlideBook
will use these capture parameters (image extent, bin factor and exposure time)
when acquiring the image stack for auto focus computation.
7. If you do not want to capture the autofocus channel, or you want to use a
different image extent, bin factor or exposure time for auto focusing, select the
Expose uncaptured auto focus channel checkbox.
Enter the exposure time and bin factor for the uncaptured auto focus channel.
These parameters will be used to determine image capture for the purposes of
autofocusing. Click the OK button and proceed with setting up the image
capture. Now the channel that is being used for autofocusing does not need to be
exposed during the capture series.
8. Selecting Expose uncaptured auto focus channel also provides the ability to
specify a region of interest that will be used for auto focusing. This is useful for
auto focusing on samples that have features with heterogeneous optimal focal
planes. In this case, a full chip image extent can be captured at each timepoint
while using only a small subregion to determine optimal focus. Specifying an
auto focus region of interest is done in the Capture dialog box:
9. When the Expose uncaptured AF channel checkbox is selected in the Capture
Preferences dialog box, the Autofocus radio button is active in the Extent,
Offset, and Binning (pixels) section of the Capture dialog box. This menu is
used to alternate between designating the image extent for captured channels
and the image extent for auto focus. To define a subregion for autofocus, simply
select the Autofocus radio button and specify a Width, Height, X Offset, and
Y Offset. Alternatively, draw a selection in the Live View and press the Update
button. For more information on defining image subregions refer to the Selecting
the Area to be Imaged section of Chapter 6 on page 92.
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7.10 Varying Capture Rates During Timelapse Capture (Sequences)
You may choose to capture at various capture rates throughout your experiment when
performing a multi-channel 2D or 3D timelapse capture. The capture rates may be changed
either automatically in a preset protocol or manually during capture.
7.10.1 Setting Capture Preferences
To use this feature, you must first set up capture preferences. To do so:
1. Open the Capture dialog (Image > Capture) and press the Advanced button.
2. In the Capture Preferences dialog, select the Sequence tab.
The elements of the dialog box are described below. A sequence is made up of a
series of stages. Each stage may have a specified duration and timelapse interval
(time between timepoints).

Begin capture with first event in sequence – if checked, capture will start
with the first event in the sequence

Loop to first stage when done – if checked, capture will return to first stage

Stages – each stage will be automatically performed in the order they are
displayed, from top to bottom
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
Not in Sequence – these stages must be manually selected in the Capture
Status dialog; they will not be performed automatically

Stage Name – defined by the user

Timelapse Interval - the delay between the beginning of one timepoint and the
beginning of the next timepoint. Available units are milliseconds, seconds,
minutes, and hours.

Duration of Stage - how long capture will occur at the defined rate. You may
define the length as timepoints (e.g., 5 timepoints), a time period (e.g., for 10
min), or continuous (ending when manually selecting the next stage)
An example of a completed dialog box is shown below.
3. To add a stage, select Add. Enter the stage information described above. The
stage will appear in the Stages list.
4. To add a stage that will be started manually in the Capture Controls, click on the
Not in Sequence list to activate it.
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5. Add a stage as described in Step 3. The stage will appear in the Not In
Sequence frame. You may use the > or < buttons to move events from the
Stages frame to the Not In Sequence frame and vice versa.
To change the sequence of stages, use the Up and Down buttons to move the
stage in the list up or down one place.
6. To remove a stage, select the stage from the list by clicking on the stage name
and then clicking Remove.
NOTE: The overall time (duration) and the number of timepoints of all sequential
stages is shown.
7. After you have set up your sequence, press OK. If you have selected to perform
an automated sequence, the appropriate parameters will be loaded into the
Capture dialog box.
7.10.2 Setting Capture Dialog Box Parameters
1. Open the Capture dialog box by selecting Image > Capture or by selecting the
Capture icon in the tool bar
.
2. If it is not already checked, check the Timelapse checkbox in the Capture
Type section of the Capture dialog box. You now have the option to define a
variety of timelapse parameters. Enter the desired values in the following fields:

# of Timepoints – The number of timepoints that will be captured.

Duration – The total length of time for the experiment. Units of time, in
milliseconds (ms), seconds (s), minutes (m) or hours (h), can be selected from
the dropdown menu.

Interval – The delay between the beginning of one timepoint and the
beginning of the next timepoint. The interval unit can be selected from the
dropdown menu. If the capture sequence at one timepoint takes longer than
the interval, SlideBook will capture the next timepoint immediately following
the preceding timepoint.
NOTE: As you type in two of these values, the third field will be calculated
automatically.
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3. To begin a capture click OK. The Capture Status Window will appear.
4. Click on the Stages tab.
This tab has two lists, Stages and Not in Sequence.
5. If you have opted to start a sequence at the beginning of capture, the first stage
in the top frame will be marked with a “*”. To manually execute a stage, select
the stage by clicking on it in the list and then click on the Execute Stage
button. The number of timepoints in the stage and the timelapse interval will be
displayed at the bottom of the window.
7.10.3 Examples for Variable Capture
7.10.3.1 Example 1
Suppose you have set up a stage with 22 timepoints and a 200ms interval, and have
checked the Begin capture with first stage in sequence checkbox in the Capture
Preferences dialog. In the Capture window, you select a timelapse capture with 100
timepoints, and a 1000ms interval. When you click Start, capture will start by executing
the sequential events in the displayed order in the Stages list. After the total sequence has
been captured (22 timepoints) the remaining 78 timepoints (100 – 22 = 78) will be captured
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with the interval set in the Timelapse Capture section of the Capture dialog box
(1000ms).
7.10.3.2 Example 2
Suppose you have set up capture as in Example 1, but did not check the Begin capture
with first stage in sequence checkbox. Capture will begin with 1000ms interval between
timepoints. To execute the stage, click on the stage to be executed and click Execute
Stage. Twenty-two timepoints will be captured. The remaining timepoints will be capture
at 1000ms interval until the total of 100 timepoints have been performed.
7.11 Saving Images to Disk (Spooled Capture)
SlideBook allows you to save your images directly to disk. This is especially important
when performing long timelapse captures that will exceed your computer’s memory limit.
To save images to disk:
1. From the Capture dialog box, select Advanced to bring up the Capture
Preferences dialog box.
2. Select the Spool tab. The following window will be displayed:
2. Select the desired spool capture mode:

Capture images to memory only – Images are saved in memory and
only become permanent after capture if Save Slide is selected.

Capture to memory and save to spool file after each timepoint –
Images are both kept in memory and saved to disk. This feature allows
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you to permanently save images as they are captured, and immediately
view the images post-capture (no need to import a spool file). This is only
useful for captures that are not larger than the memory available.
3. Select the location for storage of spool files by selecting Browse and then
navigating to the desired location.
4. Click OK to save these preferences, and then set up for your desired capture
sequence. Once your capture is finished, you may import it for viewing as
discussed in the section Importing SlideBook Spool Files on page 97.
7.12 Saving Capture Parameters
You can store and recall frequently used capture parameters. The following parameters can
be saved:






Image extent
Binning
Capture type
Channels and exposure times
Camera intensification and gain settings (if applicable)
Capture preferences (e.g., Periodic, 4D, Spool features)
To do so:
1. Begin a new capture by selecting Image > Capture. The Capture dialog will
appear.
2. Select your capture parameters. To access advanced parameters such as periodic
capture select the Advanced button. The Capture Preferences dialog will
appear.
3. Configure the desired preferences and select OK.
4. To save these preferences, select Save As from the Capture Settings dropdown menu in the Capture dialog. If you choose Save, the default parameters
will be overwritten.
The following dialog will appear:
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5. Enter a name for these preferences and select OK.
6. If you would like to load a different set of parameters, select one from the
dropdown list. If you modify your capture parameters, the name of the currently
loaded parameters will say “(mod)” next to the name. You may choose to save
them under the same name by selecting Save or under a different name by
selecting Save As as discussed above.
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8 Image Display and Manipulation (Views)
SlideBook stores images in slides and displays images using views. There are two kinds of
views: data views and display views. Data views are interactive—the user can display any
combination of different channels and masks as well as change lookup table parameters
(renormalization) and edit the image’s masks. A new data view can be spawned either from
the slide, or in one case, from another view. Display views, on the other hand, are always
spawned from a data view and are set to the channel, mask, and renormalization selections
of that view. Data views focus on interactive analysis, while display views focus on
visualization.
This chapter covers the following topics:






8.1
Introduction to Slide and Image Display in SlideBook
Using Data Views to Display Images
Manipulating Data Views Using the Info/Tool Bar
Using the Tool Menu
Using Display Views (Creating Renderings and Movies)
Exporting Views
Introduction to Slide and Image Display in SlideBook
When you start SlideBook a new slide called Slide1 is created, and unless you open
another slide, it is into this slide that all new and imported images are inserted. Typically
a slide corresponds to all of the images taken from a particular sample.
Each image in a slide is represented in a slide window as a thumbnail and some associated
data. The image may be either a single image or a set of images depending on the type of
capture that was performed (e.g., 2D, 3D, or timelapse). The thumbnail is either a
shrunken rendering of the first plane of an image (the default) or of some portion of an
image (if you have specified a particular thumbnail using the thumbnail button—see
Changing the Default Display on page 180). The information includes the following:
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
Image name - automatically generated in the case of captured images or
corresponds to a file name in the case of imported images.

Comments – entered by the user either before or after capture

Capture date and time

Capture type – 2D, 3D, 2D timelapse, or 4D
Chapter 9 – Preparing an Image for Analysis or Export

Number of channels – number of fluorophores

Dimensions – image size in pixels and planes (can be X*Y*Z/T or X*Y*Z*T)

Image size – image size in MB

Masks – number of masks stored with the image
8.1.1 Working with Slides
The Slide (.sld) file is SlideBook’s native document type. Opening, closing, and saving slide
files are performed in a manner similar to other software programs.
8.1.1.1 Saving Slides
To save a newly created or updated slide:
1. Click on the slide that you wish to save to make it the active slide.
2. Select File > Save Slide. If you have not previously saved the slide, you will be
prompted for a file name.
3. Enter a filename and select Save.
To save a copy of a slide:
1. Click on the slide that you wish to save to make it the active slide.
2. Select File > Save Slide As.
3. Navigate to the location where you would like to save the file, enter a filename,
and select Save.
8.1.1.2 Opening Slides
To open an existing slide:
1. Select File > Open Slide.
2. Navigate to the desired slide and select Open.
8.1.1.3 Closing Slides
To close a slide:
1. Click on the slide that you wish to close to make it the active slide.
2. Select File > Close Slide or click on the
in upper right corner.
8.1.1.4 Deleting Slides
Slides can be deleted using standard Windows operations for deleting files. You may not
delete a slide from within SlideBook; however, you may delete the images that are present
in the slide.
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8.1.1.5 Changing the Slide View Display
SlideBook offers several ways to view your Slide. To change your Slide View display, go to
View > Slide View Display and select one of the following options:
Large Icons
List
The default display is the Details display (shown at the beginning of the chapter).
8.1.2 Working with Images
As discussed above, each slide may contain several images. Using the same clipboard
metaphor as other applications, SlideBook lets you remove images from a slide, duplicate
images, and insert images. You can cut or copy an image from one slide and insert it into
another.
NOTE: There is currently no mechanism for multiple selection; you can only manipulate one
image at a time.
8.1.2.1 Selecting an Image
Before performing any operations on a given image, it must first be selected, or made the
active image. To make an image the active image, click on an image thumbnail in an active
slide window (or anywhere in the box surrounding an image’s information).
The icon (and attributes) of the selected image will turn blue. It is on the selected image
that all data manipulation commands (such as deconvolution), mask creation commands,
and image information commands will operate.
8.1.2.2 Removing an Image from One Slide and Placing it in Another
To move an image from one slide to another, do the following:
1. Click on the slide that contains the image you would like to move; this makes it
the active slide.
2. Click on the image you would like to move; this makes it the selected image.
3. Select Edit > Cut Image, or click on the Cut Image icon
the image from the slide.
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4. Click on the slide that will hold the pasted image.
5. Select Edit > Paste Image, or click on the paste image icon
.
NOTE: Since the image is stored internally in a proprietary format, these operations do not
use the Windows clipboard. In other words, if you quit SlideBook without pasting a cut
image, you will not be able to get to that image again.
8.1.2.3 Copying an Image
You may duplicate an image and place it in another slide, or in the same slide. It is often
useful to copy an image before performing manipulations that affect the underlying data.
1. Make the slide that contains the image that you would like to copy the active
slide by clicking on it.
2. Make the image that you would like to move the selected image by clicking on it
as discussed above.
3. Select Edit > Copy Image, or click on the copy image icon
.
4. Click on the slide that will hold the copy of the image. This may be the current
slide, or another slide.
5. Select Edit > Paste Image, or click on the paste image icon
.
8.1.2.4 Deleting an Image
To permanently remove an image:
1. Make the slide that contains the image that you would like to delete the active
slide by clicking on it.
2. Make the image that you would like to delete the selected image by clicking on it
as discussed above.
3. Select Edit > Delete Image.
CAUTION: This is a permanent removal that cannot be undone.
8.1.3 Image Display
SlideBook contains four different kinds of data views: the Main View, the Three View, the
Tile View, and the Channel View. Data views may be used not only to look at data, but to
generate statistics. Display views are strictly used for viewing data. The following display
views are available: Volume View, Surface View, and Timelapse Intensity Plot. The displays
that are most appropriate for different modes of imaging or, capture types, are shown in the
table below. Capture type is designated by the icon for any image as well as identified in
the default .sld view as shown below.
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Display
Views
Data Views
2D
Capture
Main View
Three View
3D Tile View
Timelapse Tile View
Multidimensional Channel View
3D Volume View
4D Volume View
3D Surface View
2D Timelapse Intensity Plot


3D
Capture





2D
Timelapse
4D
Capture













Next, we will discuss how to generate data views and manipulate those views using the
info/tool bar. Finally, we will discuss display views.
8.2
Using Data Views to Display Images
This section describes how to display data views. SlideBook allows multiple data views of a
single image to be open simultaneously.
8.2.1 Displaying a Main View
The Main View can be used to display any type of data. The Main View displays a single
plane at a time where the z or t axis can be scrolled using arrows or a slider.
To generate a Main View:
1. Choose File > Open Slide and navigate to the desired slide file. Double click on
the slide file to open it. A slide window will appear.
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2. Double-click anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail.
A new window will pop up that contains the image. This window is a Main View
of the image.
Alternatively, you may open a Main View using the View menu. To do this:
1. Click once anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail to highlight the image.
2. Choose View > New Main View to display a new Main View.
When working with 2D Timelapse or 4D Capture types, the Main View window will have
controls for time as shown below:
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For 4D capture types, the Main View operates similarly to Main Views for 3D
and 2D data. Additionally, there is a slider that you may click and drag to view
individual timepoints of the 4D capture. You may scroll through the z axis by
using the up and down arrow bars in the tool bar (see Scrolling through the
Invisible Axis in a 3D or Timelapse Image on page 180).
8.2.2 Displaying a Three View
The Three View is equivalent to three Main Views put together, one for each axis. It is
useful for exploring 3D, 4D, and 2D Timelapse data.
To open a Three View from an open slide:
1. Click once anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail to highlight the image.
2. Choose View > Three View to display a new Three View.
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x,z (x,t)
y,z (y,t)
x,y
NOTE: You may notice that your object may appear disproportionately small, or “squashed”
in the z direction. When performing 3D imaging, each plane that is captured is represented
by a set of pixels in the image set. Each pixel is a perfect cube. You will notice that the z
dimension in the 3D image set appears to be different than what was actually captured.
This is because the z step size that was selected during capture is not actually the same as
the length of the z dimension of the pixel. In the image set, planes are simply stacked on top
of one another with no regard for the actual z distance traveled (this is done in order to
preserve data fidelity). You may interpolate between planes and expand your z dimension
to represent the actual z distance traveled by the microscope by generating an isotropic
image (see page 213).
8.2.3 Spawning a Tile View
The Tile View can display many planes simultaneously. Both the 3D Tile View and the
Timelapse Tile View are designed to enhance interaction with 3D and timelapse data.
8.2.3.1 Displaying a 3D Tile View
The 3D Tile View is designed for use with 3D Capture data. This view will display each zplane in a montage.
From an open slide:
1. Click once anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail to highlight the image.
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2. Choose View > 3D Tile View to display a new 3D Tile View.
8.2.3.2 Displaying a Timelapse Tile
The 3D Tile View is designed for use with 2D Timelapse or 4D Capture data. This view will
display each time-point in a montage. The arrows for z-movement will be available for 4D
Capture data
From an open slide:
3. Click once anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail to highlight the image.
4. Choose View > Timelapse Tile View to display a new Timelapse Tile View.
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8.2.3.3 Displaying a 3D or Timelapse Tile View of a Portion of the Image
The Tile View is slightly different than the Main and Three Views in that it is tailored to
display a portion of the image rather than the entire image. Since the Tile View creates a
pane for every plane that it displays, a Tile View of the entire image will often be far larger
than the size of the monitor, which defeats the Tile View’s purpose of allowing
simultaneous viewing of multiple planes along the same axis. So, although a Tile View can
be created directly from the slide window, you will more likely be making a 3D selection in
either a Main View or a Three View (or even another Tile View) and spawning a Tile View
from there.
In order to spawn a Tile View from another view:
1. Make the desired view the active window by clicking on it.
2. Define the selection. There are several ways to do this, and they are discussed
Making a 2D or 3D (x,y,z or x,y,t) Selection on page 186.
3. Choose the desired Tile View from the View menu to display a new 3D or
Timelapse Tile View of the selected portion of the image.
4. To change the portion of the image that is displayed, select the hand tool from
the tool menu and click and drag in one of the panes (see table on page 184 for a
description of this function).
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You can create a Tile View that shows slices along any of the three axes simply by making
your selection in a view (or pane) that is currently set to the desired invisible axis (see
description of the axis menu in the table below). In other words, if you make a selection in a
Main View that has an invisible y-axis, and the selection spans ten units in the y-axis, the
resulting Tile View will have ten panes that display the x axis horizontally and the z axis
vertically.
8.2.3.4 Arranging your Tile View
You may wish to change the way your Tile View is displayed. For instance, the default tile
display may choose a 4 × 6 arrangement, while you desire a 6 × 4 arrangement. You may
also wish to change the background color between the tiles. To alter these settings:
1. With a Tile View displayed, select View > Settings or click on the
icon in the View window. The following dialog box will appear:
Setting
2. Select the Specify Dimensions Manually checkbox, then enter the Tiles Per
Row that you desire in the edit field.
3. In the Background Color section, click on the colored square next to View
background color.
4. Select the desired background color from the Color dialog and click OK.
5. Click OK to update your Tile View.
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8.2.4 Displaying a Multidimensional Channel View
The Multidimensional Channel View is most useful for displaying multi-channel images.
The Multidimensional Channel View displays each channel independently plus a composite
RGB image. Users can also choose to display individual channels with their default coloring
or to display multiple timepoints or z-sections of the image. To display a Multidimensional
Channel View from an open slide:
1. Click once anywhere on the thumbnail or within the shaded region that
surrounds the thumbnail to highlight the image.
2. Choose View > Multidimensional Channel View to display a new
Multidimensional Channel View.
8.2.4.1 Multidimensional Channel View Settings
The Multidimensional Channel View allows users the flexibility to create images for export
from 2D, 3D, 2D timelapse, or 4D data types. This section discusses adjusting the
Multidimensional Channel View Setting in order to maximize the information shown in the
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exported image. You may wish to choose a region before generating the Mulitidimensional
Channel View in order to improve viewing ability (see Making a 2D or 3D (x,y,z or x,y,t)
Selection on page 186). To create a Multidimensional Channel View:
1. Select an image in a slide by clicking on it.
2. Select View > Multidimensional Channel View. A Multidimensional Channel
View will appear.
3. You may then alter the display of channels, composite images, and timepoints/zplanes by going to the View Settings icon in the View window (show below) or
by going to View > Multidimensional Channel View Settings.
4. The Multidimensional Channel View Settings dialog will open. Adjust the
following parameters to achieve your desired display. Display Parameters are
shown below.
5. Once the display parameters are selected as desired, click OK.
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8.2.5 Displaying a Montage
SlideBook allows you to generate montages from images captured in SlideBook using the
montage feature (see page 131). To generate a montage:
1. Open the slide that contains montage data.
2. Select Image > Generate Montage. SlideBook will attempt to create a montage
image using only stage coordinates and pixel size information. A dialog box
similar to the following will appear.
3. You may choose to manually align the images by choosing an image from list and
then pressing any of the arrow buttons. Only the selected image will move. You
may also hold down the shift key to move all of the images to the right and below
the selected image. If you click on an image in the montage display, the
appropriate image will be highlighted in the image list. Alternatively, you may
choose to perform an auto-alignment by pressing the Auto Align key.
4. Once you are satisfied with the alignment, select OK to close the dialog box and
save your alignment settings.
8.3
Manipulating Data Views Using the Info/Tool Bar
All Data Views have, at the top, a set of buttons, menus, and readouts collectively called the
info/tool bar.
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The various features are described in the table below.
Feature
Function
X,Y, Z, (T) Position
Shows and continuously updates x,y,z (3D images) or x,y,t (timelapse
images) position of the mouse pointer. The location follows x,y,z (or t)
order regardless of the invisible axis that is displayed.
Zoom Controls
Displays and manipulates the magnification, or zoom factor of the image
Channel Menus
Allow the user to select the channels that correspond to R,G, and B or
monochrome displays; default display corresponds with filter configuration
definitions (Defining Filter Configurations on page 64)
Pixel Intensity Values
Shows and continuously updates pixel intensity values corresponding to
the mouse pointer location for a given channel
Main View: used to navigate through the invisible axis
Three and Channel View: used to navigate the z- or t-axis
Up and down buttons
4D Tile View: used to navigate z-axis
Clicking once will move the displayed plane by one; holding continuously
will scroll through the planes. Not used for 2D images.
Tool Menu
Allows the user to choose the function that the mouse and its cursor
perform, as described in the section Using the Tool Menu. This menu has
different functions when a mask is displayed (see Chapter 10).
Mask Menu
Allows the user to create and/or display a mask, or region of interest, as a
blue overlay on the image. It is usually best to display image data in only
red and green while displaying or editing masks.
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Axis Menu
Allows the user to choose the invisible axis in Main Views of 3D and
timelapse data, and in Tile Views of 4D data. The up and down buttons
are tied to the invisible axis.
Thumbnail Button
Allows the user to alter the default view of a particular image. Once the
thumbnail button is selected, subsequent views and the slide thumbnail
image will retain channel settings, renormalization information, and
invisible axis plane.
Renormalize Button
Allows the user to alter the look-up table, or renormalization parameters,
for each channel of the view
Now, we will explore several data view manipulations that require features of the info/tool
bar.
8.3.1 Altering the Renormalization Parameters (Lookup Table)
You will often want to highlight a particular range of data values in an image. Perhaps a
particular feature is dim relative to other features, so you will want to saturate the
mapping between the data and the display so that this feature will become more apparent.
Or, perhaps in one channel you have a histological marker that might otherwise overpower
the data and want to dim its contribution to the color values. To alter the range of data that
is displayed, you will choose the minimum and maximum data values that correspond to
the absence or full saturation of the color associated with each channel. You also have the
ability to apply nonlinear lookup tables to your view.
8.3.1.1 Changing Renormalization Parameters for a Single View
To alter renormalization parameters:
1. Create a Main View, Three View, Tile View, or Channel View of an image. If you
have several views open, make sure that the desired view is the active window by
clicking on it.
2. Click on the renormalize button
or select View > Renormalize. The
Renormalize Image dialog box will appear.
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A histogram is displayed showing the relative number of pixels on the y axis and
intensity values on the x axis for the specified channel. The x axis value shown
on the left is the lowest data value across the entire image. The x axis value
shown on the right is the highest data value. In the dialog box above, the CY3
channel intensities range from 0 to 13067.
3. Select the channel that you wish to renormalize from the Channel dropdown
menu.
The red and green bars allow you to select the minimum and maximum
intensities that correspond to the absence (black) or full saturation of color in the
display. Thus, any data whose value is lower than the low bar will not appear in
the view. Any data whose value is above the high bar will show as fully saturated
color. The display’s dynamic range, then, is a linear interpolation of the values in
between.
4. You may move the red and green bars and alter the display two ways.
a. Click and drag the red and green bars in the histogram window to the left or
right. The change will automatically register once you release the mouse
button.
OR
b. Enter the desired minimum and maximum intensities in the Low and High
edit fields and click on Apply to register the change and update the image.
This gives you the opportunity to see the effect of the values and make
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changes without closing and reopening the dialog. Choosing Close after
entering intensities manually will close the dialog box without registering the
change.
Again, the underlying intensity values are not affected by the renormalization
process.
5. Follow steps 3 and 4 for any other channels that you wish to renormalize.
6. Choose OK to exit the Renormalize Image dialog box. If you would like
subsequent views of the image to contain the same renormalization parameters,
click on the thumbnail button .
NOTE: You can reset the low and high values for a single channel to correspond to the
minimum and maximum intensities for that channel by pressing the Reset to Selection
Min/Max button. You can also set all channels to the global min/max (the lowest and
highest values of any channel present in the image) by pressing the Reset all to Image
Min/Max. This is particularly useful when working with transmitted light color images.
Remember that renormalization is a property of a particular view, not the entire image. It
is possible to have multiple views on the same image each with their own renormalization
values. If, however, you decide that a particular selection of values is best for most viewings
of an image, you can press the thumbnail button in the corresponding view and make those
values the default for that image (see Changing the Default Display on page 180).
8.3.1.2 Changing Renormalization Parameters for a Group of Images in a Slide
You may apply the default renormalization parameters of an open view to all images in a
slide by selecting Image > 4D Operations > Renormalize All.
NOTE: You MUST set the display as default using the thumbnail button before you apply
the settings across the images in a slide.
You may also set the renormalization to the global minimum and global maximum for each
channel for all images in a slide. To do so, select Image > 4D Operations > Renormalize
All to Global Min/Max. This function finds the lowest minimum and highest maximum
pixel value for the group of images in the slide on a per channel basis, and then uses those
values to set the renormalization of all images in the slide.
8.3.1.3 Applying Nonlinear Lookup Tables
SlideBook allows you to apply nonlinear lookup tables using a gamma correction. This
feature is particularly useful for emphasizing dim features in an image. To apply a nonlinear look-up table, simply click and drag the gray line that crosses the histogram
diagonally. This adjusts the value of gamma. Alternatively, you may enter a value for
gamma manually and select Apply. If you wish to return the gamma to 1 (no gamma
correction), simply click-and-drag the line off of the histogram and it will return to 1.
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8.3.2 Changing the Display Colors
Images may be displayed using a variety of color schemes, including RGB, monochrome,
inverted monochrome, pseudocolor, and user-defined color. SlideBook also supports a mixed
display that blends a monochrome background image (e.g., DIC) with an RGB image. The
default display is determined by the filter configuration definitions (see Defining Filter
Configurations on page 64), though you may easily change the colors that are displayed for
a given view. Generally, the filter configurations will be set to R, G, or B. However,
monochrome display may be useful for visualizing a single channel. Pseudocolor display
may be useful for visualizing one or two channels.
8.3.2.1 Changing the Channels Displayed on an RGB Image
1. Make the desired view the active window by clicking on it.
2. Select the fluorophore that you would like to display from the corresponding
drop-down channel menu in the info/tool bar.
NOTE: If your image is displayed as something other than RGB, you may change the
display to RGB by choosing View > RGB Color.
8.3.2.2 Changing an Image Display to Monochrome
1. Make the desired view the active window (by clicking on it).
2. Select View > Monochrome to change the display to monochrome.
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3. Click on the channel menu, and select the fluorophore that you would like to
display from the dropdown list
8.3.2.3 Changing an Image Display to Pseudocolor
1. Make the desired view the active window by clicking on it.
2. Select View > Pseudocolor to change the display to pseudocolor.
Two channel menus will be available. The first channel will be displayed as a
pseudocolored image ranging from saturated red to saturated blue, known as
Pseudocolor (Color).
3. Click on the first channel menu, and select the fluorophore that you would like to
determine the pseudocolor hue from the dropdown list.
The second channel menu will act to gate the pseudocolor image based on
intensity. This second channel is called Pseudocolor (intensity). Thus, an
area with a high pixel intensity for the first channel and a low pixel intensity for
the second channel will appear as dim red. An area with a low pixel intensity for
the first channel and a high pixel intensity for the second channel will appear as
bright blue.
4. If you would like to gate the first channel by the intensity of a second channel,
select a second channel from the dropdown menu.
8.3.2.4 Changing the Display to a User-Defined Color Palette
You can now display up to eight channels in colors of your choosing. This is useful for
applications such as fluorescence in-situ hybridization (FISH).
To use this feature:
1. Open a Main View, Three View, or Tile View.
2. Select View > User-Defined Color. Your image will appear in the following
format. All channels that were captured will be listed in the channel list.
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3. To change the color of one of the channels, click on the colored box next to the
channel whose color you would like to change. The following dialog box will
appear.
4. To change the color, either click on one of the Basic colors in the upper left
section of the dialog, or click on one of the Custom colors in the lower left, or
create your own color on the right side. If you would like to add a color to the
Custom colors, use the color picker on the right to select a color, then select
Add to Custom Colors.
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5. To add a channel to display, simply select the checkbox next to the channel.
You may choose to always display a channel using a color of your choosing by specifying it
in the Filter Definition (see page 66).
8.3.2.5 Displaying a DIC image as Background of an RGB Image
In an RGB image, the first three popup menus control which channel is visible in red,
green, and blue respectively. The last popup menu, labeled Bkgnd, selects a background
display channel. In multi-channel images, one channel can be chosen to be displayed in
monochrome wherever the other channels would not otherwise be visible. This can be
particularly useful for showing DIC information alongside fluorescence. To display DIC
alongside a fluorescent RGB image:
1. Create a Main View, Three View, Tile View or Channel View of an image that
contains a DIC channel and at least one fluorescent channel.
2. Make sure that the fluorescent channels are displayed as RGB. If they are not,
select View > RGB Color to change the display to RGB, and select the
fluorophores to be displayed from the channel menus.
3. Set the Bkgnd channel menu to DIC.
The DIC image will show through in any pixel that has RGB intensities that are
below the minimum intensity threshold of the fluorescent channels. If you would
like to increase the amount of DIC that shows through, you must raise the
minimum intensity threshold on all of the RGB channels (See Altering the
Renormalization Parameters (Lookup Table) on page 171).
4. To change the display so that the DIC and fluorescent channels are blended at
50% opacity, select Blend Background from the View Settings button
or
select View > Blend Background. You will not need to alter the minimum
intensity threshold on the RGB channels to see DIC.
8.3.3 Cropping an Image
You may wish to crop an image in order to reduce its size or center on a specific portion of
the image.
CAUTION: Cropping is an irreversible process. You may wish to make a copy of the image
before cropping (see Copying an Image on page 159) or select Crop to New Image.
To crop an image:
1. Open a Main or Three View of the desired image.
2. Define a 2D or 3D selection as described in Making a 2D or 3D (x,y,z or x,y,t)
Selection on page 186.
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3. Select Image > Crop to replace the existing image, or Image > Crop to New
Image to generate a new, cropped image.
8.3.3.1 Extracting Timepoints from a Timelapse Image
You may wish to extract only a subset of timepoints from a 2D Timelapse or 4D data series.
To extract timepoints:
1. Select an image in an open slide or open a Main View of the desired image.
2. Select Image> Extract Subseries to New Image. The following dialog will
appear:
3. Enter the starting and ending timepoint of the subseries you would like to
extract.
4. Check the box for Insert image at end of slide if you would like to save the
subseries in the same slide as the original data. If you do not check this box, the
image will save below the active image in the Slide View.
8.3.4 Rotating an Image
You may wish to rotate a view of the image for publication purposes.
CAUTION: Rotation is an irreversible process. You may wish to make a copy of the image
before rotating (see Copying an Image on page 159).
To rotate an image:
5. Open a Main View of the desired image.
6. Select Image> Rotate. The following dialog box will appear:
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7. Select the Image Range as described below. You may also select and deselect
images using the checkboxes in the Selected Images list.

Current Image – Operation will be performed on the selected image
only.

Current Capture Type – Operation will be performed on all images in
the slide with the same capture type (2D, 3D, 4D, etc.) as the selected
image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
8. Select the desired rotation angle and select OK. The image will be rotated as
specified.
8.3.5 Changing the Invisible Axis
SlideBook allows for 3D and timelapse data to be viewed in all possible dimensions. By
default, a Main View will display the x,y plane (invisible z axis for 3D and t axis for
timelapse). However, you can change the invisible axis to the x or y axis if you wish. To view
your image with a different invisible axis:
1. Open a Main View of the desired image as discussed above.
2. Click on the axis menu
in the info/tool bar and select the desired invisible
axis from the dropdown menu. The up and down arrows in the info/tool bar will
be tied to the new invisible axis.
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NOTE: The axis menu is not available in the Tile View or Channel View. If you would like to
view a different invisible axis in the Tile View you should first open a Main View, change
the invisible axis, make a 3D selection and then spawn a Tile View as described above
(Displaying a Timelapse Tile on page 164).
8.3.6 Scrolling through the Invisible Axis in a 3D or Timelapse Image
There are several ways to scroll through the invisible axis in Main Views, Channel Views,
and 4D Tile Views.

Using the up and down arrows
in the info/tool bar: one click on either arrow will
move one plane up or down; holding continuously will scroll.

Using menu commands: View > Prev Plane, View > Next Plane, View > Go to
Plane and View > Loop will move the invisible axis location one plane down, one
plane up, to a specified plane, or in a continuous loop, respectively. See page 27 for
instructions on how to use the Go to Plane feature.
NOTE: To scroll around Three View images, see the section titled Scrolling through a Three
View on page 187.
8.3.7 Changing the Default Display
Once you have an image that has the color scheme and renormalization parameters that
you desire, you may save those parameters so that all subsequent views retain those
parameters. To do so:
1. Using a Main View, Three View, or Tile View, adjust the color display and
renormalization parameters as described in the sections above.
2. Click on the thumbnail button
in the info/tool bar to set new default display
parameters. The thumbnail image in the slide view and all subsequent views will
display the new default parameters.
NOTE: When using a Main View, you may wish to scroll to a desired plane in the invisible
axis before clicking on the thumbnail. The selected invisible axis plane will be displayed in
subsequent Main Views.
8.3.8 Displaying Annotations
The following annotation features are available in SlideBook 4.2:






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Timestamps – available for timelapse captures
Scale Bars – bar showing relative scale/size of image
Lookup Tables – range of colors that correspond to intensities for a given
channel
Notes – formerly called Annotations in previous SlideBook versions, can be used
to record experimental actions (see Creating Notes on page 116)
Object IDs – corresponding to objects in a displayed mask
Regions – selections made using ROI tools
Chapter 9 – Preparing an Image for Analysis or Export
Timestamp
Lookup
Table
Object ID
Scale Bar
Region
8.3.8.1 Displaying Annotations
To display any of these annotations on an open view:
1. Open a Main View, Three View, or Channel View of your data.
2. Select the item you wish to display from the Annotations menu.
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3. When an item is selected for display, a checkmark will appear next to the item on
the menu.
8.3.8.2 Formatting Annotations
Several formatting options for annotations are available. Once you have displayed your
annotations, you may format by doing the following:
1. Open the Annotation Default Settings dialog by selecting Annotation Settings
from the View icon
dialog will appear:
or by going to Annotations > Settings. The following
2. Select the annotation that you would like to format. The dialog boxes for
annotations are shown below.
Scale Bars – You may define the length of the scale bar; choose color, placement
and font; and include text or not.
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Lookup Tables – You may choose the size of the lookup table (as a percentage of
the image width and height) as well as placement, text color and font.
Notes – You may choose placement, text color, or font.
Object IDs – You may select text color and font. Note that object IDs only appear if
you have a mask displayed that has been divided into objects.
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8.3.8.3 Setting Default Annotation Settings
Once you have adjusted the Annotations to the desired display, you can save the settings as
defaults by going to Annotations > Set Default Settings.
8.4
Using the Tool Menu
The tool menu of the info/tool bar allows the user to choose the function that the mouse and
its cursor perform.
The tool menu may also be accessed by selecting View > Tool. The various features are
described in the table below:
Feature
Function
Marquee Tool
Lets you select a 2D or 3D region of the image and is used when
spawning a Tile View or a volume rendering (Making a 2D or 3D (x,y,z or
x,y,t) Selection on page 186). This tool can also be used to make masks
(see Chapter 9).
ROI Selection Tool
Lets you select an ROI in order to perform further operations. Holding
shift allows you to select multiple ROIs. See Creating ROIs and Graphs to
Monitor Regions of Interest on page 118 for more information on working
with ROIs. This tool is also available from the SlideBook toolbar.
Rectangle ROI Tool
Allows you to draw rectangular ROIs on an open view by clicking and
dragging. This and all other ROI drawing tools are available from the
SlideBook toolbar at the top of the application window.
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Ellipse ROI Tool
Allows you to draw elliptical ROIs on an open view by clicking and
dragging.
Polygon ROI Tool
Allows you to draw closed polygons by clicking to draw a series of straight
connected lines. Double-clicking closes the shape.
Freehand ROI Tool
Allows you to draw freehand by clicking and holding. Shapes close
automatically when mouse button is released.
Hand Tool
Main and Three Views: lets you move the displayed portion of an image in
a window smaller than the dimensions of the displayed axes (performs
the same function as the horizontal and vertical scroll bars).
Tile View that displays a portion of an image: adjusts the position of the
display cube within the image, therefore scrolling the portion of the image
that is visible in all planes.
Main and Three Views: grows or shrinks the size of the window and
image by a factor of two.
Zoom Tool
Tile View: the overall size of the window and its panes remains constant,
but the zoom factor of the displayed image is adjusted by a factor of two.
Clicking on a view will zoom in (double the size) and clicking while holding
down the shift key will zoom out (halve the size).
Three View: updates the displayed position in the other two panes by
clicking or dragging in any of the three panes. Dragging over a pane will
continuously update the other two panes.
Point selection Tool
Main View: Ctrl-click on a region in a 2D timelapse image to display a
graph of intensity vs. time.
No function in Tile View or Channel View.
Ruler Tool
Measures a desired distance in 2D and 3D images, and velocity in 2D
timelapse images. See Making Measurements with the Ruler Tool below.
Angle Tool
Measures a desired angle in 2D and 3D images. Click and drag to draw a
single line, then make a second click at the angle you wish to measure.
The angle measurement appears next to the zoom factor.
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Now, we will explore several data view manipulations that require features of the tool
menu.
8.4.1 Making a 2D or 3D (x,y,z or x,y,t) Selection
As discussed above, the ability to make a 3D selection is useful when spawning a Tile View.
You may also want to make a 3D (or 2D) selection in order to perform volume rendering
(see page 189), crop an image (see page 177), or generate statistics for analysis (see page
227). 3D selections will be visible in all three panes of the Three View, and when scrolling
through the invisible axis in the Main and Channel Views.
8.4.1.1 Making a 2D Selection in any Data View
1. Select the Marquee tool
Tile, and Channel Views.
from the tool menu. This is the default tool in Main,
2. Click and drag to designate a 2D selection.
There are three ways to make 3D selections. 3D selections may be for 3D volumes (x,y,z) or
2D timelapse (x,y,t) images. One of the methods does not apply in Tile Views.
8.4.1.2 Making a 3D Selection in a Main, Three, and Channel View
1. Select the Marquee tool
from the tool menu.
2. Click and drag to designate a 2D selection, and continue to hold the mouse
button down.
3. Hold the shift key down while continuing to hold the mouse button and move the
mouse up or down to move the display along the invisible axis.
4. Release the shift key, and then the mouse button.
8.4.1.3 Making a 3D Selection in any Data View
1. Select the Marquee tool
from the tool menu.
2. Click and drag to designate a 2D selection and release the mouse button
3. Extend the 2D selection across the third dimension by
a. Selecting View > Select Rect in all Planes. This will extend the selection
to all planes of the third dimension.
OR
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b. Selecting View > Define Selection Cube and entering the location range of
the z or t planes that you would like to include in your selection. You must
click OK to complete the selection.
NOTE: You may also use the View > Define Selection Cube menu selection to enter x, y,
and z (or t) dimensions. The x and y dimensions are pixel locations, while the z and t
dimensions are plane numbers.
8.4.1.4 Selecting an Entire Image
If you would like to return your selection to the entire image, simply select View > Select
All. This will negate any previous selections.
8.4.2 Scrolling through a Three View
The point selection tool
allows you to easily maneuver around the Three View, and it is
the default tool for the Three View. Click the point selection tool somewhere in the pane
where z is the invisible axis (the x,y pane), and the x axis (y,z pane) and y axis (x,z pane)
will both scroll to the same coordinates that you selected in the z axis pane. Clicking and
dragging around any of the panes will continuously update the other two panes.
8.4.3 Making Distance or Velocity Measurements
This tool can be used in any of the views to measure a desired distance (2D, 3D images) or
velocity (2D timelapse images). In all cases, it is critical that both the optical parameters of
the objective (those that determine µm/pixel) are accurately defined (see Defining
Objectives on page 62) and the z or t interval is specified in the image info (see Getting and
Editing Image Information After Capture on page 101). The ruler tool makes one
measurement at a time, and each measurement replaces the previous measurement. In
other words, the ruler tool is not used for permanent measurements, but it can be used to
make scale bars or to make quick and easy measurements.
8.4.3.1 Making a Measurement on a 2D Image
This measurement can be made in any of the views.
1. Select the ruler tool
from the view’s tool menu.
2. Click and hold the mouse button on the image at the point where you would like
to begin measurement.
3. Drag the mouse to the desired end point and release the mouse button.
Alternatively, you may click and release once at the start point and click and
release at the end point.
The length of the line that you have just drawn will be displayed in microns in
the info/tool bar. Note that these distances cannot be saved to disk from within
SlideBook. In order to perform measurements that are to be saved, see Chapter
10.
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4. To remove the line from the view, click once anywhere on the view.
8.4.3.2 Making a Measurement on a 3D or 2D Timelapse Image
1. Open a Three View of the desired image by clicking once on the thumbnail
image, and then selecting View > Three View.
2. Click the mouse button on the image at the point where you would like to begin
measurement. This point may be in any of the three panes.
3. Click the mouse button on the image at the point where you would like to end
measurement. Again, this point may be in any of the three panes.
The length or velocity between the two points will be displayed in microns in the
info/tool bar area. Note that these distances can not be saved to disk from within
SlideBook. In order to perform measurements that are to be saved, see Chapter
10.
4. To remove the line from the view, click once anywhere on the view.
8.4.3.3 Making a Measurement on an Imported Image
1. Import the image as discussed in Importing an Image on page 96.
2. Make sure that the objective definitions exist by selecting Edit > Define Optics
> Objectives and looking at the available objectives in the dropdown list. If the
objective is not available, add the objective definition as described on page 62. It
is extremely important that the microns/pixel data field is reported accurately.
3. If a magnification changer or relay lens was used, make sure that its definition
exists by selecting Edit > Define Optics > Mag. Changers and looking at the
available magnification changers in the dropdown list. If the magnification
changer is not available, add a new magnification changer definition as described
on page 73.
4. Add the objective and magnification changer information to the image by
selecting Image > Get Info, pressing the Edit Info button, and then selecting
the objective and magnification changer information from the dropdown menus.
You will also want to select the capture type from the dropdown menu.
5. Select OK to exit the Edit Image Info dialog box, then OK in the Image Info
box to save the image information.
6. Use the ruler tool as discussed above to perform distance and velocity
measurements.
8.4.3.4 Using the Ruler Tool to make a Scale Bar
1. Open a Main View or Three View of the image.
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2. Select the ruler tool as discussed above to draw a line of the desired length,
observing the distance reported in the info/tool bar.
3. To preserve the line, export the view of the image as discussed below in
Exporting Views on page 196. The scale bar will be present in the exported
image. This image can then be further modified using a drawing program.
8.5
Using Display Views (Creating Renderings and Movies)
Besides data views, SlideBook can generate two types of movie displays, volume renderings
and series movies. Both must be created from an open data view (Main, Three, Tile, or
Channel View) and will use the same color assignments and renormalization values as that
data view. The volume rendering will be generated and displayed from within SlideBook,
while the series movie must first be saved and then later viewed in QuickTime. The volume
rendering can also be exported and viewed using QuickTime.
8.5.1 Displaying a 3D or 4D Volume View (Volume Rendering)
SlideBook can perform interactive volume renderings using the 3D Volume View. The
following rendering types are available. When you go to View > 3D Volume View, two
options will be available, High Speed and High Quality. High Speed will render the
image at the highest speed possible. High Quality will render the image with a more
rigorous algorithm that may improve the quality of the image with a small decrease in the
speed performance. These setting apply to all modalities of volume rendering but are most
noticeable when using Dynamic Lighting mode.
Examples of the various modes of volume rendering are shown below:

Dynamic Lighting – allows you to illuminate your object as you wish by
allowing control of the lighting angle. Useful for determining surface
characteristics.
Dynamic Lighting
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
Fixed Lighting – uses a fixed lighting pattern for shading, approximates
surface characteristics.
Fixed Lighting

Maximum Intensity Projection (MIP) – The brightest pixels through the axis
perpendicular to the screen are displayed. This algorithm requires the least
processing power and is particularly useful for rendering large data sets (and
does not require advanced graphics card).
MIP

X Ray – Similar to MIP; however, the sums of pixels through the axis
perpendicular to the screen are displayed. This mode is useful when trying to
visualize density or concentration differences.
8.5.1.1 Working with 3D Volume Views
To open a new 3D Volume View:
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1. In a Slide View, click once anywhere on the thumbnail or within the shaded
region that surrounds the thumbnail to highlight the image.
2. Select View > 3D Volume View or View > 4D Volume View and select either
High Speed or High Quality. A new window will appear:
3-D Dynamic Lighting Volume View
3. To change the channels that are displayed, use the channel menus as you would
for the Main View, Three View, or Tile View.
4. To alter the shading, grab the Lighting Tool in the upper right hand corner
and rotate it to achieve the desired lighting.
5. To zoom into the view, click on the zoom tool
down in your view to zoom in and out.
and then click and drag up and
6. To rotate the image, click on the rotate tool
and then click and drag in the
direction that you wish to move the image. You may either click and drag
directly on the image or you on the coordinate image in the lower right hand
corner.
7. To move the image in the field of view, click on the Hand Tool icon
grab and move the image by clicking and dragging on it.
and then
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8. To instantly reorient your image, you may choose to display a specific face of the
volume by choosing the Axis Tool
Front, or Back.
and selecting Top, Bottom, Left, Right,
9. To select a different rendering mode, or to alter the colors displayed, select the
Renormalization icon
. The Volume View Settings window will appear.
NOTE: The Volume View Settings window also automatically opens when you
open a 3D Volume View.
Several features are offered in addition to the ability to renormalize the image
(change the lookup tables).
10. Alter the background color using the Background Color slider and change the
brightness of the gridlines using the Gridlines slider.
11. You may alter the opacity of an individual channel using the corresponding
Opacity slider control.
12. Select the rendering mode from the Style drop-down menu.
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13. You may also change the channels that are displayed just as you would in other
standard views.
8.5.1.2 Working with 4D Volume Views
The 4D Volume View has the same functions as the 3D Volume View plus a timepoint
navigation feature.
To display a 4D Volume View, click on a thumbnail in your slide and select View > 4D
Volume View.
You may use any of the timepoint controls to move between timepoints or play the images
like a movie. Other controls such as rotate, zoom, hand, axis, and histogram function
exactly as in the 3D Volume View.
8.5.2 Displaying a 3D Surface View
You may also display your image as a surface; to do so, click on a thumbnail in your slide
and select View > 3D Surface View. A new window and corresponding renormalization
dialog will appear.
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This view has functions similar to the 3D Volume View. The rotate tool, hand tool, zoom
tool, axis tool, and lighting tool all function as described above in the section on 3D Volume
View. The 3D Surface View Settings function similarly to the Volume View Settings. To
change the intensity at which the surface is drawn, adjust the bar on the histogram of pixel
intensities.
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To display or remove surfaces, simply check or uncheck the appropriate channels in the
Surfaces list. You may also change the color of the surface by selecting the color box for
the corresponding channel. You may also change the width of the outline using the
Outline Width slider.
8.5.3 Generating a Physically Proportional Rendering of 3D Data
You may notice that your object may appear disproportionately small, or “squashed” in the
z direction. When performing 3D imaging, each plane that is captured is represented
by a set of pixels in the image set. Each pixel is a perfect cube. You will notice that the z
dimension in the 3D rendering appears to be different than what was actually captured.
This is because the z step size that was selected during capture is not actually the same as
the length of the z dimension of the pixel. In the image set, planes are simply stacked on top
of one another with no regard for the actual z distance traveled (this is done in order to
preserve data fidelity).
To generate a proportionate rendering:
1. Open a data view (Main, Three, Tile, or Channel View) of the image as discussed
above.
2. Alter the renormalization parameters and color display as desired (see above).
3. Interpolate between planes and expand your z dimension to represent the actual
z distance traveled by the microscope by generating an isotropic image (see page
213).
4. Create a Volume or Surface View as discussed in the sections on pages 189 and
193.
8.5.4 Creating Series Movies
8.5.4.1 2D Timelapse Images
Scans through time lapse images can be converted into QuickTime movies. To do so:
1. Open a Main View of the image as discussed above.
2. Alter the renormalization parameters and color display as desired (see pages 171
and 174). You may also wish to crop the image (see page 177).
3. Select View > Create Series Movie. The Series Movie Properties dialog box
will appear:
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4. Enter the speed at which you would like the movie to play in the Frame
Duration (s) edit field. A value of 0.2 seconds generally yields acceptable
results.
5. Click OK. The following dialog box will appear:
6. Select the export format and quality that you prefer and then click OK. Lossless
has the highest quality and the largest file size. A standard “Save As…” dialog
box will appear.
7. Type in a filename and click Save and you are finished.
8.5.4.2 4D Images
There are two ways to generate movies of 4D images.
1. Open a slide that contains a 4D Capture.
2. Generate a projection image as discussed in Creating a Projection Image on page
213. SlideBook will automatically generate a 2D timelapse image from the series
of projection images.
3. You may now generate a series movie as discussed above in steps 3-7.
OR
1. Select an individual z plane for the movie by scrolling to the desired z plane and
selecting the thumbnail button.
2. You may now generate a series movie as discussed above in steps 3-7.
8.6
Exporting Views
Views may be exported as a variety of different file types. These images will be 8-bit for
each color and should not be used for intensity analysis. However, an exported view may be
loaded into Adobe Photoshop (or any other illustration program) or inserted directly into a
word processor document.
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NOTE: If you would like to use the image in another analysis program for purposes of
deriving intensity information, you will need to export the image as described in Exporting
Images on page 221.
NOTE: If you would like to show annotations such as scale bars, time stamps, etc, you
should export the image from the View menu.
8.6.1 Exporting a Main View, Three View, Channel View, or Tile View as a TIFF
1. Open a Main View, Three View, Tile View, or Channel View of the image and
adjust the look of the data as desired. This may include altering the color display
and renormalization parameters or cropping the image.
2. Select View > Export > TIFF to export the view as a single TIFF (.tif) file.
3. Enter the file name and select Save. The TIFF image can now be used in other
programs.
8.6.2 Exporting a 3D or 2D Timelapse View as a TIFF or TIFF Series
Three-dimensional Main Views may be exported as a single TIFF file containing multipleplanes or as a series of TIFF images.
1. Open a Main View of the image and adjust the look of the data as desired.
2. Select View > Export > TIFF to export the view as a single TIFF (.tif) file or
View > Export > TIFF Series to export the view as a series of TIFF images
(one image for each z or t plane).
3. Enter the file name and select Save. The TIFF image(s) can now be used in
other programs.
8.6.3 Exporting Default Views of All Images in a Slide
SlideBook allows you to use a single command to export a series of tiff files, one for each
image in the slide. Each TIFF file will be a single plane that has the same look as the
thumbnail image (default view). To use this feature:
1. Alter each image to the desired look as described in this chapter. This may
include altering the color display and renormalization parameters or cropping
the image.
2. Select View > Export > Default Views of All Images as TIFFs.
3. Choose a directory for the files and select OK. The TIFF images will be saved
under the individual image names in the chosen directory and can be used in
other programs.
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8.6.4 Exporting Volume View and Surface View Movies
You may wish to export your volume or surface views so that they may be displayed in a
presentation or online publication. You may export either as a QuickTime movie, or as a
QTVR (QuickTime Volume Rendering) file. QTVR files can be rotated at arbitrary angles in
QuickTime (available at www.apple.com). To export your Volume or Surface View:
1. In the Slide View, click on an open Volume or Surface View to make it the active
view.
2. Select View > Export > Movie. The following dialog box will appear:
The dialog has the following sections:

Axis of Rotation Options – you may generate either QuickTime movies
where your volume or surface rotates around a single axis (x, y, or z) or QTVR
movies where you may rotate your volume as desired using QuickTime.
o
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Start at current plot orientation – check this box if you want to start
your movie with the view that is currently displayed. If left unchecked,
the movie will begin with a view with surfaces at right angles to the
selected axis.
Chapter 9 – Preparing an Image for Analysis or Export
o
Axis relative to screen – check this box if you’d like the image to rotate
around an axis relative to the screen. If left unchecked, the image will
rotate around the image axis.

Rotation Increment - enter the desired rotation increment in the data field.
The rotation range that you selected is computed in the increments you
provide. Choosing a smaller increment increases the smoothness of the
rotation and adds to the depth cue provided by moving the image. Projection
is an expensive computation, so choosing a larger increment will make the
projection process faster. To generate a single head-on projection of the entire
image, choose 360.

Rotation Range – select 360 to rotate the data completely. You may select a
smaller range if desired.

Show current timepoint only (disable animation) – available for 4D
images only. Allows you to render a single timepoint to conserve time and
memory.

Timelapse Options – select the timepoints you would like to render and
how you would like your timelapse movie to proceed (either step through time
as the object is rotating, or complete a rotation before proceeding to the next
timepoint).

Movie Dimensions – allows you to change the dimensions of the movie
o
o
o

Keep Aspect Ratio – keeps the current height/width ratio
Scale – change the size of the movie with respect to the original image
dimensions
Width/Height – manually enter the dimension you wish to export
Movie frames per second – adjust the speed of your movie
3. Once you have selected the appropriate parameters, select Export. A standard
“Save As…” dialog box will appear.
8. Type in a filename and then click Save and you are finished.
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9 Preparing an Image for Analysis or Export
Before performing statistical analysis on your images, you may wish to perform a variety of
pre-processing operations. In this chapter, you will learn how to:







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9.1
Manipulate Individual Channels
Crop an Image
Align an Image
Manipulate Timelapse Data
Perform Flat Field Correction
Perform Photobleach Correction
Perform Background Subtraction
Create a Projection Image
Create an Interpolated Image
Apply Filters
Perform Deconvolution
Manipulating Individual Channels
A channel denotes the image data collected for a specific fluorophore. SlideBook allows you
to manipulate channels prior to performing quantitative analysis on your images. You may
insert a channel from one image into another image, remove a channel, or create new
channels using channel math. For timelapse images, you may also create a timelapse
composite image. The timelapse composite channel adds the previous timepoints to each
timepoint. Thus, timepoint 3 in a timelapse composite channel is the sum of timepoints 1, 2,
and 3.
9.1.1 Inserting an Image as a Channel
You may wish to insert an image containing a single channel, such as a background image,
into another image that you have collected. To do this:
1. Make sure that the image you would like to insert is in the same slide as the
destination image (see Copying an Image on page 159).
2. Select the destination image (the image that will contain the newly inserted
image) by clicking on it. You may also start from an open view of the image.
3. Select Image > Channel Operations > Insert Channel. The following dialog
box will appear:
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4. Select the image that you would like to insert as a channel from the dropdown
menu. If the image that you select contains more than one channel, the channel
that is listed as channel 1 (see Getting and Editing Image Information After
Capture on page 101) will be the channel that is inserted.
5. Select the x, y and z coordinate positions for insertion in the X Pos, Y Pos, and Z
Pos edit fields. If left at 0, 0, 0, the single channel image will be inserted in the
upper right hand corner of the image in the first plane (if the destination image
has multiple planes). Images that are the same size will overlay exactly.
6. Click OK to insert the channel. The new channel can now be selected from the
channel menus in the info/tool bar and displayed in any view. The channel may
also be selected when performing channel math (see Using Channel Math on
page 202).
9.1.2 Removing a Channel
You may also wish to remove a channel that was either added improperly or does not
contain useful data. To do so:
1. Click on the image that contains the channel you would like to remove. You may
also start from an open view of the image.
2. Select Image > Channel Operations > Remove Channel. The following
dialog box will appear:
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The dialog box has the following options for Image Range:

Current Image – Operation will be performed on the selected image only.

Current Capture Type – Operation will be performed on all images in the
slide with the same capture type (2D, 3D, 4D, etc.) as the selected image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
3. Select the channel that you would like to remove from the dropdown menu.
4. Select the image extent for which you would like to remove channels. You may
select and deselect images using the checkboxes in the Selected Images list.
5. Click OK to remove the channel(s).
NOTE: Removing a channel is an irreversible process. You may wish to keep a copy of the
original image (or entire slide).
9.1.3 Using Channel Math
SlideBook allows basic mathematical operations to be performed on specified channels in an
image. This is appropriate when doing background subtraction or when calculating
bleedthrough.
To perform channel math, do the following:
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1. Make sure that the channels that are to be used in the mathematical operation
are present in a single image. You may wish to insert a channel as discussed
above.
2. Select the image by clicking on it. You may also start from an open view.
3. Select Image > Channel Operations > Channel Math. The following dialog
box will appear:
4. Setting the scope section of the Channel Math window allows channel math
operations to be applied to multiple images in the slide. Select one of the
following options:

Current Image – Operation will be performed on the selected image only.

Current Capture Type – Operation will be performed on all images in the
slide with the same capture type (2D, 3D, 4D, etc.) as the selected image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
5. Select the channels that will be used for the mathematical operations from the
dropdown list. For instance, if you would like to subtract the FITC channel from
the CY3 channel, you would select the FITC channel from the second channel
menu, and the CY3 from the first channel menu.
6. Select the mathematical operation that you would like to perform using the radio
buttons. From left to right, the operations are: addition, subtraction, absolute
difference, division, and no operation. If you select (no op), the second channel
selection will become unavailable.
7. Enter any desired Coefficients or Offsets in the edit fields. The coefficient will
be multiplied by the selected channel, and the offset will be subtracted.
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8. Enter a Channel Name in the edit field and select OK to generate the new
channel. This new channel will now appear in the channel selection drop-down
box in the image views.
9.1.4 Creating a Timelapse Composite Channel
To create a timelapse composite channel, select Image > Channel Operations > Create
Timelapse Composite Channel. Select the desired channel from the dropdown channel
and then OK. An example of a timelapse composite channel is shown below. The timelapse
composite channel shows the image data as accumulation over time. This is useful if you
wish to see the “path” of travel for migrating cells or other objects of interest.
Original Data
Timelapse Composite Channel
9.2
Cropping an Image
Before analyzing an image, you may wish to crop an image. This is especially important to
increase the speed of computationally intensive processes such as deconvolution. Please see
Cropping an Image on page 177.
9.3
Aligning an Image
Sometimes, due to misalignments of filters or lack of mechanical repeatability, data for one
channel of an image may be slightly offset from data for another channel. To solve this
problem, SlideBook offers an alignment guide that lets you adjust relative positions of any
channel in the x, y, and z axes. The alignment guide works alongside a Main View, a Three
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View, or a Tile View (see Using Data Views to Display Images on page 160) in order to shift
how each channel lies in the image with respect to the other channels.
Changing channel alignment for an image makes a permanent change to the image itself
and will be saved with the image. In other words, every view that you open from that point
on will reflect the new alignment. Of course you can re-adjust the alignment later if you
wish.
To align an image:
1. Select the image in the Slide View by clicking on it.
2. Open a Main View, Three View, or a Tile View of an image (see Using Data
Views to Display Images on page 160)
3. Select Image > Align Channels. The following dialog box will appear.
4. Select the channel that you wish to align from the dropdown menu.
5. Use the left and right arrows to move the selected channel in the x direction, and
the up and down arrows to move in the y direction, or enter an offset for any axis
directly into the corresponding edit controls.
NOTE: The view will update automatically when you use the arrow buttons, but
you must press the Apply button if you manually enter the offsets. Clicking
Cancel will ignore any changes to the edit field that were not previously applied.
6. Click OK when you are satisfied with the image alignment.
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NOTE: If you find it necessary to align certain filters consistently, your system may need
repair. Please contact Olympus America ([email protected] or 1-800-446-5967
option 4).
9.4
Manipulating Timelapse Series
SlideBook allows you to remove timepoints from a timelapse series or merge two timelapse
series into one series.
9.4.1 Removing Timepoints from a Timelapse Series
To remove timepoints from an image:
1. Click on an image’s thumbnail in the Slide View, or have an open view and select
Image > Advanced Operations > Remove Multiple Timelapse Planes. The
following dialog box will appear:
2. Enter the timepoint (or group of timepoints) that you wish to remove. You may
also choose to subsample the image by selecting Remove every nth timepoint
or Keep every nth timepoint. Alternately, you may enter a list of points to be
removed. If you display timestamps, you will see that the timepoints have been
removed.
3. Select the checkbox Place result in new image if you choose to do so, then
select OK. If you do not choose to do so, the timepoints will be irreversibly
removed from the current image.
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9.4.2 Merging Timelapse Series
If you have an experiment that was captured into multiple timelapse images, you may
merge these images by selecting Image > Advanced Operations > Merge Timelapse
Images. The following dialog box will appear:
In the example dialog above, “Time Series 2” will be appended onto “Time Series 1” and
placed into a new image. The original images will remain unchanged.
9.5
Performing Flat Field Correction
As with any optical system, specks of dust or pieces of lint will find their way into the
microscope’s optical path. There may also be some variation in the level of illumination
across the field of capture. SlideBook offers an operation called flat field correction, which
eliminates many of these constant artifacts from an image. Flat field correction is
particularly useful in brightfield and DIC (Nomarski) imaging, where such artifacts are
usually more evident than in fluorescence imaging. Flat field correction (or shading
correction) is not the same as background correction (background subtraction is strictly
subtraction). When flat field correction is applied, the desired image is multiplied by a
matrix of coefficients that is determined from a series of flat field images (see Collecting
Flat Fields below). To generate this matrix, flat field images are collected, and the average
of each pixel for the set of images is calculate to generate a single averaged image (flat field
image). The average intensity of all the pixels in the resulting averaged image is then
calculated. Finally, a coefficient matrix (pixel by pixel map) is calculated so that when the
intensity at any pixel location is multiplied by its coefficient, the generated pixel intensity
matches the average pixel intensity of the flat field image. If you are planning to perform
both background subtraction and flat field correction, perform flat field correction first.
9.5.1 Collecting Flat Fields
In order to perform flat field correction, SlideBook needs to know what an image with no
sample in focus looks like. This image should contain all the constant artifacts, but little
that will change from sample to sample. From this image, SlideBook can compute a
coefficient matrix that can be applied to an image to eliminate most of its constant artifacts.
SlideBook, in fact, can average several “empty images” to better ensure that the flat field
contains only constant artifacts of the optical system and not some peculiarity of a single
“empty image.”
Since constant artifacts might exist anywhere along the optical path, a flat field matrix is
necessary for each combination of objective, filter, and magnification changer setting. To
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keep things simple, SlideBook stores all collected flat fields in a single database and keeps
track of the optical parameters for each one.
9.5.1.1 Adding or Replacing a Flat Field
If you want to add a flat field matrix to the database (or replace an existing entry), you will
need to capture a series of images on an empty sample using the desired set of optics,
including the filter, objective, and magnification changer. To add or replace a flat field, do
the following:
1. Place a blank slide containing no sample (or a uniformly fluorescent slide if
collecting a fluorescent flat field) on the stage.
2. Bring the desired optics into place as described in Chapter 5 - Using the Focus
Window.
In order to bring the z stage of the microscope to the correct position, focus on
any irregularities (e.g., dust or scratches) on the blank slide. Then move the xy
position of the slide to a location where there are few irregularities and adjust
the microscope so it is slightly out of focus. This will minimize the contribution of
irregularities with the slide to the flat field images.
3. Select Edit > Setup Guides > Flat Field Guide to bring up the flat field guide.
The following dialog will appear:
4. Specify an exposure time and number of images to take.
Later, you will be able to adjust the exposure time so that the linear range of the
camera is being used. The number of images required depends on the user: the
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more images, the more accurately the flat field matrix will reflect only constant
artifacts, though the collection process will take more time. Typically three will
be sufficient.
5. Select Capture Image 1. SlideBook will capture an image and display it in the
upper left corner of the guide.
Adjust the exposure time such that the maximum intensity shown in the
histogram is 3000-3500 for 12 bit CCDs (on a 0-4095 scale) or 48000-56000 for 16
bit CCDs (on a 0-65535 scale)
6. If necessary, make an appropriate adjustment to the exposure time and click
Recapture Image 1. Otherwise, move the xy position of the slide slightly and
capture the next image by clicking Capture Image 2.
After the first image, it’s unlikely you’ll need to recapture any of the subsequent
images, but you will still have that option.
7. Acquire the remaining images making sure to move the slide slightly between
capture.
8. Once you’ve captured all the images, click on the Finish button to complete the
process.
9. Repeat the process for other optical combinations.
9.5.1.2 When to Update the Flat Field Database
If you try to add a flat field matrix for optics where one is already defined, SlideBook will
alert you and ask if you want to replace the existing one. SlideBook does not keep multiple
flat field matrices for identical optics.
As time passes, it is possible for a flat field matrix to become outdated. Specks may have
changed position, or other constant artifacts have either appeared or disappeared. A telltale
sign that it is time to update a flat field is when performing the flat field operation no
longer does its job—marked by white specks appearing after applying the flat field.
9.5.1.3 Displaying Existing Flat Fields
You can get a list of the existing flat fields (and their corresponding optical combinations)
by selecting Edit > Setup Guides > Display Flat Fields. To display the flat field, click on
the desired flat field icon in the table and press the Display button.
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9.5.2 Applying Flat Fields
If you wish to flat field correct an image that was captured using an optical combination
that has a corresponding entry in the flat field database, you may choose to apply the flat
field correction during or after image capture.
9.5.2.1 Applying Flat Field Correction During Image Capture
1. Bring a sample into view and focus as discussed in Using the Focus Window on
page 85.
2. Open the Capture dialog box by selecting Image > Capture or by selecting the
Capture icon in the SlideBook toolbar .
3. Set all other capture parameters as discussed in Chapters 6 and 7.
4. Select the Flat Field Correction checkbox, which is located below the exposure
information.
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5. Click OK to begin capture. The resulting images will be flat field corrected.
9.5.2.2 Applying Flat Field Correction after Image Capture
1. Select the image that you would like to correct by clicking on it. You may wish to
copy the image before applying the flat field, as it is an irreversible operation
(see Copying an Image on page 159). You may also start from an open view of the
image.
2. Select Image > Apply Flat Field. The flat field correction will be applied to the
image.
NOTE: If you do not have an entry in the flat field database that match the optics used to
capture an image, SlideBook will alert you that it is unable to perform a flat field
correction.
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9.6
Performing Photobleach Correction
Photobleach correction can be used to correct for photobleaching in 2D timelapse images.
The photobleach correction operation takes the mean intensity at each timepoint and fits a
line or curve through the mean intensity versus time data. Then, it determines a
multiplication factor for each timepoint that is used to adjust the intensities at each pixel,
resulting in constant intensities over time.
Before performing photobleach correction, you should examine your data to determine what
type of fit is most appropriate. To do so, you need to view the intensity-time profile of your
data set. You can do this by drawing a box over the entire field for a single plane, then
selecting Image > Statistics > Ratio/Timelapse Data. If the intensities decrease linearly
over time, you may perform a direct fit. If they seem to decrease exponentially, choose
exponential fit.
To perform photobleach correction:
1. Select Image > Photobleach Correction. The following dialog will appear:
2. Select the fit, and the scope of operation, as described below:

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Chapter 9 – Preparing an Image for Analysis or Export

All Images with Same Capture Type – Operation will be performed on
all images in the slide with the same capture type (2D, 3D, 4D, etc.) as the
selected image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.

All Images – Operation will be performed on all images in the slide.
3. Select the timepoints to modify and whether or not you would like to overwrite
your data.
4. Select OK to perform the operation.
WARNING: Photobleach correction is an irreversible operation. Please be sure to save a copy
of your original data.
9.7
Performing Background Subtraction
You may also select a region of interest of arbitrary size and subtract the background mean
intensities from your image using channel math. Please see the example in Chapter 2,
Creating Masks Manually (Background Subtraction Example) on page 41. You may also
permanently subtract a background ROI:
1. Make a copy of the data that you are working with.
2. Use the marquee tool to select a background region in your image.
3. Select Image > Select Background.
4. Select Image > Advanced Operations > Subtract Region Background. The
average values from your background ROI will be subtracted on a channel by
channel basis.
WARNING: The background subtraction operation described above is an irreversible
operation. Please be sure to save a copy of your original data.
9.8
Creating a Projection Image
You may wish to view 3D data as a single 2D projection image. To do so:
1. Make the desired image the active image by clicking on it in the slide view. You
may also start from an open view of the image.
2. Select Image > Create Projection Image. The following dialog box will
appear:
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3. Select the Operation as described below:

Maximum – The brightest pixel throughout the chosen axis will be
displayed.

Average – An average of all the pixels through the chosen axis will be
displayed.

Sum – The sum intensity of all the pixels through the chosen axis will be
displayed.
4. Select the Axis you would like to compress to create the projection image.
5. Select the Image Range as described below. You may also select and deselect
images using the checkboxes in the Selected Images list.

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
All Images in Slide – Operation will be performed on all images in the
slide.
6. Click OK. The projection images will be added to the bottom of the slide. The
type of projection will be displayed in the Comments section of the slide view.
9.9
Creating an Interpolated (Isotropic) Image
You may notice that your object may appear disproportionately small, or “squashed” in the
z direction (especially when using the Three View). When performing 3D imaging, each
plane that is captured is represented by a set of pixels in the image set. Each pixel is a
perfect cube. You will notice that the z dimension in the 3D image set appears to be
different than what was actually captured. This is because the z step size that was selected
during capture is not actually the same as the length of the z dimension of the pixel. In the
image set, planes are simply stacked on top of one another with no regard for the actual z
distance traveled (this is done in order to preserve data fidelity). Generating an
interpolated image will expand your z dimension to represent the actual z distance traveled
by the microscope. Creating interpolated images can also help smooth the z-axis when less
than optimal z-step size is used.
To create an interpolated image:
1. Select the image that you would like to expand by clicking on it. You may also
start from an open view of the image.
2. Select Image > Create Interpolated Image. The following dialog box will
appear:
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The dialog box recommends expansion parameters based on the calibrated pixel
size and z step size used for capture. These recommended parameters will
generate an image with the approximate dimensions of the actual sample. You
can use the recommended expansion parameters or enter custom values in the
edit boxes.
3. Select the Image Range as described below. You may also select and deselect
images using the checkboxes in the Selected Images list.

Current Image – Operation will be performed on the selected image
only.

Current Capture Type – Operation will be performed on all images in
the slide with the same capture type (2D, 3D, 4D, etc.) as the selected
image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
4. Select OK and a new image(s) will be generated.
NOTE: The m/pixel value for the objective that was used must be accurate for this
feature to work properly. Before generating the isotropic image, check the image
information to make sure the m/pixel value is set properly (see Getting and Editing
Image Information After Capture on page 101).
9.10 Applying Filters
SlideBook offers a variety of filtering options, including a user-defined kernel and semidefined Gaussian, Gaussian derivative, hot pixel, median, and mean filters. These filters
perform local, rather than global, calculations on a pixel by pixel basis. In other words, each
pixel is altered based on the intensities of the neighboring data.
9.10.1 User-Defined Convolution Kernels
This filtering option allows you to define a custom kernel. To apply a custom kernel:
1. Select the image that you would like to correct by clicking on it. You may wish to
copy the image before applying the filter, as it is an irreversible operation (see
Copying an Image on page 159 of Chapter 5.). You may also start from an open
view of the image.
2. Select Image > Filter > Convolve. The following dialog box will appear:
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3. Select the Batch as to determine if you will apply the filter to the current image
or all images with the same channels.
4. Select which channels will be filtered using the convolution kernel by checking
the desired channels in the Channel list.
5. Select the destination of the filtered channel:

Replace each channel – The selected channels will be replaced with the
filtered data.

Create a new filtered channel – New channels containing the filtered
data will be created within the image.
6. Select either a 3x3 or a 5x5 kernel size.
7. Enter the desired Coefficient and Offset in the edit field. The output value of
the kernel will be multiplied by the coefficient and added to the offset. These
parameters can be used to scale the convolved data to a desired range and to
account for negative kernel output values.
8. Enter the desired matrix elements in array of edit fields.
9. Select OK to perform the convolution.
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9.10.2 Gaussian, Hot Pixel, Mean, and Median Filters
SlideBook offers a set of standard filters for digital image processing.

Gaussian – A blurring filter that utilizes a Gaussian kernel of a given radius.
Useful for removing noise from an image.

Gaussian Derivative- An edge detection filter that uses a first derivative
Gaussian kernel of a given radius.

Mean or Median – Blurring filters that replace the intensity value of a pixel
with the mean or median intensity value of pixels within a given radius.

Hot Pixel – A nonlinear filter used to remove “hot pixels” in an image. If a pixels
intensity value is some factor greater than the mean of its neighbors it is
replaced with this mean value. A hot pixel in an image is generally caused by a
bad pixel on a CCD chip.
To apply the Gaussian, Gaussian Derivative, Mean, Median and Hot Pixel Filters:
1. Select the image that you would like to filter by clicking on it in the Slide View.
You may wish to copy the image before applying the filter, as this is an
irreversible operation (see Copying an Image on page 159). You may also start
from an open view of the image.
2. Select the appropriate filter from the Image > Filter hierarchical menu. A
dialog box will appear that is similar to that shown below:
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3. Select the batch, channel and destination options for the filter as described in the
above section User-Defined Convolution Kernels on page 216.
4. Each filter has different user-definable parameters. Enter the parameters for the
filter based on the following descriptions.

Gaussian filter – A Gaussian kernel of a designated Radius will be used
to determine a pixels intensity value.

Gaussian Derivative – A first derivative Gaussian kernel of a
designated Radius will be used to determine a pixels intensity value.

Mean or Median – A pixel will be replaced with the mean or median
intensity value of pixels within a designated Radius.

Hot Pixel – A pixel whose intensity value is higher than the mean of its
neighboring pixels by more than the designated Factor will be set to the
mean of its neighboring pixels.
5. Select OK to filter the image.
9.11 Performing No Neighbors Deconvolution
Deconvolution is the process by which SlideBook mathematically removes out-of-focus
information from a fluorescence image set. SlideBook can implement three deconvolution
approaches: constrained iterative, nearest neighbors, and no neighbors. No Neighbors
deconvolution is incorporated into the base SlideBook package. Nearest neighbors and
constrained iterative deconvolution are available as an additional SlideBook module. For
more information about nearest neighbors and constrained iterative deconvolution refer to
the supplemental SlideBook 3D Deconvolution Module manual or contact Olympus America
([email protected] or 1-800-446-5967 option 4).
No neighbors is the least computationally intensive method of deconvolution offered in
SlideBook and the only method that can be used on 2D images. While no neighbors
deconvolution can be applied to three dimensional data sets, it does not use information
across multiple planes to calculate and remove out of focus information. Rather, it functions
by creating a theoretical blurred image from the original two-dimensional image. This
blurred image is then subtracted from the original image, leaving objects that are more
likely to be in focus.
9.11.1 Running No Neighbors Deconvolution
1. Select the image that you would like to correct by clicking on it. You may also
start from an open view.
2. Make sure that the image information is correct as described in Getting and
Editing Image Information After Capture on page 101.
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3. Select Image > Deconvolve > No Neighbors. The following dialog box will
appear:
4. Select the scope of the deconvolution operation.

Current Image – Operation will be performed on the selected image
only.

Current Capture Type – Operation will be performed on all images in
the slide with the same capture type (2D, 3D, 4D, etc.) as the selected
image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
5. Select the Channels that will be deconvolved by toggling the checkbox next to the
channel name.
6. Select the deconvolution Options according to the following descriptions:

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Subtraction Constant – Adjusts the amount of intensity subtraction
used in the deconvolution algorithm. Increasing the subtraction constant
will increase the amount of deblurring but can also result in a loss of
image data.
Chapter 9 – Preparing an Image for Analysis or Export

Theoretical Spacing – Adjusts the distance between theoretical image
planes used for de-blurring. The closer the theoretical planes, the greater
the amount of de-blurring. Make sure to enter a value greater than zero
for this parameter.

Edge Padding – creates a space around the border of the image in order
to resolve fluorescent point sources that exist outside of the image.
7. Once the parameters are set, select OK to begin the deconvolution.
In addition to the user-definable parameters, the No Neighbors Deconvolution dialog box
also displays the following information:

Microscope Components – Displays the optical components that were used to
take the image.

Optical Parameters - Displays the optical parameters associated with the
microscope components used for image capture. To change these parameters
refer to Chapter 4 - Configuring Your System on page 54.
9.11.2 Memory Requirements for Deconvolution
Deconvolution is a memory-intensive process and is limited by the size of the image that is
being deconvolved. On the PC, any given program can use up to 2 GB of memory. This
results in an 800MB size restriction for no neighbors deconvolution.
9.12 Exporting Images
If you would like to perform intensity analysis or image processing in a program other than
SlideBook, you may want to export the image from the Image menu. This will export a 16bit file that retains all of the intensity data from the original SlideBook image. In contrast,
exporting an image from the View menu will generate an 8-bit file. This will result in a
loss of data assuming the images were captured on a 12-bit or higher digital camera. To
export an image from the Image menu perform the following steps.
1. Select the image that you would like to export by clicking on it. You may also
start from an open view of the image.
2. Select the appropriate file type from the Image > Export hierarchical menu. A
dialog box similar to the following will appear.
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3. Select the Image Range as described below. You may also select and deselect
images using the checkboxes in the Selected Images list.

Current Image – Operation will be performed on the selected image
only.

Current Capture Type – Operation will be performed on all images in
the slide with the same capture type (2D, 3D, 4D, etc.) as the selected
image.

All Images with Same Channels – Operation will be performed on all
images in the slide with the same channels as the selected image.
3. Press OK and to export a single multi-plane image for each channel in the
selected image(s).
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10 Using Masks for Image Analysis
Masks are the principal means to perform analysis in SlideBook. A mask is a binary
overlay on image that can be used to select an arbitrary region (or set of regions) of the
image at the same resolution as the image itself. Masks are often referred to as Regions of
Interest (ROIs) in other imaging programs. Once you have selected a region of interest(s)
using masks, you may generate a variety of morphometric measurements and intensity
statistics on those regions, including:

Morphometrics
 Area and Volume
 Center of Mass
 Estimated perimeter or surface area
 Length along major axis
 Center of area

Intensity Statistics
 Mean
 Minimum and maximum
 Integrated intensity
 Center of intensity
 Correlation (Colocalization)
Plus many others….
In this chapter, you will learn how to:







Display an Image Histogram and Mean Intensity
Create Masks and Objects
Display or Delete Masks
Perform Boolean Mask Operations
Use Masks to Perform Smooth Curve Analysis
Generate and Export Mask and Object Statistics
Display Graphs
10.1 Displaying Image Histogram and Mean Intensity
You may quickly get a statistical snapshot of your image using the Statistics > Intensity
Statistics function. A dialog box will display a histogram of the intensities of the entire
image by channel along with the mean intensity and standard deviation. You must first
open a view of your image before selecting the Intensity Statistics menu selection.
10.2 Creating Masks and Objects
A mask is a 2D or 3D set of binary values that has the same extent as the image to which it
is assigned. Once created, a mask is permanently stored with its corresponding image.
Masks are used for performing advanced selection and analysis of image data. They can
either be created and edited manually or generated automatically through threshold
segmentation and other techniques. Selecting Mask > Create creates a mask with the first
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method; selecting Mask > Segment creates a mask with the second. These menua items
can also be found under the mask icon
multiple masks.
in any View window. A single image can contain
Often you may have several distinct objects that will have intensities that are very similar,
and thus will segment together. A mask that contains several distinct entities may be
divided into objects. For instance, if an image contains a field of many cells, performing
threshold segmentation of a nuclear stain such as Hoechst will result in a mask that
contains distinct “islands.” Defining objects permits functions such as counting of objects
and generating statistics for each subregion.
We will now learn how to create masks using a variety of techniques.
10.2.1 Creating a Mask using Threshold Techniques
Threshold techniques allow the user to perform a global selection based on image
intensities. SlideBook’s segmentation process generates regions of interest that persist
throughout the entire image in all dimensions.
1. Click on an open view to make it the active view.
2. Create a mask by selecting the mask icon
> Create Segment Mask or by
going to Mask > Segment. A dialog box similar to the following will appear. Note
that it resembles the Renormalization dialog box.
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3. Select the channel that you would like to segment from the dropdown menu.
4. Enter a name for your mask.
5. Select the scope of the operation using the radio buttons. For any operation that
segments more than one image, you must select OK to segment all of the desired
images. If you select Apply, only the current image will be segmented.

Current Image – Segments the current image only

Current Capture Type (on OK) – Segments the current capture type only.
For instance, if you have a 2D timelapse image open in a slide with at least
one other timelapse image, as well as other capture types, only the 2D
timelapse images would be segmented.

All Images with Same Channels (on OK) – Segments images that have the
exact same number and type of channels. For instance, if you wish to segment
a FITC channel, and one image contains a FITC channel while another
contains both DAPI and FITC, both images will NOT be segmented. Only the
active image will be segmented.
NOTE: All images in the slide must be of the same size for this operation.
6. Set the thresholds for segmentation. You may do this in one of two ways:
a. Move the red and green bars to the right and left by clicking and dragging
them. Note that when you release the mouse button, the image will be
segmented and the mask will be displayed.
OR
b. Enter intensity values in the Low and High edit fields, then click Apply
to register the change and move the red and green bars.
SlideBook has selected, or masked, any pixel that has intensity values that lie
between the red and green bars in all of the three channels. In other words,
segmentation is performing an AND operation across the bounds defined in all
channels.
7. If working with a 3D image, scroll through the image as discussed in Scrolling
through the Invisible Axis in a 3D or Timelapse Image on page 180 and Scrolling
through a Three View on page 187. Note that the mask persists throughout the
entire 3D extent of image.
8. If you are satisfied with your mask, click on OK. If you are not repeat step 6.
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NOTE: Once you have created a mask using threshold techniques, you may use
editing tools to add or remove pixels in the mask (see Editing the Mask on page
227).
10.2.2 Creating Masks Manually
SlideBook also offers a variety of tools that can be used to manually select regions of
interest. An example demonstrating the creation and use of a manual mask is described in
the Quick Tour section Creating Masks Manually (Background Subtraction Example) on
page 41.
10.2.2.1 Creating an Empty Mask
To make a mask manually, you will first create an empty mask, then you will select your
regions of interest using editing tools.
1. Click on an open view to make it the active view.
2. Create a mask by selecting the mask icon
> Create Empty Mask or by
going to Mask > Create. The following dialog box will appear:
3. Enter a name for the mask in the edit field.
4. Choose to create the mask for the current image or for multiple images using the
radio buttons, which are defined as follows:
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
In the current image – Creates mask for the current image only.

In all images with the current capture type – Creates mask for the
current capture type only. For instance, if you have a 2D timelapse image
open in a slide with at least one other timelapse image, as well as other
capture types, only the 2D timelapse images would be segmented.

In all images with same channels - Creates mask for images that have
the exact same number and type of channels. For instance, if you wish to
segment a FITC channel, and one image contains a FITC channel while
another contains both DAPI and FITC, both images will NOT have a newly
created mask. A mask will only be created for the active image.
Chapter 10 – Using Masks for Image Analysis
5. Click OK to create a new mask for the current image.
10.2.2.2 Editing the Mask Manually
Once you have created an empty mask, you can use a variety of tools to construct a useful
mask. There are two means of editing the mask: using drawing tools from the tool menu,
and performing fine adjustments using tools from the mask menu on the info/tool bar.
1. Make sure that your mask is the currently selected mask. To do this, click on the
mask menu
and select the desired mask from the list.
2. Select a tool from the drop-down tool menu
Tool Menu on page 184).
in the info/tool bar (see Using the

Marquee tool – click and drag to draw a box outline, then select Mask >
Mark Selection to fill in your selection. To remove the box, choose Mask >
Unmark Selection.

Single pixel pencil – click and hold the mouse button to generate a
single pixel-width freehand line (NOTE: the resulting line is guaranteed to be
continuous—no gaps—as long as you have the mouse button down)

Small pencil – click and hold the mouse button to generate a thin
freehand line

Large pencil – click and hold the mouse button to generate a thick
freehand line

Small eraser - click and hold the mouse button to remove small
amounts


Large eraser - click and hold the mouse button to remove large amounts
Paint bucket – fill a shape by single clicking within any closed shape
drawn with the small or large pencil
Additionally, you may generate a 2D or 3D rectangular volume by first defining a
volume as discussed in Making a 2D or 3D (x,y,z or x,y,t) Selection on page 186,
then choosing Mask > Mark Selection.
6. Use the tool to add or remove pixels. The selected pixels will be shown in blue.
7. If you are working with a 3D or timelapse image, and would like to copy your
selections to other planes in the image, select Mask > Copy This Plane. The
following dialog box will appear:
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8. Use the radio buttons to select the desired action depending on your capture
type, and click OK.
9. To further refine your mask, you may select a variety of operations by selecting
Mask > Mask Filter. The menu items are described below. You may use these
operations in any order or as many times as desired.

Erode - Applies a kernel to the entire mask that removes a user-defined
number of voxels from the perimeter of the mask. This function can be
applied to in only the x, y dimension (2D) or in the z dimension (3D) as well.

Dilate - Applies a dilation kernel to the entire mask and thickens features.
The user defines a radius of the number of pixels the current mask will be
expanded by and whether the mask should be dilated in 2D or 3D.

Skeletonize – Reduces the mask to a set of connected junctions. This
function can only be applied to single plane (2D) images.
NOTE: These operations do not create new masks, but simply edit the current mask.
10.2.2.3 Moving the Mask
You can shift the mask in any direction by selecting Ctrl-arrow keys.
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10.2.2.4 Copying a Manually Created Mask to Other Planes or Other Images
Once you have created a mask, you may choose to copy the mask to other planes in the
image (if you have a 2D timelapse or 3D image). To do this:
1. Select Mask > Copy This Plane. The following dialog box will appear:
2. Select the desired data you would like to copy the mask to and select OK.
You may also copy the mask to all of the images of the same size in the same slide. To do
so, from an open view of an image:
1. Display the mask that you wish to copy by selecting it from the mask menu
in the info/tool bar.
2. Select Mask > Copy to all images in slide.
The mask will be copied to all of the images in the slide provided they are the same size.
10.2.3 Defining Objects
Objects are defined when you wish to get data for individual objects in a mask. The mask
statistics dialog box allows you to automatically define objects when generating statistics.
However, in order to view timelapse graphs of individual objects or to obtain a quick object
count, you will need to define objects. To do so:
1. Generate a mask that contains distinct regions, either using manual editing or
segmentation techniques.
2. Select Mask > Define Objects in Mask. The following dialog box will appear:
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The dialog box allows you to set upper and lower bounds on the size of the
regions that should be made into objects.
3. If you would like to set limits on the size of the objects, select Gate Objects by
Size and choose a minimum and maximum size. The size bounds can be specified
in either voxels or cubic microns (square microns for a 2D image).
4. Select the Generate for all similarly named masks in current slide
checkbox to perform the object definition operation on the entire slide.
NOTE: This process does not create multiple masks, but rather assigns a number to
each isolated, contiguous subregion of the existing mask.
5. Select OK. A dialog box will appear that gives the status of the object generation.
When object generation is complete, a dialog box will appear, noting the number
of objects generated. When objects are defined for a 2D timelapse image,
SlideBook will display the maximum number of objects generated at any given
timepoint.
6. Click on OK. If you now position the cursor over one of the objects in the Main
View, the object number appears to the right of the cursor location coordinates in
the info/tool bar.
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7. If you would like to remove objects, select Masks > Update Object
Definitions. The Define Objects dialog will appear as it did when selecting
Masks > Define Objects in Mask.
8. Select Gate Objects by Size and increase the lower threshold, or decrease the
upper threshold, then select OK. The number of objects generated will decrease.
This is particularly useful when working with noisy data that may contain a very
large number of objects.
NOTE: You may not decrease the lower threshold to increase the number of objects.
If you have removed too many objects, then you will need to remake your mask.
10.2.4 Splitting Objects
If you have two or more objects that are touching but exist under one contiguous region of
the mask (a mask object), you may choose to split the mask into multiple objects. This can
be achieved either manually by drawing lines along which to split the object or in an
automated fashion.
10.2.4.1 Manually Splitting Objects
To split objects manually, use the line ROI tool to draw lines where you would like to
separate the objects. Then select Mask > Advanced Operations > Split Objects Along
Lines. To see the split objects, turn the ROI display off by going to Annotations >
Regions. You may undo the split by selecting Mask > Advanced Operations > Undo
Split Objects Along Lines.
10.2.4.2 Automatically Splitting Objects
To split objects automatically, SlideBook employs a “watershed” algorithm. This is useful to
separate objects that may be joined by a thin connector as shown below:
Raw Data
Initial Mask
After Split Objects
To perform a watershed operation:
1. Generate a mask that contains distinct regions, either using manual editing or
segmentation techniques.
2. Go to Mask > Advanced Operations > Split Object Automatically. The
following dialog will appear:
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3. The slider bar controls how aggressive the splitting algorithm will be. A value of
100% refers to no splitting while 0% refers to the most aggressive splitting
algorithm. Adjust this slider to a position you feel will give you clear object
separation.
4. Click Preview. You will notice that the mask is updated on the image.
5. If the splitting is adequate, click OK. If the splitting is inadequate, repeat steps
3 and 4 until you are satisfied with the result, then click OK.
10.2.5 Removing Objects from Edge of Image
In some instances, you may wish to eliminate objects that touch the edges of your image. To
do so, select Mask > Advanced Operations > Remove Edge Objects. The following
dialog box will appear. Select the desired Image Range and select OK.
10.2.6 Tracking Objects (Particle Tracking)
Once you have defined objects, you may wish to track them over time. SlideBook offers two
methods for particle tracking, automated and manual.
10.2.6.1 Automated Particle Tracking (Tutorial)
To perform particle tracking, you may either use the Particle Tracking Protocol or
individual dialog boxes. The Particle Tracking Protocol has the advantage of being able to
save and reuse your particle tracking parameters, and apply them in a batch fashion. Below
we describe how to use the Particle Tracking Protocol. Each step in the protocol can be
accessed via a menu item, which is also noted. You may wish to practice particle tracking
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using the sample file “ParticleTrackingDemo.sld”. You may download this file at the
SlideBook download site.
1. Select the objects you wish to track by generating a mask using thresholding
and/or manual marking (see Creating a Mask using Threshold Techniques on
page 224 or Creating Masks Manually on page 226). You may need to separate
objects that are touching using mask filters (Mask > Mask Filter > Erode) or
an object splitting algorithm (see Splitting Objects on page 231).
For ParticleTrackingDemo.sld, create a mask using a threshold of 217 for the i485 channel.
2. Begin by selecting Mask > Particle Tracking > Particle Tracking Protocol.
The following dialog will appear:
The dialog box contains the list of protocol steps on the left along with a
dropdown menu for saving and loading settings. The list of steps is underneath.
Required steps must be completed before moving to the next step in the protocol.
Steps that are not required may be skipped. Once a step is complete, an X will
appear in the Done column. The right side of the dialog will update as you
progress through the protocol.
3. Select Tracking Parameters as described below.

Mask Name – Select the Mask that contains the objects you would like to
track. The menu will default to the mask that is currently displayed on your
open view.
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
Remove Objects smaller than – Check this box to selectively remove
objects smaller than a given size. Enter the size in the edit field and choose
either microns or pixels as the unit.

Track with – Select the parameter that you wish to track from the
dropdown list:

Center of Area – the coordinates of the center of the object,
unweighted by intensity values.

Center of Intensity – the coordinates of the center of the object,
weighted by intensity values. If you select this feature, you will also
need to select the channel that is used for weighting.

Mean Adjusted Center of Intensity – the coordinates of the center
of the objected, weighted by intensity values that are above the mean
intensity. This is a useful feature to track if you have delineated your
objects by hand. By using values that fall above the mean, any
imprecision in your hand delineation does not impact the center of
intensity calculation. If you select this feature, you will also need to
select the channel that is used for weighting.

Maximum Movement – enter the maximum movement that you expect
from any given object from one timepoint to the next. This will help the
particle tracking algorithm distinguish between objects that may have
similar centroid values.

Minimum Path Length – enter the minimum number of timepoints that
must be tracked in order to determine a path. For instance, if you enter “3,”
any object that cannot be followed for at least three timepoints will be
removed for the mask.
NOTE: This dialog box may also be found via the Mask > Particle Tracking >
Basic Particle Tracking menu.
4. For the tutorial, select the parameters as shown in the dialog below:
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5. Select Track and the tracking algorithm will be executed. When completed the
paths will be displayed on the image. You may click on the image and scroll
through the timepoints to see the paths. The first point in the path appears blue,
and the last appears red, with a gradation of color between. At this point, you
may wish to view the object IDs by selecting View > Annotations > Object
IDs. Also, you may wish to perform the tracking algorithm using new
parameters. You may enter those parameters and then press Track again. If you
already know the parameters in advance you may select Track and Continue.
The tracking algorithm will be executed and the protocol will automatically
advance to the next step.
NOTE: You may turn path display on and off by selecting Mask > Particle
Tracking > Display Paths.
6. Once tracking is complete, select Next. The protocol will advance to the next
step.
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Here, you may choose to calculate the following statistics for each path.
d1
d6
d2
d3
d5
d7
d4
Fig. 10.1 – Schematic of tracked object. Blue circles represent centroids




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Endpoint Displacement – the distance traveled between the first and last
timepoints of the path, as represented by the length of the red line in the
figure above.
Endpoint X Displacement – the distance traveled in the x-axis between
the first and last timepoints of the path, as represented by the length of the
dotted green line in the figure above.
Endpoint Y Displacement – the distance traveled in the x-axis between
the first and last timepoints of the path, as represented by the length of the
dotted blue line in the figure above.
Endpoint Speed – the distance represented by the length of the red line
divided by the time required to travel that distance.
Chapter 10 – Using Masks for Image Analysis







Average Speed – the total distance traveled (the sum of distances d1 to d7
as shown in the figure above) divided by the time required to travel that
distance.
Total Displacement – the total distance traveled by the object (the sum of
distances d1 to d7 as shown in the figure above).
Path Length – number of timepoints in the path.
Path MSD Path Lifetime – the total elapsed time for a given path.
Path Start Point – centroid of the first point in the path.
Path End Point – centroid of the last point in the path.
NOTE: The Path Statistics dialog box can also be found by selecting Mask >
Particle Tracking > Path Statistics.
7. Select the statistics you wish to calculate using the checkbox. Select Calculate
and Continue if you wish to progress to the next step, or simply select
Calculate if you wish to view your statistics without going to the next step, then
select Next when you are ready to go to the next step. The following dialog will
appear:
NOTE: Object statistics can also be calculated via the Mask Statistics dialog
(Mask > Statistics).
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Statistic definitions are given in the section Generating and Exporting Mask and
Object Statistics on page 243.
8. Select the statistics that you wish to calculate and either select Calculate and
Continue, or Calculate and then Next. The following dialog will appear:
9. If you have more than one image in the slide, you may choose to apply the
protocol to other images in the slide. Simply check the checkboxes of the images
you wish to analyze, and either select Apply and Close to finish the protocol, or
select Apply to perform the batch analysis. You may select whether you would
like to send results to the screen or straight to disk.
10. If you would like to save the particle tracking parameters for processing another
slide, select Save As from the Save/Load Settings dropdown menu. Enter a
name for the settings and select OK. You may now select those settings the next
time you open the Particle Tracking Protocol dialog. You may also delete settings
or modify the settings and then choose Save to save the settings with the same
name.
10.2.6.2 Performing Manual Particle Tracking
1. Open a main view of the image containing objects you wish to track.
2. Go to Mask > Particle Tracking > Manual Tracking. The following dialog
box will appear:
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3. Select either the medium or large pencil tool from the tool menu. The view will
go to the first timepoint automatically.
4. Click near the center of the first object you would like to track. The view will
automatically move to the next timepoint.
5. Continue clicking on the object you would like to track until the path is complete.
When complete, either choose New Path to go back to the first timepoint and
start over, or OK to close the dialog. You may now generate object statistics as
described in Generating Statistics for Masks and Objects on page 243.
10.3 Displaying or Deleting a Mask
If you have just issued either the Mask > Create or Mask > Segment commands, the new
mask is already selected and displayed. If not, you must select the mask you want to edit
using the mask pop-up menu
in an open view of the image. Note that while you are
editing from within a particular view, the mask is owned by the image, not just the
particular view. Any changes you make to the mask can be seen in any other view in which
the same mask is displayed.
To delete a mask:
1. Select Mask > Delete. The following dialog box will appear:
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2. Choose the mask(s) you wish to remove using the radio buttons and dropdown
menu. You may also choose to delete the mask from the current image or from all
images in the slide.
3. Choose OK to remove the mask.
10.4 Boolean Mask Operations
Masks can be combined or altered using Boolean operations. For instance, the intersection
of two masks can be generated by an AND operation. The Mask Operations dialog lets you
either generate a new mask that contains the result of a Boolean operation or replace an
existing mask with the result of an operation.
To perform Boolean mask operations:
1. Open a view that contains the masks you would like to manipulate.
2. Select Mask > Mask Operations. The following dialog will appear:
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3. Using the radio buttons, select whether the operation will take place for the
current image or the entire slide.
4. Select the masks to be used from the Left Mask and Right Mask lists.
5. Select the operation to be performed using the radio buttons.





AND – selects pixels that are in both masks
MINUS – removes any pixels that are present in the right mask from the left
mask
NOT – selects all pixels that are not marked in the left mask
OR – selects pixels that are in either of the two selected masks
XOR – adds both masks and removes any common pixels
6. Select the destination for the mask operation using the radio buttons. If you
want the result to replace the contents of either the left or right mask, select the
corresponding radio button for that mask. If you want to generate a new mask,
select the New Mask radio button and give the new mask a name in the edit
field.
7. Click OK to perform the Boolean operation. You may display the new or edited
mask as described above.
10.5 Using Masks for Smooth Curve Analysis (Kymograph)
You may use masks to identify regions to be used for smooth curve analysis, also known as
kymograph analysis. Smooth curve analysis allows you to follow time-dependent lateral
intensity changes along a static object. For example, an action potential moving along a
neural spine could be displayed as an x vs. t image using smooth curve analysis. You could
then extract velocity and displacement information from this image. To perform smooth
curve analysis:
1. Open a Main View or Three View of your timelapse data.
2. Create a new mask as discussed above in Creating an Empty Mask on page 226.
3. Select the single pixel pencil tool from the tool menu
the desired object.
and draw a line along
4. Copy the mask to all planes in the image by selecting Mask > Copy This Plane.
The following dialog box will appear:
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5. Use the radio buttons to select the desired action, and click OK.
6. Select Mask > Advanced Operations > Smooth Curve Analysis. The
following dialog box will appear:
7. Adjust the dimensions as necessary (please see Appendix A for a detailed
description of Smooth Curve parameters) and select OK. A new image will
appear in your slide.
8. Open a main view of the Smooth Curve Image and select the y axis from the axis
menu
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on the info/tool bar. You are now able to display a 2D view of your 3D
Chapter 10 – Using Masks for Image Analysis
selection. You may perform velocity measurements of timelapse data with the
ruler tool (see Making Distance or Velocity Measurements on page 187).
10.6 Generating and Exporting Mask and Object Statistics
You can answer many quantitative questions about a mask through mask statistics.
SlideBook lets you compute three types of mask statistics: statistics for the entire mask,
statistics for each timepoint in a time lapse image, and statistics for objects (defined above
on page 223). SlideBook allows for a wide range of statistics to be generated, from simple to
sophisticated.
10.6.1 Counting Objects in a Mask
You may wish to count the individual objects, in your mask. If you do not wish to obtain
other statistics, you may count objects by generating objects (see page 229). You may also
get an object count by generating the Object Count statistic (see next section). This is
especially useful if you wish to count objects for multiple images in a slide.
10.6.2 Generating Statistics for Masks and Objects
SlideBook allows you to generate a wide variety of statistics for regions of interest in 2D
and 3D data.
To generate statistics from an open view of an image:
1. Display the mask of interest by selecting it from the mask menu
.
2. Select Statistics > Mask Statistics. The following dialog box will appear:
The dialog box has the following sections:
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
Image Scope – allows for generation of statistics in multiple images. If you
are working with a 2D or 3D image, the first radio button will be selected by
default. If you are working with a 2D timelapse or 4D data set, the second
radio button will be selected by default. You may select the third radio button
if you have multiple images with the same mask name.

Mask Scope – allows you to select whether you want statistics for the entire
mask or on a per object basis. If you have chosen to obtain statistics for
multiple images, the Object option will be unavailable.

Primary Mask – defaults to the mask that is currently displayed but can be
altered via the dropdown menu. The primary mask will automatically be
made into objects. You may also remove objects under a specified pixel size
(gating by size, see page 229). To get statistics for all pixels in the mask,
leave this box unchecked.

Secondary Mask – can be selected to obtain additional statistics (see
below).

Features – lists all available statistics that may be calculated. Note that the
list of features will depend on the dimensions of the mask. For instance, area
will be displayed as a statistic for 2D masks, while volume will be displayed
for 3D masks.

Output Grouped By – these choices are only activated if you are performing
object statistics on 2D timelapse or 4D images. Sample output is shown
below:
Output Grouped By Statistic:
Chapter 10 – Using Masks for Image Analysis
Output Grouped By Object:
Output Grouped by Timepoint:
3. Check all the statistics you want to compute. SlideBook offers the following
options for statistics for a single mask:
Morphometry

Object Count – number of objects. This statistic is only available when you
select Entire Mask in the Mask Scope section of the statistics dialog.

Volume (voxels) / Area (pixels) - total number of voxels. Note that in a 2D
or time lapse image, you will always get an area measurement.
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
Volume (µm³) / Area (µm²) - volume in cubic microns (or square microns in
2D or timelapse images). Note that the image must have the correct objective
and optovar selected for these values to be accurate (see Defining Objectives
on page 62 and Defining Magnification Changers on page 73).

Surface area (µm²) / Perimeter (µm) – the area of exposed voxel faces for
3D objects (or exposed pixel faces in 2D or time lapse images). Note that
these values should only be used as rough estimates, as area or perimeter
measurements are highly affected by the resolution of the image.

Center of Area/Volume – the coordinates of the center of the object,
unweighted by intensity values.

Major Axis Coordinates – the coordinates of the two edge voxels that lie
along the major axis.

Major Axis Length – the length of the longest line that runs through the
center of volume, also called the ‘axis of minimal inertia’.

Minor Axis Length – the length of the minor axis (in 2D) or both minor axes
(in 3D).

Longest Chord Length – the distance between the two furthest voxels in
the object.

Major/Minor Shape Factor – The ratio of major axis to first minor axis (i.e.
width vs. height).
Intensity
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
Center Of Intensity – the coordinates of the center of the marked object
weighted by intensity, calculated for each channel.

Mean Adj Center Of Intensity – the coordinates of the center of the object,
weighted by intensity values (minus the mean intensity of the object).

Mean Intensity – average marked voxel intensity value for each channel.

Sum intensity – sum of all marked voxel intensity values for each channel.
Note that this value is equivalent to the product of the mean intensity and
the volume or area.

Minimum Intensity – the minimum voxel value in the object.

Maximum Intensity – the maximum voxel value in the object.

Median Intensity – the median voxel value in the object.
Chapter 10 – Using Masks for Image Analysis

Variance – the variance of intensities of the marked voxels.

Standard Deviation – the standard deviation of intensities of the marked
voxels.

Background Intensity – the average background intensity in the channel.
Cross-Channel

Pearson’s Correlation – the correlation of intensity in one channel with
another; a 0 value signifies no correlation, while a value of 1.0 signifies
perfect correlation. A negative value signifies anti-correlation.
This statistic can be used for measuring colocalization. The equation used is
shown below. Ri is the intensity in channel 1 for pixel I, and Gi is the
intensity in channel 2 for the same pixel. Rav and Gav are the average (mean)
intensity values over all pixels.

Mander’s Coefficients – these coefficients indicate the percent of protein 1
colocalized with protein 2 (M1) and the percent of protein 2 colocalized with
protein 1 (M2) as given by the equation below. The value T (threshold) is
considered to be zero intensity. Thus, adequate background subtraction must
be performed.
SlideBook offers the following statistics to describe relationships between objects
that are present in two different masks. You must first set the Mask Scope to
Object, then select a secondary mask to view these statistics.
Cross-Mask

Mask Overlap (objects) - for each object in the primary mask, this returns
a count of the number of overlapping objects in the secondary mask.

Mask Overlaps (voxels) – for each object in the primary mask, this returns
the number of overlapping pixels/voxels in the secondary mask.

Mask Overlaps (largest object volume) – for each object in the primary
mask, this returns the area or volume of the largest overlapping object in the
secondary mask.
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
Mask Contains Other Masks – returns the IDs of the objects in the
secondary mask that overlap with the primary mask.
4. Select Display to generate a new window that shows the selected statistics.
Alternatively, you may directly export the data by pressing Export (see below).
If you have generated object data, you may click on any of the object numbers in
the table, and the corresponding object will be highlighted in pink in the data
view.
5. To save the data, select the Export button. A dialog box will appear that will
allow you to choose the file destination.
6. Select a destination and a file name, then select Save. The file will be saved as a
tab-delimited text file (.txt) file, which can be easily importable into a
spreadsheet program, such as Excel.
10.7 Displaying Graphs
SlideBook allows you to view intensity profiles of selected data. You may view line intensity
or timelapse intensity profiles.
10.7.1 Viewing a Line Intensity Profile
SlideBook allows you to generate a line intensity profile using the Statistics menu. To view
the intensity profile of a line, do the following:
1. From an open view, use the ruler tool to draw a line (see Making Distance or
Velocity Measurements on page 187). You may draw this line in two or three
dimensions.
2. Select Statistics > Line Intensity. A dialog box similar to the following will
appear:
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3. Select the channel that you would like to view from the Channel menu. The line
intensity profile displays pixel distance on the x axis and intensity on the y axis.
4. To export the graph as a tiff, click on the tiff icon
. You will be prompted to
enter a filename. To export the data as a text file, click on the notepad icon
.
You will be prompted to enter a filename.
5. Click Close to close the dialog box.
10.7.2 Viewing Timelapse Intensity Profiles
SlideBook also allows you to view timelapse intensity profiles. There are four methods for
viewing profiles.
10.7.2.1 Method 1 – Using Masks
1. Select the regions that you would like to profile using masks (see above).
2. If there are multiple objects in the mask for which you would like to plot
intensity profiles, generate objects by selecting Mask > Define Objects in
Mask (see above for a discussion of objects).
3. Select Statistics > Ratio/Timelapse Data to generate a timelapse intensity
graph:
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4. Use the radio buttons in the upper left hand corner to either Graph all regions
or Graph all channels.

Graph all regions - displays all regions for a single channel. If you have
multiple fluors, you may select the channel to display from the Channels
list.

Graph all Channels – displays all channels for a single region. If you have
multiple regions defined, you may select the region to display from the
Regions list.
5. To export the data, click Export. You will be prompted to enter a filename.
6. Navigate to the desired file location, enter a filename, and choose Save. The
intensity profile data will be saved as a text file that can be opened in programs
such as Excel.
7. Click Close to close the dialog box.
10.7.2.2 Method 2 – Point and Click
From a main view, select the point selection tool from the tool menu
. Press Ctrl and
left click on any object in the image. An intensity-time profile will appear as shown below:
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Chapter 10 – Using Masks for Image Analysis
The intensity displayed is the average intensity of an area around the point where you
clicked of the specified radius. You can alter the radius edit field, and choose to export the
statistics as a tab-delimited text file (.txt).
10.7.2.3 Method 3 – Using ROIs
If you have drawn regions of interest to monitor timelapse capture, you can create graphs of
these regions post-capture.
1. Open a view of the desired image.
2. Select any of the region tools or go to Annotations > Regions to display the
ROIs.
3. Select View > Graph. The following dialog will appear:
4. Select the type of graph you wish to display and the channels that you wish to
display.
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5. Select which lines you would like to display (only applies when graphing
intensities, not ratios).
6. Click OK.
The graphs can be explored as described in Creating Graphs to Monitor Regions of Interest
on page 120.
NOTE: You can add ROIs by using any of the ROI tools to draw regions. Please see Creating
ROIs and Graphs to Monitor Regions of Interest on page 118 for more information on
creating and deleting ROIs.
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Appendix A: Smooth Curve Analysis
Appendix A: Smooth Curve Analysis (Kymograph)
Smooth curve analysis allows you to follow time-dependent lateral intensity changes along
a static object. For example, an action potential moving along a neural spine could be
displayed as an x vs. t image using smooth curve analysis. You could then extract velocity
and displacement information from this image.
SlideBook’s Smooth Curve Analysis is a mathematically rigorous and flexible operation
with several adjustable parameters. These parameters are defined using the following
figure.
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r1
114
r2
112
110
108
106
104
102
100
84
86
88
90
92
94
Figure A1: Representation of Smooth Curve Analysis (Kymograph)
The above figure is a schematic of a pixel map of a 2D image. When performing kymograph
analysis, you first define a curve along a static object that will serve as a “backbone” for
your analysis. Once this curve is defined, you may perform a smooth curve analysis.
Smooth curve analysis uses the following parameters:

Spline parameters – Knot Interval (discrete pixels), the default is set to five. A
spline is fit between two “knots.” Multiple splines are used to fit a given curve. The
knots are shown in pink in the figure above.

Measurement dimensions
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o
Radius Perpendicular to Curve (pixel units) – shown as dimension r2 in
the figure above
o
Radius Parallel to Curve (pixel units) – shown as dimension r1 in the
figure above
o
Increment Along Curve (pixel units) – center to center distance between
rectangular areas used to calculate smooth curve. In the above figure, the
boxes are drawn every two pixels units along the curve. The resultant smooth
curve analysis with have half the resolution of the original image.
Once these parameters are defined, the smooth curve algorithm averages the areas at each
timepoint and creates a new image in two dimensions (distance in x vs. time in y).
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