Applied Precision Core, personalDV Restoration Microscopy System User's Manual

®
DeltaVision Core
and personalDV
Restoration Microscopy
System User's Manual
Revision D
®
built with precisionware
AppliedPrecision
®
ii
DeltaVision Core and personalDV User's Manual
Legal Notices
Revision D of the DeltaVision Core and personalDV Restoration Microscopy System User’s Manual.
Part number 04-720104-000 Rev D.
© 1999-2008 Applied Precision, Inc. All rights reserved. No part of this manual may be reproduced,
transmitted, stored in a retrieval system, or translated into any language in any form by any means
without the written permission of Applied Precision, Inc.
Information in this document is subject to change without notice.
DeltaVision, softWoRx, and Applied Precision are registered trademarks of Applied Precision, Inc.
CoolSNAPHQ is a trademark and Cascade is a registered trademark of Roper Scientific.
All other registered names and trademarks referred to in this manual are the property of their
respective companies.
Applied Precision, Inc
1040 12th Ave. NW
Issaquah, WA 98027
(425) 557-1000 FAX: (425) 557-1055
Other Reference Documents
User instructions for DeltaVision Core include the following publications:
Document
Purpose
Available for…
Online Help
Provides reference information
for softWoRx and procedures
that show how to use softWoRx
tools
All softWoRx workstations
Product Notes
Provide examples and tips for
using softWoRx
All softWorRx users (online at
SoftWoRx
Imaging
Workstation User's
Manual
Shows how to process, visualize,
and analyze data.
All softWoRx workstations
Getting Started
with QLM
Shows how to acquire
photokinetic data with the QLM
module
Acquisition workstations that have
the optional QLM module
www.appliedprecision.com)
In addition to the above Applied Precision documentation, you will be provided with
manufacturers’ manuals for the microscope components. You may also be provided with
manufacturers’ manuals for cameras and other devices, depending on your system configuration.
(Some manufacturers do not provide manuals for these devices.)
AppliedPrecision
Contents
iii
Contents
Preface .............................................................................................ix
About This Manual................................................................................................................ix
Document Conventions ......................................................................................................... x
Lists .................................................................................................................................... x
Notes, Warnings and Cautions .....................................................................................xi
User Interface Description Conventions......................................................................xi
Contacting Applied Precision, Inc......................................................................................xii
Customer Service Hotline .............................................................................................xii
Corporate Office .............................................................................................................xii
1 Introduction...................................................................................1
What is DeltaVision? .............................................................................................................. 1
History ............................................................................................................................... 2
What Can You Use DeltaVision for? ..................................................................................... 3
Standard Data Collection Options................................................................................. 4
Live Cell Imaging ............................................................................................................. 5
Laser Photo-bleaching and Photo-activation ............................................................... 6
TIRF (Total Internal Reflection Fluorescence).............................................................. 6
What should you know to use DeltaVision? ........................................................................ 6
2 Safety .............................................................................................7
UV Exposure ........................................................................................................................... 8
Bright Light Exposure ............................................................................................................ 8
Burn .......................................................................................................................................... 8
Shock ........................................................................................................................................ 8
Xenon Lamp Safety ................................................................................................................ 9
Explosion ........................................................................................................................... 9
High Voltage..................................................................................................................... 9
UV, Visible, and IR Radiation ........................................................................................ 9
Thermal Hazards.............................................................................................................. 9
Disposal ............................................................................................................................. 9
Damage Prevention .............................................................................................................. 10
Warning Labels ..................................................................................................................... 11
3 Getting Started............................................................................13
Before you start............................................................................................................... 14
Getting Familiar with DeltaVision....................................................................................... 15
Controlling the Light path ............................................................................................ 15
Focusing........................................................................................................................... 15
Choosing Filters.............................................................................................................. 16
Using the Keypad and Joystick .................................................................................... 18
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Turning DeltaVision On ........................................................................................................19
Acquiring and Saving Data .................................................................................................21
Setting Up the Sample for Image Acquisition............................................................21
Finding the Sample ........................................................................................................26
Acquiring an Image........................................................................................................27
Saving Image Data................................................................................................................28
Setting up a Personal Data Folder................................................................................29
Saving a Single, Multi-Channel Image ........................................................................29
Turning DeltaVision Off........................................................................................................30
4 Setting Up and Running Experiments .......................................33
Creating and Running an Experiment Macro...................................................................33
Sectioning Specimens for 3D Images .................................................................................35
Guidelines for Designing and Running 3D Experiments .........................................39
Optical section spacing ..................................................................................................39
Selecting Filters .....................................................................................................................42
Choosing which filters to use .......................................................................................43
Setting up Time-lapse Experiments ...................................................................................45
Point Visiting .........................................................................................................................47
Marking Points ...............................................................................................................48
Editing a Point List.........................................................................................................50
Loading a Point List and Specifying Points to Visit ..................................................50
Monitoring Point Visiting Experiments ......................................................................51
Collecting Panel Images over Large Areas........................................................................53
Determining Border Rolloff Voxels .............................................................................53
Collecting Panel Images ................................................................................................54
Using the Multiplexed Wavelength Option......................................................................55
Setting Up the Multiplexed Wavelength Option .......................................................55
Designing a Multiplexed Wavelength Experiment ...................................................60
5 Acquiring Data from Live Specimens ......................................65
Using Autofocus in Experiment Macros ...........................................................................66
Tracking Cells........................................................................................................................67
Guidelines for specifying Cell Tracking Options ......................................................70
Acquiring 3D Z Projections with OAI................................................................................73
Continuous Z Sweep versus Traditional Projections ................................................73
Using Continuous Z Sweep ..........................................................................................74
Acquiring a Reference Image ..............................................................................................76
6 Data Collection Techniques......................................................79
Finding a Specimen and Recording its Position...............................................................79
Finding Exposure Time........................................................................................................82
Using Köhler and Critical Illumination .............................................................................84
Monitoring Data Acquisition ..............................................................................................85
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Viewing Deconvolved Image Previews ...................................................................... 85
Selecting Viewing Modes.............................................................................................. 86
Displaying Statistics and the Histogram .................................................................... 88
Editing Experiment Macros ................................................................................................ 88
7 Facility Requirements & Components ......................................91
Electrical and Environmental Requirements .................................................................... 92
Electrical Requirements................................................................................................. 92
Environmental Requirements ...................................................................................... 93
Overview of Components ................................................................................................... 95
Optical Components ............................................................................................................ 96
Fluorescence Microscope .............................................................................................. 97
Optical Filters.................................................................................................................. 97
Cameras ........................................................................................................................... 97
Light Sources .................................................................................................................. 99
Desktop Components......................................................................................................... 101
Flat-Panel Display Monitor......................................................................................... 101
The Keypad and Joystick ............................................................................................ 101
Vibration Isolation Table............................................................................................. 102
Cabinet Components.......................................................................................................... 102
Instrument Controller / Microscope Interface Chassis (IC/MIC) .......................... 103
Workstation................................................................................................................... 104
DVD-R Recording ........................................................................................................ 104
Other Standard Components ............................................................................................ 104
The Repeatable Slide Holder ...................................................................................... 104
Slide Holder Adapter .................................................................................................. 105
Calibration Kit .............................................................................................................. 105
The Fiber Optic Module .............................................................................................. 106
The Tool Kit................................................................................................................... 106
Software......................................................................................................................... 107
Optional Components........................................................................................................ 108
The Environmental Chamber ..................................................................................... 109
Additional Filter Modules .......................................................................................... 109
Quantifiable Laser Module Components ................................................................. 110
Total Internal Reflection Fluorescence (TIRF) Module........................................... 110
EM CCD Camera.......................................................................................................... 111
Analysis Workstations................................................................................................. 112
Multiplexed Wavelength Module.............................................................................. 112
Software......................................................................................................................... 112
Consumable Parts............................................................................................................... 113
8 Changing Cameras and Filters .............................................. 115
Changing Cameras ............................................................................................................. 115
Using Live Cell or Custom Filter Wheel Modules ......................................................... 117
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Changing Filter Wheel Modules.......................................................................................118
Calibrating the Filter Wheels.............................................................................................125
9 Maintenance ............................................................................131
Shutting Down and Starting the System .........................................................................131
DeltaVision Power Switches.........................................................................................132
Guidelines for Using Switches ...................................................................................133
Shutting Down the System .........................................................................................133
Starting the System.......................................................................................................133
Replacing the Xenon Bulb..................................................................................................134
Replacing the Transmitted Light ......................................................................................138
Aligning the Illumination Path .........................................................................................140
The Fiber Optic Module ..............................................................................................141
Before You Check or Adjust Illumination Path Alignment ...................................142
Checking Illumination Path Alignment ....................................................................143
Path Alignment.............................................................................................................144
Replacing IC/MIC Fuses ....................................................................................................150
Cleaning ...............................................................................................................................151
Moving the System .............................................................................................................151
Appendix A: The Immersion Oil Kit .............................................153
The Oil Calculator...............................................................................................................153
Appendix B: Troubleshooting ......................................................155
Diagnosing System Problems............................................................................................155
Troubleshooting the Controller..................................................................................155
Troubleshooting the Workstation ..............................................................................156
Analyzing Reasons for Poor Image Quality....................................................................156
DeltaVision Problem Report Form.....................................................................................159
Appendix C: Acquiring a PSF ......................................................161
Before You Start ............................................................................................................161
Acquiring a PSF...................................................................................................................161
Tools ...............................................................................................................................162
Finding Beads ...............................................................................................................164
Selecting the Correct Immersion Oil..........................................................................165
Converting PSF to OTF ......................................................................................................166
Appendix D: Reference Information...........................................171
Standard Filename Extensions..........................................................................................171
Standard Fluorescence Filters ...........................................................................................172
Live Cell Filter Sets .............................................................................................................172
Reference List ......................................................................................................................173
Microscopy ....................................................................................................................173
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Contents
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Linux Operating System ............................................................................................. 180
Image Processing.......................................................................................................... 180
Optics ............................................................................................................................. 180
Appendix E: Resolve3D and Keypad Options .......................... 181
The Resolve3D Window .................................................................................................... 182
The Resolve3D Menu................................................................................................... 183
The Resolve3D Toolbar ............................................................................................... 186
Image Control Fields ................................................................................................... 187
Stage Position Control Fields and Buttons ............................................................... 189
Image Intensity and Scale Values .............................................................................. 193
The Message Pane ........................................................................................................ 193
Resolve3D Shortcuts .................................................................................................... 194
The Design Experiment Tab.............................................................................................. 194
Experiment name and Enable Fast Acquisition....................................................... 196
Sectioning Setup ........................................................................................................... 196
Channels Setup............................................................................................................. 198
Time-lapse Setup .......................................................................................................... 199
Point Visiting Setup ..................................................................................................... 200
Panel Collection Setup................................................................................................. 201
The Design/Run Experiment Dialog Box ........................................................................ 202
The Settings Dialog Box ..................................................................................................... 204
Action Buttons .............................................................................................................. 210
Keypad/Joystick Operation ............................................................................................... 211
Index
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AppliedPrecision
Preface
This manual shows how to use the DeltaVision system to acquire images. It also
shows how to maintain the system.
„
About This Manual describes the information in each chapter.
„
Document Conventions explains the typography, notes, and other conventions
used in this manual.
„
Contacting Applied Precision, Inc. provides information about how to contact
customer support.
About This Manual
This manual provides instructions for scientists who are using DeltaVision to
acquire data. It also includes instructions and references for maintaining the
system.
The Introduction provides a brief summary of the DeltaVision system features.
Safety warnings and guidelines are provided in Chapter 2.
Chapters 3 - 6 show how to use the system to acquire data.
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DeltaVision Core and personalDV User's Manual
„
Chapter 3, Getting Started, describes how to turn the system on, acquire an
image, and run an experiment macro.
„
Chapter 4, Setting Up and Running Experiments, shows how to set up
experiment macros for 3D sectioning, Time lapse, Multiple wavelengths,
Paneling (for stitching), and Point Visiting.
„
Chapter 5, Acquiring Data From Live Specimens, provides information on how to
use the DeltaVision Core system to collect images from live specimens.
„
Chapter 6, Data Collection Techniques, describes how to determine the proper
exposure time and provides guidelines for finding the areas of interest on a
sample.
The remaining chapters provide information on how to maintain and configure
the system.
„
Chapter 7, Facility Requirements and Components, lists requirements and
describes the DeltaVision system components.
„
Chapter 8, Changing Cameras and Filters, describes how to replace cameras and
how to install or replace filters.
„
Chapter 9, Maintenance, shows how to change and align the xenon lamp, align
the light path, clean the system, and change fuses.
The appendices include reference information and procedures for configuring the
system.
Document Conventions
To make the information provided in this manual as easy as possible for you to
locate and use, the following conventions are observed.
Lists
•
Round bullets indicate options in procedures.
1. Numbered items are sequential steps for completing a procedure.
„
Square bullets indicate items in a list.
X Arrows indicate single step procedures.
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Preface
xi
Notes, Warnings and Cautions
Note Indicates information about the previous paragraph or step in a procedure.
!
Important Indicates important or critical information about the previous paragraph
or step in a procedure.
Tip Indicates helpful advice.
WARNING: Indicates important information regarding potential injury.
WARNING: Indicates risk of explosion.
WARNING: Indicates risk of shock.
Indicates important information regarding potential damage to
CAUTION:
equipment or software.
User Interface Description Conventions
Boldface indicates the names of buttons, menus, dialog box options, and fields.
Initial Capitals indicate the names of windows, dialog boxes, and tabs.
ALL CAPITALS SAN SERIF indicates the name of a key on your keyboard or
keypad, such as ENTER, DELETE, or STEP INCREASE.
Uniform width font indicates text to enter on a command line or in the GUI.
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Contacting Applied Precision, Inc
If you have questions about DeltaVision, first refer to this manual or consult the
online Help system. If you don’t find the information you need, contact us at one
of the following addresses.
Customer Service Hotline
Phone: 800-862-5166
email: [email protected]
Hours: 8:00 AM – 5:00 PM, Pacific Time, Monday – Friday
Corporate Office
Applied Precision, Inc.
1040 12th Avenue NW
Issaquah, WA 98027
USA
Phone: (425) 557-1000
Fax: (425) 557-1055
Internet Address: www.appliedprecision.com
Technical Publications email address: [email protected]
AppliedPrecision
1
1
Introduction
This chapter provides an introduction to DeltaVision.
„
What is DeltaVision? introduces the DeltaVision system and provides a history
of its development.
„
What Can You Use DeltaVision for? lists the supported imaging modes and
summarizes the data acquisition options that are supported by DeltaVision.
„
What should you know to use DeltaVision? summarizes the background and
experience required to run the system.
What is DeltaVision?
The DeltaVision Restoration Microscopy System can be used to collect and analyze
three-dimensional microscope images, acquired over long periods of time and on
multiple samples. With the sophisticated softWoRx image analysis and modelbuilding software, the system is a comprehensive package for biological image
data collection, interpretation, and display.
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DeltaVision Core and personalDV User's Manual
History
The original restoration microscopes were designed and developed in the
laboratories of Dr. John W. Sedat and Dr. David A. Agard at the University of
California, San Francisco. Their first working system actively collected images as
early as 1983. At that time, a small deconvolution (128x128x64) required overnight
processing on a million dollar mainframe computer.
During the evolution of the UCSF microscope, it became clear that
micropositioning was a critical part of the optical sectioning process. In particular,
controlled movement of the focal plane relative to the specimen (the Z axis) was
identified as a key to reliable deconvolution. To accomplish adequate Z scans, Dr.
Sedat built a microscope stage using Applied Precision’s Nanomotion™
micropositioning technology.
Before long, Dr. Sedat contacted Applied Precision cofounder Ron Seubert for
detailed information about Nanomover performance. The relationship between
UCSF and Applied Precision grew steadily. Later, in 1993, Applied Precision
licensed the image restoration technology from UCSF and began development of
DeltaVision. Collaboration between Applied Precision and UCSF still continues, for
the benefit of both parties.
In October of 1993, Applied Precision shipped the first Applied Precision/UCSF
hybrid to Michael Paddy at the University of Florida. In 1994, Applied Precision
designed, built, and delivered a DeltaVision prototype to Paul Goodwin at the Fred
Hutchinson Cancer Research Center, Seattle. In August 1994, the first commercial
AppliedPrecision
Chapter 1: Introduction
DeltaVision microscope was shipped to Bethe Scalettar at Lewis & Clark College,
Portland, Oregon. All three of these systems are still active. The DeltaVision
software has grown continuously since 1983, with contributions from scientific
programmers, faculty, and graduate students at UCSF. Applied Precision’s
contribution to the software started in earnest in 1994.
The advances in computer and camera technology in the early 1990s resulted in
the emergence of optical sectioning technology. For example, in 1993 Applied
Precision’s benchmark deconvolution (512 × 512 × 64) required 3 hours of
processing time on a $35,000 workstation. (The benchmark deconvolution is 16
times larger than the original deconvolutions performed in 1983.) Although only a
few laboratories were able to afford $35,000 for a computer, the lower cost
warranted commercialization of a deconvolution microscope. In 1996, the same
deconvolution required about 1/4 of an hour with a $14,000 workstation, and ½ of
an hour with an $8000 machine. This thirty-fold increase in performance/price
allowed a more widespread acceptance of deconvolution microscopy.
Advances in computing power and data storage have continued to benefit
DeltaVision users. In 2003, a $2500 workstation could perform the standard
benchmark in less than four minutes, representing another twenty-fold increase in
performance/price since 1996. The current workstations can now perform this
benchmark in less than 30 seconds (under 20 seconds for DeltaVision Core and
under 30 seconds for personalDV). Vast amounts of data generated by these
experiments can be stored on local hard drives, by burning DVD's, or by
transferring the data to other locations using high-speed network connections.
What Can You Use DeltaVision for?
DeltaVision Core and personalDV use research grade microscopes to collect optical
images in one of the following imaging modes:
*
„
Fluorescence
„
Brightfield
„
Phase contrast*
„
Differential Interference Contrast (DIC)
Phase contrast requires additional equipment that is available from Olympus distributors.
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DeltaVision Core and personalDV User's Manual
The following table summarizes the capabilities of the DeltaVision Core system.
Capability
Description
Digital Microscopy
Fluorescence Imaging.
Also capable of Brightfield, Phase Contrast, and
DIC imaging.
Automated Optical
Sectioning, Time-lapse,
Point visiting
Optical sectioning, filter changes, and shutters
are coordinated by the controller.
Quantitative Processing
Image processing and 3-D reconstructions of
multi-dimensional data files.
Image Display and Analysis
3-D reconstructions can be visualized, rotated,
and enhanced.
DeltaVision supports a wide array of imaging applications, including: Cytoskeletal
Studies, RNAi experiments, Live Cell Imaging, Cell Cycling Studies, Protein
Translocation, and Protein Pathway Analysis.
Standard Data Collection Options
DeltaVision supports the following types of data acquisition:
3D Imaging
To acquire “3D data,” you can set up DeltaVision to acquire a series of images
along the Z axis. The softWoRx workstation provides a sleek interface that allows
you to control the optical sectioning through a specimen. Behind the scenes, macro
language provides automated computer control of sample position, optical filters,
and shutters. After image data acquisition, a series of image processing algorithms
improve image resolution. Three-dimensional information can be reconstructed
and then visualized in a variety of ways that allow quantitative measurement and
analysis.
Multiple Filters
You can acquire images through several filters and combine them into one image
file.
Panel Collection
Panel collection acquires a series of images with adjacent fields of view. You can
stitch these images together to create images that are much larger than a single
field of view. This is especially useful when you want to collect data at a high
magnification over a large area.
AppliedPrecision
Chapter 1: Introduction
Time-Lapse
You can run macros to acquire time-lapse images and use the data to create timelapse movies. This is especially useful for studies of live samples or for
experiments that use lasers.
Point Visiting
Point Visiting allows you to acquire data from several areas of interest during a
single experiment. You can select which points to monitor on your sample and
save them in a list that contains the exact stage coordinates of each point. When
you run the experiment, DeltaVision reads the coordinates for the points in the list,
moves the stage to each point, and captures an image. This process is repeated at
specified time-lapse intervals. For live specimens, this significantly improves the
lab efficiency of experiments by allowing you to monitor multiple points of
interest in a single session.
Autofocus
You can use Autofocus to automatically focus when you are viewing a sample or
when you are running an experiment.
Köhler and Critical Illumination
You can easily switch between Critical and Köhler Illumination. Köhler
Illumination provides very even specimen illumination across the field of view.
You will typically use Köhler Illumination for most of your data collection. Critical
Illumination directs the entire light source to the size of the detected area. It is
useful for low abundant probes that require more light.
Live Cell Imaging
You can set up a controlled environment to acquire data from live specimens.
DeltaVision supports an optional environmental chamber that you can use to
control temperature and inject carbon dioxide.
DeltaVision also includes software that is specially designed to acquire data from
live specimens:
„
Cell tracking is used for point visiting time-lapse experiments. It automatically
changes the coordinates of a point to follow a cell as it moves.
„
Real-Time Z Sweep Acquisition (or Optical Axis Projection) allows you to
quickly acquire 2D projections of specimens.
„
Real-Time Deconvolution provides previews of deconvolved images as they
are acquired.
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DeltaVision Core and personalDV User's Manual
„
Reference Imaging is useful for acquiring reference images that can be used for
Differential Interference Contrast (DIC) and other techniques.
Laser Photo-bleaching and Photo-activation
If your system has the Quantifiable Laser Module (QLM) hardware module, you
can use DeltaVision to run and analyze laser photo-bleaching and photo-activation
experiments. The QLM supports up to three lasers and provides software to
control and to analyze the data that is obtained from these experiments.
Note The QLM hardware is not an available option for personalDV.
TIRF (Total Internal Reflection Fluorescence)
If your system has the TIRF hardware module, you can use DeltaVision to run and
analyze TIRF experiments. TIRF is used in a number of research applications, such
as cellular protein and vesicle trafficking, focal cellular adhesions, single
biomolecule dynamics, studies in neuroscience, and cell-to-cell communications.
The ability to excite molecules on the surface of a specimen while eliminating the
fluorescence from the depth of the sample makes TIRF a valuable tool for
examining cell surface structure and protein dynamics.
Note The TIRF hardware module is not an available option for personalDV.
What should you know to use DeltaVision?
This document assumes that you are familiar with the basics of microscopy.
Correct operation of the microscope is fundamental to obtaining quality images
with DeltaVision. In addition, an understanding of image processing basics will
help you use the system to its full potential. To manage the computer systems,
some familiarity with Linux workstations and IBM-type personal computers is
helpful.
We have taken care to ensure that DeltaVision is straightforward to use, reliable,
and complete. Please report errors and problems with DeltaVision to Applied
Precision using the e-mail address [email protected], or alternatively,
use the problem report form in Appendix D: Reference Information.
AppliedPrecision
2
2
Safety
The precautions detailed in this chapter must be carefully observed to prevent
possible personal danger:
„
UV Exposure discusses potential for UV exposure from the xenon arc lamp.
„
Bright Light Exposure warns about bright light exposure from the transmitted
light source installed in the microscope.
„
Burn provides guidelines for avoiding burns from the xenon arc lamp. (The arc
lamp reaches very high temperatures.)
„
Shock includes warnings about potential shock hazards. (Hazardous voltages
are present even when the system is disconnected from the AC main power
outlet.)
„
Damage Prevention describes actions that can damage the system.
„
Warning Labels describes the system labels.
Additional safety guidelines for maintenance and alignment are detailed in
Chapter 9: Maintenance.
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DeltaVision Core and personalDV User's Manual
UV Exposure
Since the xenon arc lamp emits ultraviolet (UV) light, there is a danger of exposing
your eyes and skin. Loss of eyesight could occur if unfiltered light from the xenon
arc lamp reaches your eyes.
To prevent UV exposure:
•
Open the shutter only when an excitation filter is engaged.
•
Do not open the xenon arc lamp housing during operation. See Chapter 9,
Maintenance for detailed instructions on changing the xenon arc lamp bulb.
Bright Light Exposure
While the transmitted light source installed in the microscope does not present
possible UV exposure, it could cause discomfort under certain conditions.
„
You must be aware of the eyepiece filter wheel when viewing a specimen
through the microscope oculars. Make sure that the proper filters are in place
so that your eyes are not suddenly exposed to a bright flash of light when the
transmitted light shutter is opened.
„
Do not look through the eyepiece while switching filters. Your eyes can be
exposed to unfiltered light during the filter transition.
Burn
The xenon arc lamp reaches a very high temperature when lit. Never touch the
housing during operation. Never remove the housing during operation or before
allowing it to cool completely. Carefully follow the directions found in Chapter 9,
Maintenance, for changing the lamp.
Shock
Hazardous voltages are present even when the system is disconnected from the
AC main power outlet.
To replace the transmitted light source LED, follow the instructions in the manuals
that are included with your microscope. To replace the xenon arc lamp, follow the
instructions found in Chapter 9: Maintenance. Refer also to the following section,
“Xenon Lamp Safety,” for additional related safety issues. No other system
components contain user-serviceable parts and do not warrant disassembly.
If the High Res Camera coolant fluid is leaking, shut down the system and contact
Applied Precision Customer Service. (See Customer Service Hotline on page xii.)
AppliedPrecision
Chapter 2: Safety
Xenon Lamp Safety
Ceramic xenon lamps are under high pressure and emit high levels of radiation.
Proper handling procedures and safety precautions should be observed to assure
the safety of the users of this product. Only operate this lamp within the
recommended operating specifications as detailed in this manual. Refer to
“Replacing the Xenon Lamp” in Chapter 9 of this manual for details on the proper
procedures for lamp replacement.
Explosion
These lamps are under high pressure. Use of face shields or safety glasses during
handling is recommended. Avoid applying excessive shock or stress to the lamp
during handling.
High Voltage
The ignition voltage of the xenon lamp presents a very high voltage hazard. Do
not touch the lamp during operation. To avoid the risk of electrical shock, the
input power should be disconnected prior to servicing the lamp.
UV, Visible, and IR Radiation
Xenon lamps emit high levels of radiation that can cause severe skin burns and
permanent eye damage. Avoid direct exposure to the emitted or reflected beams.
Thermal Hazards
Xenon lamps can get very hot during and after operation (up to several hundred
degrees C). To avoid potential for serious burns, do not touch the lamp during
operation. Do not touch the lamp after operation until the lamp has adequately
cooled.
Disposal
It is recommended that the xenon lamp’s internal pressure be relieved prior to
disposal. This is accomplished by squeezing off the tip (see illustration below)
with needle-nosed pliers until the pressurized gas escapes.
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DeltaVision Core and personalDV User's Manual
Pinch off the tip
with needle-nosed
pliers to relieve
pressurized gas
before disposal.
Relieving xenon lamp's internal pressure before disposal
Pressurized xenon lamps should not be incinerated, but disposed of in a landfill.
Damage Prevention
The following actions could damage the system:
„
Moving the stage to the home position with the objective up could break or
scratch the objective. The stage could be driven into the objective and may
potentially scratch the top lens or compress the lens housing, causing a leak,
crack, or lens misalignment.
„
Disconnecting cables before the system is completely shut down will damage
one or more of the electronic circuit boards.
„
Disconnecting cables to the camera when the power is on can damage the
camera.
„
Touching optical filters or the polychroic beam splitter contaminates them with
oil and can lead to premature failure or poor image quality. For cleaning
information, see Chapter 9: Maintenance.
„
Bending the fiber optic cable into a coil with a diameter less than 24" will
damage the cable.
„
Using improperly rated replacement fuses can create a fire hazard and may
result in damage to components. Use only the fuse types listed on the
component or in the manual.
AppliedPrecision
Chapter 2: Safety
„
11
Leaving a camera out of the tray when it is not in use presents an opportunity
for the camera to fall to the floor and break. Always place the camera in the
camera tray when it is not in use.
Warning Labels
Warning labels have been applied to the components of the system that pose a
potential hazard to the user. The labels have been duplicated here and carefully
explained. Please read this section carefully.
Note For a description of DeltaVision components, see Chapter 7, Facility
Requirements and Components.
DeltaVision Warning Labels
Hazardous Voltage Warning Label
This label indicates the danger of electric shock.
This label is found on the xenon arc lamp
housing.
Caution or Warning Label
This label indicates a danger of personal injury
or possible damage to equipment. It is
accompanied by an explanation of the
specific danger. This label may be found on the
microscope, the High Res camera, the Fast
Camera, or the workstation.
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AppliedPrecision
3
3
Getting Started
This chapter shows how to get started with DeltaVision.
„
Before you start includes a checklist of things that you need to have before you
can acquire images for your sample.
„
Getting Familiar with DeltaVision describes the key controls that you will use to
direct the light path, focus, and select filters. It also introduces the keypad and
joystick.
„
Turning DeltaVision on shows how to turn on the system.
„
Acquiring an Image shows how to place the slide and the objective, find the
sample, and acquire an image.
„
Saving Image Data describes how to set up a personal data folder and how to
save an image file.
„
Turning DeltaVision Off shows how to turn off the system.
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Before you start
Before you start, make sure that you:
•
Select the proper oil for your objective. The immersion oil kit includes 18 oils
with refractive indexes that range from 1.500 to 1.534, in increments of 0.002.
(For personalDV, the kit includes 6 oils that range from 1.512 to 1.522.) To
calculate the best refractive index for your application, follow the instructions
in Appendix A: The Immersion Oil Kit on Page 153. If you are working at
standard temperature and pressure, the oil with a 1.516 refractive index is
generally a good place to start. For work at 37 degrees C, use the oil with a
1.520 refractive index.
•
Know your login ID and password.
•
Prepare your sample using the recommended practices that are documented in
the following references. (If you are untrained in sample preparation, consider
attending a course like the one shown below.)
Books on Cell Biology Methods
Current Protocols in Cell Biology. Bonifacino, Dasso, Lippincott-Schwartz,
Harford, Yamada ed. Wiley Press, http://www.wiley.com/legacy/cp/cpcb/
Cells, A Laboratory Manual. Spector, Goldman, Leinwand ed. Cold Spring Harbor
Press, 1998.
Video Microscopy: The Fundamentals (2nd Edition). Inoue and Spring, Plenum
Press, 1997.
Digital Microscopy (3rd Edition), Methods in Cell Biology Vol. 81. Sluder and
Wolf, Academic Press, 2007.
Papers on Sample Preparation
Rines DR, He X, Sorger PK.Quantitative microscopy of green fluorescent proteinlabeled yeast. Methods Enzymol. 2002;351:16-34.
Hutchins JR, Moore WJ, Hood FE, Wilson JS, Andrews PD, Swedlow JR, Clarke
PR. Phosphorylation regulates the dynamic interaction of RCC1 with
chromosomes during mitosis. Curr Biol. 2004 Jun 22;14(12):1099-104.
Courses in Microscopy and Cellular Imaging
In Situ Hybridization, Immunocytochemistry, and Live-cell Imaging. Dernberg,
Hu, and Murray Course Directors, Cold Spring Harbor, October (annual).
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Chapter 3: Getting Started
15
Analytical and Quantitative Light Microscopy, Sluder and Wolf Course Directors,
Marine Biological Laboratory, Woods Hole, MA, May (annual).
Getting Familiar with DeltaVision
Before you acquire an image, become familiar with the key DeltaVision controls for:
•
Controlling the Light Path
•
Focusing
•
Choosing Filters
•
Using the Keypad and Joystick
Most of the manual controls for controlling the light path, focusing, and choosing
filters are similar to those that you will find on any microscope. Additional
controls for moving the stage and acquiring images are provided by the keypad
and joystick.
Controlling the Light path
DeltaVision provides a transmitted LED light and a xenon arc lamp. The
transmitted light works the same as the light source for a traditional microscope,
with the light path directed on the specimen from above. The xenon arc lamp
provides excitation light directed through the back of the microscope and focused
on the specimen from below. You can use the Beam Selector to direct either light
path from the specimen to the eyepiece or to the camera.
Transmitted LED Light
Camera
xenon Arc
Lamp
Fiber Optic
Cable
Beam Selector
Focusing
There are three manual focus controls and a Focus Lock on the microscope. These
controls are similar to those on other systems.
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Eyepiece
Focus
Fine Z
Focus
Focus
Lock
Coarse
Z Focus
Not currently
used
Not currently
used
Eyepiece Focus Use the Eyepiece Focus (on the left ocular) to focus the eyepiece.
Focus Lock Use the Focus Lock to lock or unlock the Z focus.
Fine Z Focus Use the Fine Z Focus knob to move the objective in very small
increments. It is used to focus on the focal plane.
Coarse Z Focus Use the Coarse Z Focus knob to move the objective in large
increments. It is typically used to lower the objective when the system is initialized
or to move the objective up to the slide until the oil is touching the slide.
Choosing Filters
Choosing and controlling filters is a key for any fluorescent probe experiment.
When a fluorescent probe is excited by a specific wavelength, it emits light at
another wavelength. Choosing the right filters for the dyes in your sample allows
you to obtain a complete set of data specifically from your probe without
interference from other wavelengths.
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Chapter 3: Getting Started
17
Eyepiece Filter Wheel
Neutral
Density Filter
Emission
Filter
Excitation
Filter
DeltaVision provides five different types of filters for controlling the fluorescent
light path:
•
Neutral Density filters reduce the amount of light that illuminates your
sample when you are using fluorescence. DeltaVision provides six filters that
block from 0 to 99.9% of all light.
•
Excitation filters block all but one band of wavelengths to provide a specified
range of light to excite the fluorescent probes in the sample.
•
Polychromatic Beam Splitter reflects the excitation wavelengths to the sample
and transmits the emission wavelengths from the sample. DeltaVision ships
with a standard polychromatic beam splitter for DAPI, FITC, TRITC, and Cy5.
Other beam splitters ship with optional live-cell sets.
•
Emission filters allow only a single band of light from the excited probe to
reach the camera.
•
Eyepiece filters are emission filters that allow only a single band of light from
the excited probe to reach the eyepiece and your eyes.
These filters are arranged in sets that are associated with specific dyes. (For
example, a dye such as DAPI is typically used with a DAPI Excitation filter, a
DAPI Emission Filter, a DAPI Eyepiece Filter, and a 100% Neutral Density filter.)
You can choose filter sets manually by rotating the eyepiece filter wheel. The filter
sets are synchronized so that when you change an eyepiece filter, the neutral
density filter, excitation filter, and emission filter automatically change.
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Using the Keypad and Joystick
The keypad and joystick are used to move the stage, open shutters, acquire
images, and control other acquisition options. Key controls are shown below.
Key controls on the Keypad
Acquire Image
Acquires an image and displays it on the monitor. Use this key when you are
scanning through your sample and using the eyepiece to find a region of interest,
or when you want to get a quick look at the specimen on the monitor.
Ex Shutter
Opens or closes the Excitation (i.e., Fluorescence) shutter. You will use this control
frequently to open and close the shutter. Because the shutter is designed to protect
your eyes from exposure to ultraviolet light, it automatically closes each time that
a filter wheel is moved. It must be reopened with the EX SHUTTER button.
Trans LED Source
Toggles the transmitted light LED between off and on. (Subsequent to changing
the transmitted light source from halogen to LED for DeltaVision Core and
personalDV, an actual shutter is no longer necessary.)
Slow, Medium, and Fast
Control the speed that the stage is moved by the joystick or keypad arrows. It’s
usually best to start with medium.
The Joystick
Controls stage movement. Use the joystick to move the stage in the direction that
you point with the joystick (for example, moving the joystick up moves the stage
away from you, moving joystick left moves the stage to the left, and so on).
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Chapter 3: Getting Started
19
Turning DeltaVision On
Use the following instructions to turn the system on for day-to-day use. For
instructions that show how to turn on DeltaVision after a system shutdown, see
Shutting Down and Starting the System on Page 131.
To turn on DeltaVision:
1. Turn on the power strip bar.
Note For personalDV, there is no power strip bar. Begin the power-up process with
Step 2.
2. Turn on the IC/MIC.
IC/MIC Power Switch
Workstation Power Switch
Power Strip Bar
The Main Power Switches in the
DeltaVision Cabinet (acrylic cover not shown)
3. If the monitor is off, turn it on.
4. If the Workstation is off, turn it on and wait for it to boot up.
5. Log on to the Workstation.
6. Remove any slides from the stage.
7. On the desktop, double-click the DV icon to open softWoRx.
8. On the softWoRx menu, choose File | Acquire (Resolve3D) and follow the
prompts, allowing the system to initialize.
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9. Release the Focus Lock by turning it clockwise (when facing the lock) until it is
loose.
10. Lower the objective by turning the Coarse Z Focus knob away from you
(clockwise) when facing the knob.
Always lower the objective before you initialize the system
CAUTION:
to prevent damage to the objective lens.
11. A dialog box is displayed prompting you to lower the objective before
continuing. After you lower the objective, select Initialize to initialize the
system.
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The Resolve3D window, the Data Collection Window, and the Filter Monitor
dialog box open on the desktop.
Filter Monitor
Resolve3D
Acquisition
Parameters
Data
Collection
Window
Resolve3D
Stage
Controls
The Resolve3D window includes acquisition parameters and controls for
moving the stage. The Data Collection window displays images as they are
acquired and the Filter Monitor displays the filters that are selected.
Note You will use the Resolve3D window throughout the data collection process.
For more about this window, see The Resolve3D Window on
Page 182.
Acquiring and Saving Data
To acquire an image of your sample, you will need to:
•
Set up DeltaVision by placing the slide and selecting the appropriate filters.
•
Find the sample so that it is visible in the eyepieces.
•
Acquire an image with the camera.
Setting Up the Sample for Image Acquisition
Setting up DeltaVision for imaging includes placing the sample on the stage so that
it is in contact with the immersion oil and selecting the appropriate filter set for the
fluorescent probe used to label your sample.
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To prepare for image acquisition:
1. Rotate the Coarse Focus knob so that the top of the knob rotates away from
you (while you are sitting in front of the microscope) to move the objective all
the way down.
2. Rotate the objective turret to select an objective.
Use the objective turret to select an objective
Tip Use a high power objective (e.g., 60X) to find the sample. The approach
for finding a sample with a fluorescence microscope is different than what
you would use for a transmitted light microscope. With transmitted light, a
typical approach is to find the sample with low powered objectives and then
increase power after you find the sample. With a fluorescent light however,
the sample is often not visible at low powers. The best approach for finding a
sample with fluorescent light is to start with a high power objective.
3. In the Resolve3D Lens list, select the same objective.
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Chapter 3: Getting Started
CAUTION: Make sure that the objective is selected in the Resolve3D Lens list.
4. Rotate the dichroic filter wheel to select the appropriate dichroic mirror. For
standard filters, use position 1.
5. In the Resolve3D Window, click the Info button to open the Lens Information
window.
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Refractive
Index
Optical
Conditions
6. In the Optical Conditions fields, enter the conditions for the sample.
7. Note the displayed value in the Recommended Refractive Index field and use
an oil with that refractive index.
Before you start each data collection session, calculate the oil.
CAUTION:
Your oil selection should be the same if the sample and conditions are the
same.
8. Place a drop of oil on the objective. Be sure to use the proper oil (see Page 14).
CAUTION: Do not touch the glass dropper to the objective.
9. Place a drop of oil on the coverslip. Be sure to use the proper oil (see Page 14).
10. Mount the slide on the Repeatable Slide Holder with the coverslip down.
11. Use the Adjustment knob on the Repeatable Slide Holder and the joystick on
the Keypad to center the coverslip over the objective.
Tip If you have the Repeatable Slide Holder, you can record the position of
your slide. This is useful when you are performing a Point Visiting experiment
and you need to remove the slide before you are finished with the experiment
(see Page 104).
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25
12. Rotate the Coarse Focus knob toward you to move the objective up until the
objective is just in contact with the oil. From this point on, use only the fine
focus knob to raise and lower the objective.
13. Rotate the eyepiece filter wheel (below the oculars on the scope) to select the
filter for the probe that you used to stain your sample. If your sample has more
than one probe, select the one with the brightest fluorescence (typically DAPI).
The selected filter is displayed on the Filter Monitor window.
The filter names are displayed in the Filter Monitor dialog box on the right side
of the workstation screen. As you rotate the filter wheel, the filter name next to
EP (eyepiece) changes and the EM and EX (emission and excitation) filters
change automatically to match.
The displayed colors match the wavelength of the filters. If a QLM module is
installed (not available on personalDV), the Filter Monitor also displays the
wavelengths of the lasers that are available on your system.
Note You must move the filter wheel to initialize the Filter Monitor.
Standard Filter Set
Appropriate
Probes
Filter
Name
Excitation
Emission
DAPI, Hoechst,
Coumarin
DAPI
UV, 350nm
Blue, 455nm
Fluorescein, GFP,
CY2, Al488
FITC
Blue Green,
490nm
Green, 525nm
Rhodamine, Texas
Red, Cy3,
TRITC
Green, 555nm
Orange, 605nm
CY-5®
CY-5
Red, 645nm
Infrared, 705nm
Filter
Name
Excitation
Emission
CFP
CFP
(optional)
Deep Blue, 430
nm
Blue, 470nm
YFP
YFP
(optional)
Blue Green, 500
nm
Yellow Green,
535nm
Live-cell Filter Set
Appropriate Probes
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mCherry
mCherry
(optional)
Yellow, 572nm
Red, 632nm
EGFP
EGFP
(optional)
Blue, 470nm
Green, 525nm
See Optical Filters on Page 97 for more information.
Finding the Sample
To set the focal plane for imaging, you will need to find the sample in the
eyepieces and then focus on the sample.
To find the sample:
1. With the sample on the stage and the filters selected (shown in the previous
procedure), set the Beam Selector to Eye.
2. Turn off the lights in the lab. If you cannot turn off the lights, place a box over
the sample to reduce the amount of ambient light.
3. Open the Excitation shutter by pressing the EX SHUTTER button in the lower
left corner of the keypad.
You should see light through the objective. The light on the stage should be the
same color as the Excitation filter that you selected. (For example, if you
selected DAPI, the light should be very deep violet. It may be hard to see.) Be
sure the eyepiece filter position matches the desired excitation filter position.
Note The Excitation shutter is designed to protect your eyes. Each time the
filter wheel position is changed; the shutter automatically closes and must be
reopened with the EX SHUTTER button.
4. Focus to find the focal plane. Turn the Fine Focus knob toward you to slowly
raise the objective until you see a cloud of emission color. Then use the joystick
to move the stage. If the cloud moves, you have found the sample. If not, you
may be seeing colors from lens effects. Continue to focus until the image is
sharp and clear.
5. Use the joystick to move the stage around. Change the speed of the movement
with the SLOW, MEDIUM, and FAST keys on the Keypad. When you find a
sample, place it in the middle of the field of view.
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On the Stage View, note the stage trails that show where you have moved the
stage in XY.
Acquiring an Image
To acquire a DeltaVision image, you’ll need to direct light to the camera and work
with the images that are displayed in the Data Collection window until you are
satisfied. You can then save the image as a DeltaVision file or create and run
experiments, as described in Creating and Running an Experiment Macro on Page 33.
To acquire an image:
1. On the keypad, press EX Shutter to close the shutter.
2. Switch the beam selector to camera.
3. In the Resolve3D window Exposure field, enter an exposure time (in seconds).
A good starting exposure time is 0.1 second (see Finding Exposure Time on Page
82 for details).
Tip You can also click the Find button on the Resolve3D window to find a
good exposure time. Use Find carefully. Overuse of this option can photobleach the specimen. You need to be particularly careful when determining
exposure times for live cells. When using Find for live cells, start with lower
exposure times by changing the Target Intensity Value to around 200 counts.
4. Click
to acquire an image.
Tip You can also use Auto Focus to focus the sample as follows: On the
Resolve3D window, click AF to auto focus the sample. If your image is far out
of focus, you may need to click AF more than once.
5. To focus, use the mouse to slide the Stage Z Control bar up or down.
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Z Control
and then click on the object that you want to
6. To center the image, click
center in the Image window.
Tip You can also acquire an image by clicking the ACQUIRE IMAGE button, rightclicking on the stage view, or choosing File | Acquire on the Resolve3D menu.
7. To enlarge the thumbnail image displayed in the stage view, drag the mouse
down over the zoom wheel.
The Zoom
Wheel
Note Thumbnails appear only when the Show Stage Thumbnails option is selected
on the Misc tab in the Resolve3D Settings window.
8. To clear the thumbnail image, click
.
Saving Image Data
After you acquire an image, use these instructions to set up a personal data folder
and use the Snapshot utility to acquire a single image.
AppliedPrecision
Chapter 3: Getting Started
Setting up a Personal Data Folder
You can set up a personal data folder in which to save your images. After you
create and save this folder, DeltaVision automatically uses it as the data folder
when you log on to the system.
To set up a personal data folder:
1. At the top of the Resolve3D window, click the Settings button to open the
Resolve3D Settings window. Then click the Files tab.
2. In the Data folder field, enter the directory (e.g., /data1/myData) in which
you want to save your images. To create a new folder, type the name of the
folder after /data1 (e.g., /data1/myNewData) and select Save Settings.
!
Important Store all files in the /data1 directory unless you are instructed to do
otherwise by your system administrator.
Tip If you are using an existing folder for your data, you can drag and drop the
folder from the Linux File Manager to the Data folder field.
3. To reset the default Data folder when you log out, select the Data folder is
temporary option.
Saving a Single, Multi-Channel Image
Sometimes it is desirable to acquire a single, multi-channel image. The Snapshot
utility helps you quickly create a 2-D, multi-wavelength image without having to
run a full experiment.
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To acquire a single Multi-channel image:
1. From the Resolve3D window, choose File | Snapshot to open the Snapshot
dialog box.
Tip Alternatively, you can open the Snapshot dialog box by right-clicking on the
Resolve3D window and choosing Snapshot from the shortcut menu.
2. In the Image File Name field, enter a file name. Then select which channels to
save. (Snapshot uses the exposure conditions that are displayed in the
Resolve3D window for those channels.)
Note SoftWoRx adds the _R3D.dv extension to the file name. If you enter a file
name without a directory path, the file will be located in the current data folder.
You can specify to place the file in another directory by including the path in the
file name (e.g., /tmp/myfile).
3. Click Do It.
The Image window opens and displays the new image. The image is saved as a
DeltaVision file that you can open in softWoRx.
Turning DeltaVision Off
Use the following instructions to turn off DeltaVision on a daily basis. For
instructions that show how to shut down the system, see Shutting Down and
Starting the System on Page 131.
To turn off DeltaVision:
1. Turn off the xenon light source using the bulb
main menu. Clicking the icon will switch it to the off
icon on the Resolve3D
state.
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31
2. Save all data on the workstation.
3. On the softWoRx menu bar, select File | Exit. Then exit all other workstation
applications.
4. Log out of the workstation account.
5. Press the IC/MIC power switch to turn off the IC/MIC. Wait 30 – 60 seconds for
the IC/MIC to power down.
Note If the IC/MIC fails to turn off within 30 – 60 seconds after pressing the
power switch, press and hold the IC/MIC power switch for 5 seconds to turn off
the device.
6. Clean the objective with a clean, unused cotton swab or lens paper to remove
all of the oil on the objective. Then apply chloroform to a cotton swab, gently
roll it over the objective lens once, and discard it.
After you finish imaging, Always clean the objective with a clean,
CAUTION:
unused swab or lens paper. Never reuse swabs or lens paper.
7. Lower the objective by turning the Coarse Focus knob.
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AppliedPrecision
4
4 Setting Up and Running Experiments
A DeltaVision experiment is a macro that runs a set of instructions for collecting
image data. You will use experiments to acquire almost all of your data. This
chapter shows how to set up and run experiments that use several DeltaVision data
collection options. It includes:
„
Creating and Running an Experiment Macro
„
Sectioning Specimens for 3D Images
„
Selecting Filters
„
Setting Up Time-lapse Experiments
„
Point Visiting
„
Collecting Panel Images Over Large Areas
„
Using the Multiplexed Wavelength Option
Creating and Running an Experiment Macro
DeltaVision Experiment macros are files that include settings and commands for
acquiring data with DeltaVision. Macros allow you to automate data collection.
After you use the DeltaVision interface to create a macro, you can run the macro to
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DeltaVision Core and personalDV User’s Manual
automatically collect data. Macros can be very simple or they can be very complex
and include numerous options. After you create a macro, you can use it as a
template to modify and create other macros.
To create and run an experiment macro:
1. At the top of the Resolve3D window, click the Experiment button to open the
Design/Run Experiment window.
2. Click the Design Experiment tab.
3. In the Experiment Name field, enter a name for the macro.
Note By default, softWoRx uses Resolve3D as the experiment name. This is a
special disposable name that does not require confirmation to overwrite. If you do
not intend to reuse an experiment, using Resolve3D for the experiment name is the
simplest method.
Refer to Sectioning Specimens for 3D Images, immediately following this section,
for the specific components of designing an experiment.
4. From the File menu, choose Save. The macro name appears in the Design/Run
Experiment window title bar.
5. Click the Run Experiment tab.
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35
6. In the Image file name field, enter the file name.
Notes
#1 Do not use file names that include spaces or special characters (e.g., %, /, \, “,
!, $, #, ~, *, &, @, ’, +, = ).
#2 The image file name has an _R3D.dv extension. This is the unprocessed (i.e.,
raw) Resolve3D image. A file with a .log extension is also created. The .log file is a
text file containing information about the image file, including the macro and
objective that were used to collect the image.
7. In the toolbar at the top of the screen, click the green arrow button
the scan.
to start
The macro collects the image and saves it in your data folder (see
Setting up a Personal Data Folder on Page 29). To process the image, use the
deconvolution module in the softWoRx program, as described in the softWoRx
Imaging Workstation User’s Manual.
Sectioning Specimens for 3D Images
You can acquire images of multiple sections. This data can be used to create 3D
projections, view cross sections, and create volumetric and line models.
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When you set up a Z scan on DeltaVision, you normally begin by defining a focus
range to use. This is accomplished by marking the top and bottom of the sample.
You then focus on a plane of interest. The focus range is taken above and below
the plane of interest.
If you are new to 3D microscopy, you may have never had to deal with the
specimen's three-dimensional size (except in relation to the rest of the specimen).
Manually operated microscopes provide very little information about real
distances in Z, or even X and Y. Once you start viewing your specimens in threedimensions, however, you will become accustomed to the relationship between
the microscope stage, sample, and optics, and will find that designing a Z scan is
usually straightforward.
Use the following procedure to create and run a 3D experiment. To determine the
Focus point when scan starts, Optical section spacing, Number of optical sections,
and Sample thickness, refer to Guidelines for Designing and Running 3D Experiments
on Page 39.
Note The softWoRx software reports all distances in microns.
To design and run a 3D experiment:
1. Manually acquire images of your specimen and adjust acquisition parameters
and settings until you are satisfied with the images. This includes focusing on
the specimen and determining what channels to use.
2. In the Resolve3D window, use the Z Slider on the right side of the Resolve3D
window to find and center the object of interest. Drag the slider up to find the
top of your sample. (When you release the mouse button, an image is acquired
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37
and displayed in the Data Collection window.) Drag the slider and acquire
images until you are satisfied with the position. Then press the button to
mark that location. Next, use the slider to find the bottom of the sample. When
you are satisfied, press the button to mark that location.
Note To reduce the risk of running the objective lens into the sample, the Z slider is
limited to 5 μm at a time.
Z slider
Use the Z slider to find the top and bottom of a sample. The sample thickness is
indicated by the wide line on the Z slider (shown on the right image).
Note The Z stage view displays the relative position of the objective lens to the
sample for an inverted microscope. Dragging the Z slider down focuses the
microscope closer to the cover glass (negative direction). Dragging the Z slider up
moves the focus from the cover glass toward the microscope slide (positive
direction).
3. Click Experiment to open the Design/Run Experiment window and click the
Design Experiment tab. Then click the Sectioning tab.
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4. Specify the focus point of the microscope at the start of the experiment and
move the stage to that focus point as follows:
Choose one of the following *focus
points in the Focus point when scan
starts list.
Then click the corresponding button in the Resolve3D
window to move the stage to that focus point
Top of the sample
Middle of the sample
Bottom of the sample
* For most applications, the Middle of the sample provides the best results.
Be sure to set the focus consistently with this setting before you
CAUTION:
start the experiment. (For example, if you specify to use the Middle of Sample
option, be sure to focus midway between the top and bottom of the
sample.) DeltaVision generates the sectioning commands based on this
relative starting point. If this is not done correctly, you can lose important
data.
5. Specify the separation (in microns) between each optical section in the Optical
section spacing field. If you are using a 60X objective, start by using the
default value (.2 μm). If you are using another objective, click the Lens button
on the Resolve3D window to open the Lens Information dialog box and use
the value in the Recommended Z Step size field (at the bottom of this dialog
box). As a general rule of thumb, the Z step size is approximately 1/3 of the Z
resolution for each objective.
6. Specify either the thickness or the number of sections as follows:
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•
To get the thickness of the specimen, as defined in Step 2, click Get
thickness. softWoRx adjusts the number of sections to span the specimen
thickness.
•
To specify the number of sections, enter the number of sections in the
Number of optical sections field.
7. Save and run the experiment.
Guidelines for Designing and Running 3D
Experiments
Use the following guidelines to determine values for the fields on the Sectioning
tab under the Design/Run Experiment window, Design Experiment tab.
Focus point when scan starts
This list allows you to specify the focal plane of the microscope at the start of an
experiment.
Choose one of the following options:
„
Middle of Sample The focal plane is midway between the top and bottom of
the sample. This is the default and is usually the "plane of best focus" for the
sample. This is also the best point to set for Point Visiting.
„
Bottom of Sample This represents a plane where the Z stage is farthest away
from the objective.
„
Top of Sample This represents a plane where the Z stage is closest to the
objective.
Figure 1: Z Top and Z Bottom
Slide
Total Z
Motion
Top Focal Plane
Current Stage position
Bottom Focal Plane
Coverslip (#1.5 or ~0.17 mm)
Objective
Optical section spacing
This parameter indirectly defines the size of the incremental Z stage movement
(the distance between focal planes).
When DeltaVision acquires an image, the objective collects information within the
depth of field above and below the focal plane.
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Depth of Field
Focal Plane
Optical Section Spacing
The Optical section spacing specifies the upper and lower boundaries for which
information is collected. It is centered on the focal plane and includes the
information above and below that plane.
When you select an Optical section spacing, DeltaVision uses a Z step size that is
the same distance as the Optical section spacing. (The Z step size is the vertical
distance that the stage moves between focal planes.)
Figure 2: Optical Section Spacing
Focal
Planes
Z Step Size
Optical Section
The standard step size for a 1.40 NA lens is 0.2 μm, although other step sizes are
usually fine. Due to the inherent Z resolution of a 1.40 NA lens, there is little need
to take steps finer than 0.1 μm.
When scanning a PSF, use 0.1 μm steps. For low power lenses, the step size should
be appropriately larger. The Lens Info dialog for each lens contains the suggested
Z Step size for each lens (derived using Nyquist sampling). This value is
approximately 1/3 of the theoretical Z resolution for each objective.
Number of optical sections
This field defines the number of optical sections in the Z series of the experiment.
Enter the number of sections necessary to span from the lower to the upper
boundaries of the sample.
The required number of Z sections depends upon the thickness of your specimen.
A typical scan consists of between 16 and 64 sections, but there is no set number.
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For best results, use at least three optical sections. Using one or two optical
sections, however, will also provide acceptable results.
The maximum number of optical sections is limited by memory and time. Applied
Precision's benchmark deconvolution consists of 64 optical sections that have XY
dimensions of 512x512.
If you find it necessary to acquire many Z sections, use the following formula to
figure out how much random access memory (RAM) your computer needs to
efficiently perform the deconvolution. Less RAM results in slower performance.
Available RAM (bytes) > {(X size) x (Y size) x (Z size) x 4}
Sample thickness
This field displays the thickness of the optical section range. It is approximately
one section thicker than the actual stage movement, as shown below.
Range of Stage
Movement
Sample thickness
(Optical Section Range)
For optimal deconvolution, the Z sections should extend beyond the volume of
interest by 1-3 sections.
Sample thickness = Optical section spacing x Number of optical sections
A number of methods have been used to determine the necessary Z scan settings.
The best method for your work depends largely on preference and experience, and
there is not really a single best way to approach the problem. Listed below are
some of the methods that have been used:
„
Exact Method: Use the exact method when you want to make a perfect Z
scan. Use the position buttons in the middle of the Resolve3D window to find
and center the object of interest as follows:
Use the
button to move the Z focus to the top of the sample and mark the
button to move to the bottom
top location with the button. Then use the
of the object and mark the bottom location with the button. On the
Sectioning tab, use the Get thickness button to calculate the thickness of the
sample. (The thickness is entered into the Sample Thickness field). Note that
the experiment created by Design Experiment uses relative motion for Z and
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not absolute coordinates. All motion in Z is relative to the starting point and
does not refer to an absolute Z position.
„
Note The top and bottom can be viewed by pressing the
button or the
button respectively. The middle section can be viewed by pressing the
button.
Overscan Method: As the name implies, the overscan method involves using
a Z range that is much greater than the range of interest. For example, design
an experiment that scans double the expected Z range. Previous experience is
obviously helpful when using this method.
When using this method, it is recommended that you figure out which Z
sections contain the desired information before performing the deconvolution.
Cut out unnecessary Z sections during the deconvolution step of the image
processing by specifying the section numbers of the Z start and Z end. This
will help to ensure that the sample is imaged.
„
Previous Experience: This method is essentially the same as the overscan
method, except that one strives for a more exact scan. For example, if you are
scanning an erythrocyte with 7μm diameter, you could focus on the center of
the cell, and then scan 4μm above and 4μm below the cell. Of course, if you
underscan the object, then you might miss important details, so it helps to be
conservative.
Selecting Filters
From the Channels tab of the Design/Run Experiment dialog box, you can define
which filter sets to use in the experiment for imaging. DeltaVision acquires images
from each filter set (channel) and creates a single DeltaVision file that contains all of
the channels that were collected. There can be up to five channels per Experiment.
FITC (left) and DAPI (center) filtered images are combined in the final DeltaVision file (right)
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Choosing which filters to use
Your choice of filters will be determined by the types of fluorescent probes with
which you have labeled your sample and the filters available on your system. The
excitation and emission spectrum of your filters should match the spectra for the
absorption and emission of the fluorescent probes you want to use.
The following two tables show which DeltaVision filters match each particular
probe. The interchangeable Standard and Live Cell filter modules are available for
both DeltaVision Core and personalDV. The Standard filter module filter sets are
typically used for fixed specimens while the Live Cell filter module sets are
commonly used for live specimens.
Figure 3: Standard DeltaVision Module Filter Sets
Probes
Filter Name
Excitation
Emission
DAPI, Hoechst,
Coumarin
DAPI
UV, 350nm
Blue, 455nm
Fluorescein,
GFP, CY2, Al488
FITC
Blue, 490nm
Green, 525nm
Rhodamine,
Texas Red, Cy3,
TRITC
Green, 555nm
Orange, 605nm
CY-5®
CY-5
Red, 645nm
Infrared, 705nm
Figure 4: Live-cell Module Filter Sets
Probes
Filter Name
Excitation
Emission
Cyan GFP
CFP
430nm
470nm
Yellow GFP
YFP
500nm
535nm
mCherry
mCherry
572nm
632nm
EGFP, sgGFP
EGFP
470nm
525nm
You can select other combinations of excitation and emission filters. You can also
add your own custom filter sets. To determine the optimal filters for your
application, compare the excitation and emission spectrum of your filters to the
spectra for the absorption and emission of the fluorescent probes that you want to
use.
The spectra for the standard DeltaVision filter set and the optional Live Cell set are
provided in Appendix D.
Notes
#1The lists of probes in Figure 3 and Figure 4 (above) do not include all of the probes
that can be used with these filter sets.
#2 Additional filter sets may be purchased from Chroma Technologies or Semrock.
#3 The CY-5 filter set does not include an eyepiece filter due to the low sensitivity of
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the human eye at this wavelength.
#4 The standard configuration for the six position filter wheels includes: DAPI, FITC,
TRITC, CY-5, a polarizer, and a block position.
For instructions on how to change filter wheel modules and calibrate filter wheels,
see Changing Filter Wheel Modules and Calibrating the Filter Wheels in Chapter 8.
To specify which filters to use:
1. On the Design/Run Experiment window, click the Design Experiment tab.
Then click the Channels tab.
2. Select a check box on the far left next to an Exp field. The EX Filter field
controls which excitation filter will be placed into the excitation pathway
between the %T filter and the fiber optic module. The %T Filter attenuates the
intensity of the excitation light. The EM Filter field controls which filter will be
placed in the emission pathway between the microscope and the camera. The
EX Shutter field specifies which light source to use for that channel.
3. On the EX Filter list for the same line as the check box you selected, select the
excitation filter. The EM Filter (emission filter), %T Filter (neutral density
filter), and eyepiece filters for the selected filter set are automatically specified.
(DeltaVision specifies the most recent filters that were selected for that filter set
in the Resolve3D window. If no filters have been selected, DeltaVision specifies
default filters for that filter set.)
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Chapter 4: Setting Up and Running Experiments
4. To change the exposure time, enter a value in the Exp field. Values in the Exp
field are in seconds. For example, 0.100 is 1/10th of one second. If you do not
enter a value in this field, the most recently selected exposure value is
automatically set for each filter.
5. To select a different emission filter or to attenuate the intensity, choose a filter
from the EM Filter or the %T lists.
6. You can choose which light source to use for each channel by selecting a
shutter from the EX Shutter field. EX is the standard fluorescence light source,
TRANS is the transmitted light source, EX2 is an optional second fluorescence
light source, and LASER is an optional laser source (using the QLM module).
7. If you are repeating an experiment and you have entered new exposure times
or %T in the Resolve3D window, click Refresh exposure conditions.
8. Repeat Steps 2-6 for each set of filters that you want to use.
9. Save and run the experiment.
Setting up Time-lapse Experiments
Time-lapse images are very useful for analyzing live specimens.
Time-lapse images showing cell mitosis
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To design a time-lapse experiment:
1. Select the Time-lapse tab under the Design Experiment tab on the Design/Run
Experiment window.
2. Activate the Time-lapse checkbox.
3. If you want the DeltaVision lamp to turn off when the experiment is completed,
activate the Lamps Off when finished checkbox.
Note Use discretion when activating the Lamps Off when finished checkbox
feature. Cycling the DeltaVision lamp on or off more than is necessary can reduce
the xenon bulb life.
4. Enter the desired time interval in the Time-lapse field.
Note The minimum time interval is limited by acquisition time. If you specify a time
that is less than the acquisition time, the experiment proceeds at the quickest
possible rate.
5. Set up the remaining parameters for your experiment in one of the following
ways:
•
Enter the desired number of time points in the Time Points field. The Total
Time field displays the total time that will elapse during the experiment.
•
Enter the total time in the Total Time field. The Time Points field displays
the number of time points for the experiment.
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Chapter 4: Setting Up and Running Experiments
Point Visiting
You can use point visiting to monitor areas of the slide that are in different fields
of view. Instead of recording one cell or field in a single experiment, multiple sites
can be imaged in a single experiment, increasing data collection efficiency.
In practice, the number of sites is limited only by the minimum acceptable time
interval between each time point at a single site. This makes time-lapse imaging
much more efficient, and allows you to collect enough data to generate statistically
significant results. In addition, variability between cells within an experiment can
be assayed, eliminating uncertainty as to the behavior of cells in a single
experiment.
Point Visiting monitors points that are in different fields of view
To set up an experiment that visits points, you must:
„
Mark the points to visit with the Keypad or with Resolve3D and save a point
list.
„
Edit the point list if necessary.
„
Load the point list and specify which points to visit.
Notes
#1 If you are placing your slide directly on the microscope stage (without the
Repeatable Slide Holder), you can store the X, Y, and Z coordinates of the stage
positions you mark; however, if the slide is removed from the stage or the Z focus
knob is adjusted, these coordinates no longer apply.
#2 If you are using Applied Precision’s Repeatable Slide Holder, you can remove the
slide and then place it in the same position when you want to revisit the points. To
do this, you must record the position (A-G) of the slide before you remove it. Then
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put the slide in the same position when you return it to the slide holder and update
the Z coordinates.
Marking Points
There are two ways to mark points within a sample: using the keypad or using
Resolve3D. An efficient way to mark points is to use both methods together. First,
mark the points using the keypad, which is quick and approximate, and save the
point list. Then, in Resolve3D, specify a more exact location for each point.
The points marked using the keypad are automatically communicated to
Resolve3D and displayed in the Stage view. You can view the list of points by
opening the Point List dialog box.
To mark points using the keypad and Resolve3D:
1. Use the arrows on the keypad or the joystick to move to the desired point.
2. Press the POINT MARK key on the keypad.
The number of the marked point is displayed in the Stage view.
Marked point in the Stage View
3. Repeat steps 1 and 2 to mark the desired points.
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As you mark points, the stage trails and points (along with their point
numbers) are displayed in the Resolve3D Stage view.
Point
Numbers
4. To view all of the points, use the Zoom Wheel to zoom out on the stage view.
To pan the stage view, use the
button.
The stage view after zooming in (left) and panning (right)
5. In the Resolve3D window click
to open the Point List dialog box.
6. Click on a point to select it and then click Visit Point to move the stage to that
point.
7. Use the XY Stage Controls on the Stage View to move the stage to the exact XY
position.
XY Stage
Controls
8. Use the Fine Z Focus knob to focus while looking at the sample through the
eyepieces. Then acquire an image to execute an LMC move and verify that the
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image is in the best focal plane. Repeat this step if necessary until the image is
in focus.
!
Important Do not use the microscope Focus knob after you complete Step 8.
Instead, use the Resolve3D Z controls (the Z buttons on the Resolve3D window or
the Z slider) or the AF (autofocus) button.
9. On the Point List dialog box, click Replace Point to replace the old coordinates
with the new exact coordinates for the point.
10. Repeat Steps 6-9 to replace all of the points in the list with the exact X, Y, and Z
coordinates.
Editing a Point List
To delete a point using Resolve3D:
1. In the Point List dialog box, select the point that you want to delete.
2. Click Delete Point.
To save the point list:
1. In the Point List dialog box, click Save List.
2. In the Prompt dialog, enter the desired name for the list, using eight characters
or less. Since there is really no method of displaying a directory of these
names, write the name of the list down to keep a record of it.
3. Press Enter or click OK.
To visit a point using Resolve3D:
Use one of the following methods to visit a point:
•
In the Stage view, double click on the point.
•
In the Point List dialog box, click the point that you want to visit and click
Visit Point.
Loading a Point List and Specifying Points to Visit
If you are using a previously saved point list, you will need to load the point list in
Resolve3D before you specify which points to visit.
If you are using a point list that is already open in the Point List dialog box, you
can specify which points to visit.
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Tip You can mark and then visit a point list without saving it when you are setting up
an experiment.
To load a point list into Resolve3D:
1. On the Resolve3D dialog box, click
Point List dialog box.
or choose View | Points List to open the
2. Click Open List.
3. In the Prompt dialog box, enter the name of the desired list and click OK.
Note You may need to load a list when the list of points has been cleared from
the Point List dialog box and you want to reload the saved list.
To specify which points to visit:
1. Make sure that the point list is loaded in the Point List dialog box as shown
above.
2. Open the Design/Run Experiment window and click the Design Experiment
tab. Then click the Point Visiting tab and check the Visit Point List option.
The Point Visiting dialog box is linked to the point list that is open.
3. Enter the points that you would like to visit in the Visit Point List field. (For
example, entering 1-3 specifies to visit points 1, 2, and 3. Entering 1-3, 5-7, 9
specifies to visit points 1-9, but not 4 or 8.)
Monitoring Point Visiting Experiments
You can set up DeltaVision to display a separate Data Collection window for each
point in your point visiting experiment.
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With the Point Track Display Option enabled, each point in a
point visiting experiment is displayed in a separate window
To set the Point Track display option:
1. On the Resolve3D window, click the Settings button to open the Resolve3D
Settings dialog box.
2. Click the Display tab.
3. In the Image Display mode list, select Point Track.
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Chapter 4: Setting Up and Running Experiments
4. Click the Save Settings button, then click Done to close the Resolve3D Settings
dialog box.
Collecting Panel Images over Large Areas
Panel collection macros are useful when you want to scan a large area with a
relatively high magnification lens. You can use the panels as a means of reviewing
a large area of a slide, or as data that you want to stitch together to form a single,
large image.
A set of panel images (left) can be stitched together to create a single image (right)
Image files that are collected with Panel Collection Setup enabled have a .pnl
extension. These images must be stitched to create DeltaVision image files. (See
Stitching in the softWoRx Imaging Workstation User’s manual.)
CAUTION: Do not try to reuse panel collection experiment macros. These
macros are sensitive to microscope settings such as image size and
magnification because the number of panels required depends upon many
factors.
Determining Border Rolloff Voxels
Before you collect panel images, you must first determine the number of voxels in
the Border Rolloff that is used in Deconvolution.
Note Conceptually, a voxel is a three-dimensional pixel. So, while a pixel generally
refers to XY data, a voxel includes information from the Z (or depth) plane.
To determine Border Rolloff Voxels:
1. Determine the size of the panels that you plan to collect. (Smaller panels work
best because they have the flattest intensity distribution.)
2. From the main softWoRx window, choose Process | Deconvolve to open the
Deconvolve window. In the Input field, enter the file name of an image that is
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similar to the panel size. (You can acquire and save a blank image of that size
for this purpose.)
3. Click the More Options button to open the More Deconvolution Options
window and record the value that is displayed in the Border Rolloff (voxels)
field.
Collecting Panel Images
To collect 3D panel images:
1. From the Resolve3D window menu, choose View | Point List. In the Point List
dialog box, choose Mark Point and mark points in two opposite corners to
define the area of your final composite image.
2. In the Resolve3D window, click Experiment to open the Design/Run
Experiment window.
3. Click the Design Experiment tab. Then set up the Sectioning and Channels
information in the same way that you would for standard data collection.
4. In the Point List dialog box, select one of the points that you marked in Step 1
and click Visit Point.
5. Click the Panels tab under the Design Experiment tab. Then select the Collect
Panels option.
6. Click Get Start under the Panels tab. Then visit the other point that you
marked in Step 1 and click Get End. (Alternatively, you can enter the
coordinates that are displayed in the Point List dialog box.)
7. In the Overlap (pixels) field, enter at least twice the number of border rolloff
voxels as you recorded when you determined border rolloff voxels (see Page
53). If you generally adjust the pixel size to <1 or use rotation when stitching,
use a larger overlap. (The spacing information is automatically updated.)
8. Save and run the experiment.
For instructions that show how to use softWoRx to stitch panels together, see the
softWoRx Imaging Workstation User's Manual.
Note You cannot easily use Panel Collection with time-lapse or point-visiting
experiments. It is possible only by editing the header.
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Chapter 4: Setting Up and Running Experiments
Using the Multiplexed Wavelength Option
The optional Multiplexed Wavelength module for the DeltaVision system allows
you to perform nearly simultaneous two-channel imaging without the drawbacks
associated with true simultaneous two-channel imaging. This option uses two
shuttered illumination sources and a dual-band emission filter to eliminate filter
wheel movement between channels, and therefore greatly reduces the time
required for the DeltaVision system to acquire a set of two-channel images. The
combined light path ensures no registration artifacts are introduced and
independent excitation of probes helps to ensure minimal crosstalk.
Conceptual view of Multiplexed Wavelength functionality
Before you use the Multiplexed Wavelength option, you must first have it installed
and configured correctly. Your Applied Precision representative will assist you in
setting up this option and help you ensure that all hardware and software to
support Multiplexed Wavelength functionality is installed properly.
After the option has been installed and configured, the menus, tools, and other
infrastructure necessary to use the feature will be available on your workstation.
Setting Up the Multiplexed Wavelength Option
Before you begin designing your Multiplexed Wavelength experiment, you’ll need
to perform the steps described in the following procedures to activate a
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Multiplexed Wavelength filter set and prepare the DeltaVision system for
Multiplexed Wavelength operation.
To activate the Multiplexed Wavelength filter set:
1. To change the active filter set to a filter set that is Multiplex capable, select
Settings in the Resolve3D main menu.
Settings
The Resolve3D Settings window is displayed.
2. From the Resolve3D Settings window, open the Misc tab.
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Misc tab
3. In the Resolve3D Settings window, select the Excitation, Emission, and
Eyepiece filter sets you want to use. When these fields are changed, the
<<<Pending Activation message is displayed in the window (as shown).
Select the EX, EM,
and EP filter sets
Message
displayed when
filter wheel
settings are
changed
4. Click Activate Filter Sets. The following confirmation window is displayed.
Note If you select filter sets for the Excitation filter wheel and Emission filter wheel
fields and then click Done in this window, your selections are retained until you
either activate the filter sets or exit Resolve3D.
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Note For the filter sets to be activated, the selected multiplexed filter set filters
must exist in the currently installed excitation and emission filter wheels. To
determine the proper filter sets, refer to the “Changing Filter Wheel Modules”
section in Chapter 8.
5. If the selected filter wheels are installed on your DeltaVision, click Next to
continue.
Note If you click Skip from this window, the selected filter wheels are activated
immediately and the remainder of the activation wizard is skipped.
6. Insert the selected secondary filter insert (mCherry is used in this example)
into Filter Slot 1 of the secondary light path and click Next to continue.
The system gathers the information for this window (in this case, “position 4
(500 LP)”) from the MXWSetup.ini file, not from the Instrument Controller.
7. Move the beam combiner to the appropriate position and click Next to
continue.
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Chapter 4: Setting Up and Running Experiments
Again, softWoRx gets the information for this window (in this case, “position 2
(GFP/mCherry)”) from the MXWSetup.ini file, not from the Instrument
Controller.
8. Click Finish to complete the Multiplexed Wavelength filter activation process.
After the selected filter set is activated, the Design/Run Experiment window
will look similar to the following.
At this point, you have completed activating the Multiplex Wavelength filter
set. You should now continue with the steps in the next procedure for viewing
a sample with the Multiplexed Wavelength operation.
To view a sample using the Multiplexed Wavelength option:
1. Rotate the eyepiece filter wheel to the POL or BLANK position.
2. From the Resolve3D main menu, select the Excitation filter currently in the
primary light path (CFP or GFP).
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Select the EX filter
currently in the
primary light path.
3. Use the EX button on the keypad to open the primary EX shutter and view the
primary light path.
4. Use the EX2 (formerly CAMERA SHUTTER) button on the keypad to open the
EX2 shutter and view the secondary light path.
Note With the Multiplexed Wavelength option, you can view both selected
wavelengths simultaneously by opening both EX shutters at the same time.
Designing a Multiplexed Wavelength Experiment
Use the following procedure to begin the design process for Multiplexed
Wavelength experiments.
To design a Multiplexed Wavelength experiment:
1. From the Resolve3D main menu, click the Experiment button to open the
Design/Run Experiment window.
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Experiment
Button
2. If the Multiplexed Wavelength option is enabled on your DeltaVision system,
you’ll see the Multiplexed tab in the Design/Run Experiment window. Click on
the Multiplexed tab to view the options for Multiplexed Wavelength
experiments.
Multiplexed
Tab
The Multiplexed tab of the Design/Run Experiment window is displayed as
shown.
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Select
Checkbox
3. From the Multiplexed tab, select the Do Multiplexed Channel Imaging
checkbox.
If the currently active filter set is not Multiplex capable, the following window
is displayed.
You will need to change the active filter set to continue. Press OK to return to
the Design/Run Experiment window. To change the active filter set for
Multiplexed Wavelength experiments, see the procedure for activating a
multiplexed wavelength filter set in “Setting Up the Multiplexed Wavelength
Option.” (Also see “Setting Up Filter Wheels” in Chapter 8 for additional
information on changing the active filter set.)
If the currently active filter set is Multiplex capable, the Design/Run
Experiment window is displayed and will look similar to the following.
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Chapter 4: Setting Up and Running Experiments
At this point, you have completed the initial portion of the Multiplexed
Wavelength experiment design setup. You should now continue with the
standard steps for the remainder of the experiment design, such as Sectioning,
Timelapse, and so on.
Note As soon as you activate the Do Multiplexed Channel Imaging checkbox, all
conventional imaging settings are disabled. This is also true of all multiplexed
settings when you reactivate conventional imaging.
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AppliedPrecision
5
5 Acquiring Data from Live Specimens
This chapter shows how to use the following features to image live cells with
DeltaVision.
„
Set up experiment macros to automatically focus before acquiring each image.
This is useful for cells that move during the experiment.
„
Use Cell Tracking to follow cells as they move laterally and move the stage to
keep them in the field of view.
„
Use Optical Axis Integration (also referred to as Z Sweep Acquisition) to
acquire a 2D projection. This method collects and integrates one continuous
image through an extended Z movement. Z Sweep Acquisition is much faster
than the traditional technique of collecting an individual image at each focal
plane. It also reduces the risk of specimen damage and has less total camera
read noise.
„
Acquire reference images that can be used for Differential Interference
Contrast (DIC) and other types of analysis.
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Using Autofocus in Experiment Macros
You can set up your experiment macro to automatically focus before each image is
acquired. This is especially useful for long time-lapse experiments that are
susceptible to changes in environmental conditions. Autofocus allows you to
acquire focused images without closely monitoring the experiment. The
DeltaVision Autofocus algorithm is a software-based method that uses both image
contrast and peak image intensity to determine when the specimen is in focus.
To optimize the focus position for DeltaVision, you must find the Z position where
the contrast is greatest. However, this position may not always be at the optimal,
in-focus plane of the sample. Autofocus allows you to adjust several parameters
within the standard algorithm so that the final Z position is most favorable for the
specific sample. Limiting the step size and maximum Z range of travel can limit
the effects of objects that are not of interest. In cases where the contrast outside of
the actual middle of the sample is highest, you can create an offset so that the
middle of the sample is located during the Autofocus process.
The Autofocus option works the same way as the
button on the Resolve3D
window. Use the following instructions to include this option in the experiment
macro.
To set up an experiment that automatically focuses before acquiring points:
1. At the top of the Resolve3D window, click the Experiment button to open the
Design/Run Experiment window.
AppliedPrecision
Chapter 5: Acquiring Data From Live Specimens
2. Click the Design Experiment tab and then click the Time-lapse tab.
3. Select the Autofocus before imaging option. When this option is selected, the
software automatically determines the parameters for the Autofocus process.
4. To change the various Autofocus parameters, click the Autofocus Options
button. The Resolve3D Settings dialog box is displayed with the Autofocus tab
selected.
5. Deselect the Automatically determine parameters option. The remaining
parameters on the dialog box become active.
The Autofocus parameters in this dialog box are described in Settings Dialog
Box Autofocus Options on Page 208.
Tracking Cells
Cell Tracking moves the stage laterally to follow cells as they move during a timelapse experiment. With the Enable Cell Tracking option selected, DeltaVision
automatically keeps cells in the field of view as they move during a time-lapse
experiment.
After you specify an ROI around an object of interest, the software determines the
center of the cell and establishes a recognizable pattern within the ROI. In
subsequent images, the software recognizes this pattern and recalculates the center
of the cell on-the-fly. The position of the new center is compared with the position
of the previous center and, if the cell has moved beyond a specified threshold, the
system automatically moves the stage and re-centers the cell in the field of view.
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Time Point 1
Time Point 2
Time Point 3
Time Point 4
At Time Points 1 and 2, the cell is moving through the ROI (the inner square), but the center is
still within the Move Threshold (the circle). At Time Point 3, once the center of the cell
touches the threshold boundary, the stage moves to re-center the cell within the ROI. (Note
the new stage position displayed in the lower-right corner of Time Point 4.)
To use Cell Tracking:
1. Set up a time-lapse experiment as shown in Setting up Time-lapse Experiments
on Page 42.
2. On the Time-lapse tab on the Design/Run Experiment window, select the
Enable Cell Tracking option.
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Chapter 5: Acquiring Data From Live Specimens
3. Click Cell Tracking Options.
4. Manually acquire an image for each channel and determine which single
channel best identifies the features of your specimen. In the Reference
Channel list, specify that channel. (The channels are numbered in the order in
which they are listed on the Design Experiment Channels tab.)
Note DeltaVision uses the reference channel for image recognition.
5. In the Move Threshold field, enter the distance in microns that the cell must
move to trigger stage movement.
6. Specify the area around the cell (the region of interest or ROI) that you want
DeltaVision to use for image recognition. For more about this option, see ROI
Percent on Page 72.
7. If you are performing an experiment on a single cell, use the stage controls to
center the cell (laterally) in the field of view. If you are performing a point
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visiting experiment, make sure that the points that you are monitoring are
centered before you start your experiment.
8. Click the Run Experiment tab and then click Start Scan to run the experiment.
Guidelines for specifying Cell Tracking Options
You can specify several parameters that DeltaVision uses to track cells:
„
Tracking Method
„
Reference Channel
„
Move Threshold
„
ROI Percent
Tracking Method
The tracking method is the method that DeltaVision uses to determine the center of
the feature. This center is recalculated after each image is acquired. You can choose
from two tracking methods:
„
Center of Intensity calculates the center of the feature based on intensity
values.
„
Center of Geometry calculates the center of the feature based on its geometry.
Center of Intensity
Center of Geometry
Reference Channel
DeltaVision uses the Reference Channel for pattern recognition. If you are
acquiring more than one channel, choose the channel that has the most distinctive
features.
Note The term, Reference Channel should not be confused with Reference
Image, which is something very different. Reference images are described
later in this chapter in
Acquiring a Reference Image.
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Chapter 5: Acquiring Data From Live Specimens
Channels are numbered as they appear in the Experiment Designer window. In
the following example, DAPI is Channel 1 and FITC is Channel 2.
Move Threshold
The Move Threshold is the distance the center of the cell (as defined by the
tracking method) must travel before the system resets the stage. When the cell
moves beyond this threshold, the stage moves so that the center of the cell retains
its original position in relationship to the center of the field of view.
Live cells, by their very nature, are constantly on the move. When choosing a
Move Threshold, it is important to select one that is small enough to keep the cell
in the field of view, yet large enough to buffer out needless stage movement that
could result from numerous small cell movements.
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Cell tracking lags cell movement. Cell movement exceeds the threshold between Time
Points 1 and 2 (left). In response, DeltaVision moves the stage so that it is centered on the
cell position at Time Point 2. The stage is at this position when Time Point 3 is acquired (right).
ROI Percent
With rare exceptions, few live cells are isolated on the substrate. Most are
genetically “programmed” to seek attachment and communication with other
cells. When performing live cell microscopy, not only is it important to filter out
unnecessary stage movement, it is also important to define an appropriate region
of interest within the field of view. This region of interest must be large enough to
include the entire cell or structure of interest, yet small enough to exclude other
cells or structures that may tend to wander in and out of the field of view
throughout the experiment.
The ROI Percent parameter defines how much of the field of view the software
will use for image recognition. In the software, ROI units are specified as a
percentage of the width of the field of view. For example, a 50% ROI has a width
that is 50% of the width of the field of view.
In the first image, the width of the ROI is defined as 50% of the width of the field of view. In
the second image, the stage has moved to keep the cell of interest within the ROI. Perimeter
cells have also moved, almost entirely out of the field.
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73
Tip If DeltaVision loses the cell because it moves out of the field-of-view, you can
update the point coordinates by manually finding the cell and centering it in the
field-of-view. The point list is automatically updated.
Acquiring 3D Z Projections with OAI
Continuous Stage
Movement
Multiple Stage Stops
Optical Axis Integration (or OAI), also referred to as Continuous Z Sweep, is
useful for acquiring 3D Z projections of live specimens. Instead of collecting an
individual image at each focal plane, OAI collects and integrates one continuous
image through an extended Z movement.
Optical Sectioning
Real-Time Z Sweep
Instead of acquiring multiple images, Real-Time Z Sweep acquires one image during a
continuous stage movement and instantly creates a 3D Z Projection.
Real-Time Z Sweep has significant advantages for applications such as Leading
Edge Motion Analysis, Fast Organelle Dynamics, Microtubule Dynamics, and
Fluorescence in situ Hybridization (FISH). It is especially useful for studies of
objects that are moving in 3D space (e.g., kinetichores in a cell nucleus or other
rapidly moving structures).
Continuous Z Sweep versus Traditional Projections
If you are acquiring data objects that you plan to use for 2D projections,
Continuous Z Sweep provides several advantages over creating 2D projections
from multiple optical sections:
„
Complete Z data acquisition collects all data in the interval of interest (with 2D
imaging or Z section sampling, some data is lost.)
„
Fast data acquisition provides accurate image registration of rapidly moving
objects.
„
Low total exposure time reduces the risk of damage to the specimen.
„
Low total read noise (the camera is only read once) improves signal-to-noise
ratio.
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Note For samples that contain a large amount of fluorescence throughout 3D
space (for example, a tumor spheroid that has a lot of fluorescence), optical
sectioning may provide better results than OAI.
The following 2D image of endosomes in a HeLa cell (left) and a 3D Z projection
(right) of the same area were acquired under similar conditions. The additional
data in the 3D Z projection include objects that moved out of the depth-of-field of
the 2D image during the data acquisition process.
endosomes in a HeLa cell (left)and an instant 3D Z projection (right) of the same area
Using Continuous Z Sweep
To set up a Continuous Z Sweep Experiment:
1. Set up your experiment as shown in Creating and Running an Experiment Macro
on Page 33.
2. In the Resolve3D window, use the Z Slider on the right side of the Resolve3D
window to find and center the object of interest. Drag the slider up to find the
top of your sample. (When you release the mouse button, an image is acquired
and displayed in the Data Collection window.) Drag the slider and acquire
images until you are satisfied. Then press the button to mark that location.
Next, use the slider to find the bottom of the sample. When you are satisfied,
press the button to mark that location.
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Z slider
Use the Z slider to find the top and bottom of a sample. The sample thickness is
indicated by the wide line on the Z slider (shown on the right image).
3. Click Experiment to open the Design/Run Experiment window. Then click the
Sectioning tab.
4. Select the Z Sectioning and Enable OAI Scan options.
5. Click the Run Experiment tab and click the green arrow button
toolbar at the top of the screen to start the experiment.
in the
Note When deconvolving OAI images, select the More Options button on the
Deconvolve window, and then select the Deconvolve Projections option in the
More Deconvolution Options dialog box.
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Acquiring a Reference Image
You can use an alternate filter or the transmitted light to acquire a reference image
that can be combined with other images. This option is useful for Differential
Interference Contrast (DIC) analysis. It can also be useful for other types of
reference images.
A reference image of yeast cells
You can specify to acquire the reference image at the top, middle, or bottom of the
sample.
Top of Sample
Middle of Sample
Bottom of Sample
Note It is important to optimize the reference image settings prior to defining them
in the Experiment Designer.
To create a reference image:
1. Set up a 3D Sectioning experiment (see Sectioning Specimens for 3D Images on
Page 35).
2. In the Design/Run Experiment window, click the Channels tab and select the
channels for your experiment (see Selecting Filters on Page 42).
3
Select the Reference Image option.
3. In the Z position list, choose whether to acquire the reference image at the top,
middle, or bottom of the sample.
AppliedPrecision
Chapter 5: Acquiring Data From Live Specimens
4. Under the Reference Image, select which filters to use.
5. In the EX Shutter field, select the type of light to use for the image. EX is the
Excitation (or the arc lamp) and TRANS is the transmitted light.
This experiment specifies using the DAPI and FITC filters to acquire images of each
section and using the transmitted light to acquire a reference image in the middle of
the sample.
6. Save and Run the Experiment.
After DeltaVision acquires the data, it creates two image files. One file contains
the reference image data and the other file contains all of the other data.
Tip After you acquire the images, you can use the Image Fusion tool in softWoRx to
combine the reference images with the rest of the images.
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6
6
Data Collection Techniques
This chapter provides guidance and suggestions for:
„
Finding a Specimen and Recording Its Position
„
Finding Exposure Time
„
Using Köhler and Critical Illumination
„
Monitoring Data Acquisition
„
Editing Experiment Macros
Finding a Specimen and Recording its Position
Recording the position of your slide is useful when you are conducting a point
visiting experiment and you need to remove the slide before you are finished. Use
the following instructions to find a sample and record its position on the slide with
the Repeatable Slide Holder. When you resume your experiment, you can place
the slide in the position that you recorded.
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Note If you are using an oil immersion lens, you must apply immersion oil to the
sample. See Appendix A: The Oil Immersion Kit, for more information about the oil
calculator.
To find and center the specimen:
1. Choose the objective by rotating the objective turret. Be sure to select the same
objective in the Resolve3D window.
2. Secure the slide. Since DeltaVision is an inverted microscope, the sample must
be placed with the cover slip facing down toward the objective. If you are
using a standard microscope slide (1” x 3” or 25 mm x 75 mm), you can use the
Repeatable Slide Holder to hold the slide. To do this, pull the return spring to
the left and place the Slide Cover Slip down onto the Slide Holder so that the
upper right corner is pressed laterally against the brass locators (shown as A
and B in Figure 5) and the upper left edge is pressed laterally against the brass
locator C. Then gently release the return spring to secure the slide. Push down
on the slide to make sure that it is fully seated in the holder.
Figure 5: The Repeatable Slide Holder
C
A
Return Spring
B
3. Choose the desired eyepiece (EP) filter.
4. Select the desired excitation (EX), neutral density (%T), and emission (EM)
filters. (These parameters should automatically be in place after selecting the
eyepiece filter, unless non-standard arrangements are needed.)
5. Rotate the Beam Selector at the base of the microscope to direct the light
collected by the objective to the eyepiece.
6. To use the transmitted light to locate the specimen, open the transmitted light
shutter by pressing TRANS SHUTTER on the keypad. This allows white light to
transilluminate the sample. (The intensity for the transmitted light is
controlled using the %T field on the Resolve3D menu.)
7. Set the stage speed using the keypad SLOW, MEDIUM, or FAST Buttons.
Medium is typically the best speed to start with. (These buttons control the
stage speed when you are using the joystick or keypad to move the stage.)
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Chapter 6: Data Collection Techniques
8. Open the EX shutter and find the desired focal plane optically using either the
focus knobs on the microscope or the Resolve3D Z stage controls.
Note If you are using an Olympus or Nikon microscope, rotate the top of the
focus knob toward you to move the objective up and away from you to move it
down (when you are sitting in front of the microscope).
9. Use one of the following methods to maneuver the slide and find an area of
interest:
Keypad
The buttons on the keypad are used for movement in the X, Y, and Z
directions. Using the arrow keys on the keypad causes the stage to move by
steps. The size of each step is doubled each time the STEP INCREASE button is
pressed and halved each time the STEP DECREASE button is pressed. By
adjusting the step size to the frame size, you can create a condition where each
press of a step arrow will move the sample one frame. This is a convenient
way to scan a large area for rare events (e.g., mitosis). Since the step
movements are rapid, using the arrow keys in this way can be much less
fatiguing than using the joystick.
Joystick
The joystick controls stage movement in the X and Y directions.
Workstation
The Resolve3D module allows you to finely control stage motion. The arrow
buttons move the stage in discrete increments that are indicated by the values
in the dX, dY, and dZ text boxes. Resolve3D provides the only mechanism
from which to obtain discrete step sizes.
Focus Knob
The manual focus knob on the microscope base moves the nosepiece/objective
in the Z direction.
CAUTION: To record and maintain accurate stage coordinates, focus by
moving the stage with the Resolve3D controls instead of using the focus
knobs to move the objective. Resolve3D cannot track the movement of the
objective.
10. Tighten the Focus Lock on the left side of the focusing knob (see Page 15).
11. Use the keypad to find the approximate center of the specimen and place it in
the center of the field of view using stage controls.
12. Direct light to the camera port using the Beam Selector knob on the microscope
base.
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13. Record the position of the slide on the Position Indicator (the letter scale at the
bottom of the Repeatable Slide Holder).
Position Indicator
Finding Exposure Time
The exposure time correlates with the signal intensity level for the image
acquisition. Finding the appropriate exposure time for each filter in an experiment
is crucial to acquiring the best image data. Many factors must be considered.
Although you want to see the maximum intensity in each wavelength, you must
not saturate the camera or photo-bleach your sample.
High-Speed CCD Camera saturation occurs when the intensity values reach 4145
counts. The 12-bit High-Speed camera has an intensity range of 0 to 4095 counts.
(The EM CCD 16-bit High-Speed camera has an intensity range of 0 to 65535
counts.) For successful deconvolution, a minimum intensity of 50 counts above
background is recommended.
Note If you are using 0.5X Gain, the saturation is less than 4095 counts. For the EM
CCD camera (a 16-bit camera), saturation is over 50,000 counts.
Photo-bleaching occurs when the fluorescent dyes lose their emission intensity as
they are exposed to illumination. Certain dyes are more susceptible to photobleaching than others. The potential for excessive photo-bleaching is increased
with increased exposure time and decreased by use of a higher value neutral
density filter.
There are two ways to decrease illumination, and therefore the likelihood of
photo-bleaching:
„
Decrease exposure time.
„
Increase neutral density value.
Note For live cell imaging, cells tolerate shorter exposures better than longer
exposures, even at increased illumination intensity.
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The response of increasing the exposure time is roughly linear. Therefore, if an
exposure time of 0.2 sec results in a maximum intensity level of 1000 counts, then
an exposure time of 0.4 sec will result in a maximum intensity level of
approximately 2000 counts.
Use the following instructions to find the best exposure time for each excitation
wavelength. This procedure must be performed for each excitation wavelength
used in the experiment.
To find exposure time:
1. Set the neutral density filter to 100%.
2. Set the exposure time to a low value (e.g., 0.1 sec).
3. Set dZ to the desired value to sample different images throughout the sample.
4. Click the dZ textbox.
5. Enter the desired value for the Z step.
6. Move through the area of interest by pressing
move the stage in the Z direction.
and
in the Stage dialog to
7. Click Acquire. If performing a 2D experiment, go to Step 13.
8. Continue performing steps 6 and 7 to obtain a sampling of Z sections. Find the
maximum intensity in these sample images by observing the value of the Max
field in the Resolve3D window. The focal plane with the highest maximum
intensity value is the plane of optimal focus.
Note There could be a higher intensity value in the image data that you did not
sample. Therefore, do not set the exposure time to the limits of the sampled data
because a higher intensity, un-sampled plane may saturate the camera.
9. Move to the plane of optimal focus. This is the Z section with the highest
intensity value.
10. From the Resolve3D menu, choose View | Point List.
11. Click Mark Point. The Z value is the third coordinate listed for the point.
12. Document the Z value with the brightest intensity.
13. Increase exposure time, but consider the amount of exposure the specimen can
tolerate. Intensity values increase in direct proportion to exposure time (e.g.,
doubling the exposure time doubles the intensity values).
14. Repeat for all desired wavelengths.
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Using Köhler and Critical Illumination
DeltaVision Core allows you to easily switch between two types of illumination:
•
Köhler Illumination is the most commonly used form of illumination. It
provides very even specimen illumination across the field-of-view. The light
uniformly drops off as the distance from the focal plane increases.
•
Critical Illumination directs the entire light source to the size of the detected
area, and not the entire sample. With Critical Illumination, more light is
directed to the focal plane and the out-of-focus light drops off more rapidly
than in Köhler Illumination, based on the size of the field-of-view. Critical
illumination also provides better axial (Z) and lateral (X,Y) contrast.
You will typically use Köhler Illumination for most of your data collection.
However, Köhler Illumination can be insufficient for faint signals. For low
abundant probes that require more light and better contrast, use Critical
Illumination.
To switch to Critical Illumination:
X Loosen the Locking Knob and pull the Focus Control back (away from the
stand) until it snaps into place in the Critical Illumination position. Then
tighten the Locking Knob.
Critical
Illumination
Spring
Focus
Control
Locking Knob
In Critical Illumination position, the Focus Control is extended
so that the Critical Spring is in the groove.
CAUTION: When you are adjusting the Fiber Optic Module, be careful not to
disconnect the Fiber Optic cable or to bend it in a diameter that is less
than 24".
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To switch to Köhler Illumination:
X Loosen the Locking Knob and push the Focus Control toward the stand until it
snaps into place in the Köhler Illumination position. Then tighten the Locking
Knob.
Focus
Control
Köhler Illumination Spring
Locking Knob
In the Köhler Illumination position, the Fiber Optic Module is
pushed in so that the Köhler Illumination Spring is in the groove.
Monitoring Data Acquisition
You can set options that control how images are displayed as they are acquired.
Viewing Deconvolved Image Previews
You can use 2D deconvolution to get a more accurate representation of what data
will look like after deconvolution.
These images show how images are displayed during data acquisition,
with and without Real-Time 2D Deconvolution.
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To view previews of deconvolved images:
1. From the Resolve3D window, click Settings to open the Resolve3D Settings
dialog box. Then click the Display tab.
2. Select the Deconvolve preview images option.
Note This option does not provide a full iterative deconvolution, but it allows
you to preview images as you collect them.
Selecting Viewing Modes
You can select from several modes for displaying images in the Data Collection
window (or windows) as they are collected. You can choose other modes to
display images in color, display each point in a point visiting experiment in a
separate window, or to display each channel in a separate window, as follows.
Auto Grayscale mode displays a separate window for each channel.
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To select the display mode:
1. From the Resolve3D window, click Settings to open the Resolve3D Settings
dialog box. Then click the Display tab.
2. On the Image display mode list, select a display mode.
Table 1: Image display modes
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Mode
Description
None
Displays images in the current window.
Scratch
Displays all images in the default Data Collection window (Window
21).
Auto
Grayscale
Displays images in a separate window for each emission filter.
Auto Color
Displays images in color as they are collected. When using Point
Visiting, images are automatically displayed in separate windows for
each point. This option should be used only when you are running an
experiment.
Point Track
Opens a separate window for each visited point in a point-visiting
experiment.
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Displaying Statistics and the Histogram
You can choose to display statistics and a histogram of the intensity values of each
image as it is collected. These values are displayed at the bottom of the Resolve3D
window. The Min, Max, and Mean values are the minimum, maximum, and mean
intensity values. The histogram displays the intensity distribution.
To display statistics and the histogram:
1. From the Resolve3D window, click Settings to open the Resolve3D Settings
dialog box.
2. Click the Display tab. Then select from the following options.
To
Select
Calculate image intensity
statistics
Calculate Statistics.
Calculate and display an
image intensity histogram
Automatically scale the
histogram width for each
image that is analyzed.
Note: You can improve readout speed by disabling this option.
Calculate histogram
Auto Histogram Range
Note: This option changes only the display of the histogram. It
does not change the image data.
Editing Experiment Macros
The Experiment Macro Editor is used to create or edit Resolve3D experiment
macros (command scripts) that control the DeltaVision microscope. Most
experiment macros can be generated using the Design/Run Experiment window,
but you may need to create custom macros for certain types of experiments.
The best way to get started is to modify an existing macro with the Experiment
Macro Editor. One approach is to use the Design/Run Experiment window to
generate a macro and then edit it. Another approach is to use a reference macro.
To open the Experiment Macro Editor:
1. On the Resolve3D window, click Experiment.
2. In the Design/Run Experiment dialog, choose File | Edit to open the
Experiment Macro Editor.
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Status Area
Commands list
Macro Text Area
The Experiment Macro Editor
Menu Bar
The menu bar File menu contains commands to open and save experiment macro
files. The Save & Quit option saves the macro file with the current name. The Edit
menu has commands for manipulating text. The Search menu provides search and
replace capabilities and the Help menu displays additional information.
Search & Replace
These text fields are used to specify a search and replace pattern. (You will need to
enter strings in these fields before you use the Search | Replace Text menu
command.)
Status Area
The text area below the Search and Replace fields is used by the Macro Editor to
supply you with status and hint information.
The Macro Text Area
The Macro Text Area below the Status Area is the main working area of the Macro
Editor.
Commands List
This is the list of available commands. When you highlight a command in the list
by clicking it, you are provided with information about the command in the Status
Area. If you double-click or select the Use Item button below this list, the
command is inserted in the Macro Text Area at the current cursor location.
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To edit a reference macro:
1. On the Resolve3D Experiment Macro Editor window, choose File | Open
Reference Macro.
2. In the Open Experiment Macro File dialog box, select a macro file and click OK
to open it.
3. Use the mouse to select the part of the reference macro that you want to copy.
4. On the Reference Macro menu, choose Edit | Copy.
5. On the Resolve3D Experiment Macro Editor, choose Edit | Paste to copy the
macro into the macro editor.
6. Edit and save the file as a new experiment macro.
AppliedPrecision
7
7 Facility Requirements & Components
This chapter describes the main components of the DeltaVision System. It includes
the following sections:
„
Electrical and Environmental Requirements describes the DeltaVision operating
and facility requirements.
„
Overview of Components shows the location of the key system components.
„
Optical Components describes the system light sources, cameras, and filters.
„
Desktop Components provides information about the monitor, the keypad and
joystick, the vibration isolation table that supports the microscope, and other
components.
„
Cabinet Components describes the combined Instrument Controller and Microscope
Interface Chassis (IC/MIC) and the Workstation.
„
Other Standard Components describes the Repeatable Slide Holder, the Fiber
Optic Module, the tool kit, and standard software.
„
Optional Components describes the Quantifiable Laser Module (QLM), the EM
CCD camera, The Rainbow option (dual light sources for DIC), and the
softWoRx Explorer application.
„
Consumable Parts lists the fuses and other components that you will need to
replace to maintain the system.
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Electrical and Environmental Requirements
An important aspect of collecting high quality images is meeting the proper
electrical and environmental requirements for the system.
Electrical Requirements
Line Requirements
Operating Frequency: 50/60 Hz
Operating Power: 100-127 VAC requires 8A
200-240 VAC requires 4A
Transients: Transient over-voltages in accordance with Installation
Category II in IEC 664
Maximum Power: 1200 VA
Power Cord Set Requirements
The power cord set received with DeltaVision meets the requirements for use in the
country where you purchased the equipment.
General Requirements
The requirements listed below are applicable to all countries:
„
The length of the power cord set can be a maximum of 9.75 feet (3.0 m).
„
All power cord sets must be approved by an acceptable accredited agency
responsible for evaluation in the country where the power cord set will be
used.
„
The power cord set must have a minimum current capacity of 10A for 230
VAC systems or 15A for 100-120 VAC systems, as required by each country's
power system.
„
The appliance coupler must meet the mechanical configuration of an
EN60320/IEC 320 Standard Sheet C13 connector, for mating with the appliance
inlet on the Isolation Transformer Assembly.
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Chapter 7: Facility Requirements and Components
Country-specific Requirements
The following table shows the accredited agency and power cord set requirements
for each country.
Country
Accredited
Agency
Australia
EANSW
Austria
OVE
Belgium
CEBC
Denmark
DEMKO
Finland
SETI
France
UTE
Germany
VDE
Italy
IMQ
Norway
NEMKO
Sweden
SEMKO
Switzerland
SEV
United Kingdom
BSI
United States
UL
Canada
CSA
Japan
JIS
Power Cord Set Requirements
The flexible cord must be <HAR> Type HO5VV-F, 3conductor, 1.0 mm2 conductor size. Power cord set
fittings (appliance coupler and wall plug) must bear
the certification mark of the agency responsible for
evaluation in the country where it will be used.
The flexible cord must be Type SJT or equivalent, No.
14 AWG, 3-conductor. The wall plug must be a twopole grounding type with a NEMA 5-15P (15A, 125V)
configuration.
The appliance coupler, flexible cord, and wall plug
must bear a "T" mark and registration number in
accordance with the Japanese Dentori Law. The
flexible cord must be Type VCTF, 3-conductor, 2.00
mm2 conductor size.
Environmental Requirements
An important aspect of collecting high quality images is having the proper
environment for the system. The following important environmental requirements
are outlined in this section.
General Environmental Requirements
Floor space:
DeltaVision Core - 3 ft × 6 ft (90 cm × 180 cm). Include 18 in (45 cm) space behind
instrument rack.
personalDV - 22in x 54 to 62in (depending on whether or not the keyboard is kept
in a slide-out tray). Include 18 in (45 cm) space behind instrument rack.
Maximum System Weight: 940 lbs (425 kg).
Service: Indoor use only.
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Temperature: 65 - 77 °F (18 - 25 °C), daily variation of no more than 3 °F (1.8 °C).
The actual room temperature should be stable to within 1 degree (Fahrenheit or
Celsius) per hour. Fluctuations in temperature will affect microscope optics, which
can cause the specimen to drift approximately 1 μm per 0.1 degree Celsius.
Humidity: Stable humidity levels under 50%, with daily variations of less than
10%. High humidity can result in condensation on the CCD camera window that
obscures image formation. Excessive humidity may also reduce filter life and may
result in chromatic aberrations in the images.
Altitude: up to 6550 ft (2000 m).
Pollution: POLLUTION DEGREE 21 in accordance with IEC 664.
Ingress Protection Level: IP20
Communication Recommendations
Connect the workstation to a local area network for data storage. To connect to a
network, you will need an IP address, domain name server address, and network
mask. A connection to the Internet will provide access to Applied Precision's web
site.
Applied Precision, INC is not responsible for damage or harm to
CAUTION:
the workstation or scanner due to network security breaches.
Having a telephone in the same room as the system will facilitate communication
with Technical Support.
Air Movement
Air movement around the microscope can cause specimen drift on the scale of
several microns. Two common sources of air movement are window air
conditioners and open windows. Central air conditioning is recommended.
However, the system should not be placed in the direct path of the incoming air.
Vibration Isolation
The vibration absorbing design of the system minimizes motion artifacts from
internal vibration due to shutters, filter wheels, and stage movement. The system
is also designed to damp out external vibration as well. Avoiding locations near
refrigerators, elevators, ventilation equipment, and other sources of vibration will
improve image resolution.
IEC 61010-1: 2nd ed. International Electrical Commission defines POLLUTION DEGREE 2 as
follows: “Normally only non-conductive POLLUTION occurs. Occasionally, however, a temporary
conductivity caused by condensation must be expected.”
1
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Ambient Illumination
For best results, minimize ambient illumination during data collection. A lighttight room is recommended. Ensure that there are no light sources pointed
downward into the lens.
A small desk lamp located near the workstation is recommended for preparing
and monitoring experiments.
You can press the BLANK SCREEN key on the keypad to darken the monitor for
improved image quality. Pressing any key on the keyboard restores monitor
function.
Dust
It is important to minimize dust on the microscope components because dust on
components can cause spots on microscope images. Minimize contamination by
maintaining a clean room and covering the microscope when it is not in use.
Overview of Components
The main standard DeltaVision components for both DeltaVision Core and
personalDV are shown below. Detailed descriptions of these components and
descriptions of optional components are included in the following sections.
Standard DeltaVision Core Components
Optical
Components
Vibration
Isolation
Table
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Desktop
Components
Cabinet Components
(IC/MIC, Workstation,
and any additional
optional components)
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personalDV Components
Optical
Components
IC/MIC and
Workstation
Desktop
Components
Optical Components
Xenon Arc Lamp
White LED Trans Light
Excitation Filters
Neutral Density Filters
Eyepiece Filters
Camera
Emission Filters
Note Optical components are shown for a typical installation, which includes the
Olympus IX71 Microscope. Other microscope configurations vary slightly. The
Olympus IX71 is currently the only microscope available.
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Fluorescence Microscope
The microscope is an advanced research grade epifluorescence inverted
microscope. Each objective is qualified by Applied Precision to guarantee the
highest possible quality. The point spread function (PSF) is measured in order to
uphold the image quality. The microscope supports a Differential Interference
Contrast (DIC) module (this is an optional component). A transmitted light shutter
is included with your system to enable automated DIC and Brightfield image
acquisition.
Optical Filters
The system uses a four-band polychroic beam splitter and filter wheels rather than
a simple dichroic filter cube. The excitation, neutral density, and emission filters
are selected in one of three ways:
„
Selecting options in the Resolve3D acquisition software.
„
Selecting a mode from the keypad.
„
Rotating the eyepiece filter wheel.
Note The eyepiece filter wheel is operated manually but reports its position to the
instrument controller, which in turn adjusts the excitation and emission filters.
Depending upon the environment, filters last between one and three years. To
ensure optimal performance, filters should be periodically inspected and replaced
if necessary. Consult Chapter 9: Maintenance for a detailed explanation of this
procedure.
The following standard DeltaVision Filter set is included in all DeltaVision Core
systems.
Table 2: DeltaVision Standard Filter Set
Filter
Name
Excitation
CWL/BP
Emission/Eyepiece
CWL/BP
DAPI
350/50
455/50
FITC
490/20
525/36
TRITC
555/25
605/52
Cy5®
645/30
*705/72
*Emission filter only
Cameras
DeltaVision Core comes standard with the CoolSNAP HQ2 CCD (charge coupled
device) camera, referred to as the High-speed Camera, and also supports an
upgrade option of the Cascade II EM CCD camera. For more about the Cascade II
camera, see EM CCD Camera on Page 111.
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personalDV comes standard with the CoolSNAP ES2 Camera.
High-Speed Camera
The High-Speed CCD camera (CoolSNAP HQ2 ) collects digital image data from
the microscope. This camera is designed to collect image data at a high frame rate.
At the fastest speed, the camera can collect 30 frames per second of a 64 × 64 pixel
image. Increased acquisition speed is useful when collecting images of live cells
that deteriorate over time.
The High-speed Camera
The High-speed Camera is air-cooled. The cooling apparatus is incorporated into
the camera assembly; therefore, when the High-speed Camera is turned on, the
cooler is also operating. When using cooled cameras in humid conditions, it is
possible for condensation to form on the CCD camera window. If this happens, a
mottled pattern is superimposed on the images. You will need to lower the
ambient humidity level to avoid condensation.
Notes
#1 The High-speed Camera controller is built into the camera head. The High-Speed
Camera has no user-serviceable parts inside.
#2 If no images are acquired over a 4-hour period, the High-speed Camera
automatically shuts down.
CAUTION: Do not disconnect cables to the High-speed Camera when the
power is on. Be sure to leave a 1 in (2.5 cm) minimum space around the cooling
fan.
Use only the PCI cards, cable, and power supplies that are designated for this
system.
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Chapter 7: Facility Requirements and Components
High-speed Camera Power Supply
The High-speed Camera power supply provides electric power to the camera. It is
housed on the bottom shelf of the cabinet.
ES2 Camera
The CoolSNAP ES2 CCD Camera is basically the same as the CoolSNAP HQ2
Camera, except the ES2 is not as deeply cooled. This makes it more affordable, but
allows for slightly higher noise. However, the noise difference is virtually
unnoticeable unless you are using long exposure times (~8s).
Light Sources
DeltaVision provides two light sources: a xenon arc lamp for the main light source
and a white LED for transmitted light.
Xenon Lamp
Illumination for the microscope is delivered from the xenon arc lamp to the
specimen through a fiber optic cable.
The light passes through the fiber optic module (FOM), where approximately 1%
of the light is diverted to the Photo sensor. The remaining 99% of the light is
delivered to the microscope. For instructions on how to replace the xenon bulb, see
Replacing the Xenon Bulb on Page 134.
WARNING: Do not disconnect the arc lamp power cable when the
power
is on.
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WARNING: The xenon arc lamp presents potentially harmful risks to the
user, including the possibility of UV exposure to skin and eyes. Before
operating the microscope, consult Chapter 2: Safety for important
information regarding arc lamp operation.
Note The illumination path alignment is critical to acquiring the highest possible
image resolution. See Page Path on Page 144 for a complete description of the
alignment procedure.
Photo sensor
The Photo sensor measures illumination intensity by sampling a small percentage
of the light from the arc lamp. The photo sensor signal is recorded by the
controller and then used to correct for variations in the xenon lamp intensity
during an experiment.
The Corrections tool in softWoRx normalizes each image based on its photo sensor
value. These corrections are automatically applied during deconvolution. This
enables quantifiable intensity comparisons between images, even if the brightness
of the excitation light varies between the images.
LED Transmitted Light
An LED transmitted light source is also provided.
LED Transmitted Light Source
If at any point you need to replace the transmitted light, see the instructions in
Replacing the Transmitted Light on Page 138.
Note Either light source (the xenon lamp or the LED transmitted light) can be used
with the eyepieces or the cameras.
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Chapter 7: Facility Requirements and Components
Desktop Components
Desktop Components include the flat panel display monitor, the keyboard, the
mouse, the keypad, and a joystick. DeltaVision Core includes a vibration isolation
table. personalDV includes a bench-top microscopy isolation platform.
Flat-Panel Display Monitor
All DeltaVision systems are equipped with Flat-Panel LCD monitors. These
monitors offer a very high level of performance in several areas pertaining to the
quality of displayed images, the most critical of which is contrast ratio.
Note For instructions that show how to adjust the monitor, see the Flat-Panel Display
manual.
CAUTION: DeltaVision is configured to work with the monitor that is
included with the system. Other monitors are not necessarily supported.
The Keypad and Joystick
Many of the functions accessible through Resolve3D are also available on the
keypad/joystick (see Keypad/Joystick Operation on Page 211).
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Vibration Isolation Table
Within the vibration isolation table is a breadboard surface that is supported by
mechanical vibration isolators. These components provide optimal performance
without an external air source. The isolators are sized for the system weight as
delivered. If significant additional weight is added to the breadboard, higher
capacity isolation may be required. Contact Applied Precision for more
information.
Note The vibration isolation table is not included with personalDV. Instead,
personalDV includes a bench-top isolation platform to provide similar stability for the
microscope. The DeltaVision vibration isolation table is available for personalDV as
an optional upgrade.
Cabinet Components
Note The component cabinet is included with DeltaVision Core only. The cabinet is
not available for personalDV.
The cabinet contains all of the electronic control equipment and provides surfaces
for the keyboard and the Flat Panel Display. The front two wheels should remain
locked and the cabinet left in place. There are many cables connecting the
microscope that can be damaged if they are pulled. The standard cabinet
components are shown below.
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Cabinet Components
Keyboard/Mouse Tray
IC/MIC
Workstation
QLM (optional)
Note Configuration of cabinet components may vary slightly.
Instrument Controller / Microscope Interface
Chassis (IC/MIC)
The Instrument Controller (IC) is the portion of this computer that interfaces with
all of the microscope hardware (including the microscope stage motors, filter
wheel motors, and cameras). It coordinates all activities related to positioning the
stage and collecting images. Data from the camera feeds through the controller to
the workstation. The controller also receives instructions from the workstation and
issues commands to the motors through the Microscope Interface Chassis.
The Microscope Interface Chassis (MIC) side of the computer provides power and
control for the filter motors, stage drive, and shutters. It also contains the Photo
sensor, which is connected to the microscope through a fiber optic cable.
Notes
#1 The Instrument Controller / Microscope Interface Chassis has no user-serviceable
parts inside.
#2 Occasionally, you may need to replace fuses on the back panel of the chassis.
Consult Chapter 9 for a description of this procedure. Unless there is an obvious
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reason why the fuse blew, you should contact Applied Precision when you need to
replace fuses.
Workstation
The workstation hosts the softWoRx application that is the primary interface used
to control the system. The workstation can also provide a server for data storage
when used with the optional Data Management Software (DMS).
Note The workstation has no user-serviceable parts inside.
DVD-R Recording
The DVD-R/CD Drive supports DVD-R format recording (DVD + R and CD-ROM
are not supported.)
Other Standard Components
Other standard components include the Repeatable Slide Holder, the Slide Holder
Adapter, the Fiber Optic Module, the keypad, and the joystick.
The Repeatable Slide Holder
The Repeatable Slide Holder holds the slide on the stage. It also allows you to
move the slide across the stage and to mark the slide position when you remove
the slide.
The ability to move the slide across the stage allows you to view the full slide on
the 1" x 1" stage area. With the slide held against the three brass locators by the
Return Spring, you can use the Slide Adjustment Knob to move the slide laterally.
Marking the position of your slide is useful when you are conducting a point
visiting experiment and you need to remove the slide before you are finished. You
can use the Position Indicator (the letter scale at the bottom of the slide holder) to
record the position of your slide. When you resume your experiment, you can
place the slide in the position that you recorded.
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Figure 6: The Repeatable Slide Holder
Brass Locators
Return
Spring
Position Indicator
Slide
Adjustment
Knob
Note If you are using a Petri dish or any other format that is not similar to a 1" x 3"
slide, you will need to remove the Repeatable Slide Holder or use the Slide Holder
Adapter.
For more about using the Repeatable Slide Holder, see Finding a Specimen and
Recording its Position on Page 79.
Slide Holder Adapter
The Slide Holder Adapter holds a chambered coverglass. It is mounted on the
Repeatable Slide Holder. NUNC Lab-Tek™ II chambered coverglasses with
one, two, four, or eight wells are available. The Slide Holder Adapter also
supports Petri dishes from 25 – 40 mm in diameter.
Calibration Kit
The Calibration Kit is provided for calibration operations on the DeltaVision
system. The kit includes the following six slides:
•
Three plastic fluorescent calibration slides – for flat-field calibration:
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•
•
•
Blue plastic (EX 408nm, EM 440 nm) – Good for DAPI, Hoechst, etc.
Orange plastic ((EX 488nm, EM 519nm) – Good for FITC, GFP, TRITC,
CY-3®, etc.
• Red plastic (EX 590nm, EM 650nm) – Good for CY-5®, etc.
Silicon mirror slide – for light path alignment and troubleshooting.
•
Silicon grid slide – for pixel size measurement.
•
100nm Rhodamine® bead slide – for PSF measurement.
The Fiber Optic Module
Use the Fiber Optic Module to align the light path from the fiber optic cable to the
Fluorescent Illuminator. This module allows you to adjust the tilt, horizontal, and
vertical orientation of the light path.
Fiber Optic Module
The Tool Kit
A tool kit is provided that includes tools for maintaining the system. This kit
includes:
•
Ties and belts for suspending cables, screws, and other fasteners
•
Cleaning material (Q-tips and lens paper)
•
A hex wrench set (5/64" – 3/16")
•
A set of metric L keys (1mm –5mm)
•
Α #1 Phillips screwdriver
•
A 5mm T Handle hex key
•
An acrylic bulls eye level
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Chapter 7: Facility Requirements and Components
•
A micrometer nut wrench
•
An Applied Precision Hotline sticker
Software
DeltaVision Core includes softWoRx for Linux (1 copy), softWoRx Suite for Windows
(1 copy), DMS Server (1 copy, 3-seat), and softWoRx DMS (2 copies).
personalDV includes softWoRx for Linux (1 copy) and softWoRx DMS (1 copy).
Note Either system can be ordered with a number of different software
configurations. Talk with your Applied Precision representative to determine the best
configuration for your applications.
softWoRx
softWoRx is the Linux software application that runs the acquisition workstation.
The software allows you to perform the following tasks:
„
Acquire image data
„
Set up and run experiments
„
Deconvolve data
„
Measure point spread functions
„
Calculate optical transform functions
„
Process 2-D and 3-D images
„
Perform quantitative analysis
„
Archive data
„
Configure task chains
„
Manage user accounts
softWoRx Suite for Windows
softWoRx Suite is a Windows-based ensemble of software developed by Applied
Precision in collaboration with Bitplane AG. It provides sophisticated multidimensional data visualization, analysis, image restoration, image correction, and
image viewing management, all within an easy-to-use streamlined browser
interface.
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softWoRx DMS
softWoRx DMS (Data Management Solution) provides a functional infrastructure
for the storage of biological images and their associated metadata in a centralized
database. Like softWoRx Suite, softWoRx DMS includes the Browser and the
Explorer programs, but unlike softWoRx Suite does not have the ability to perform
any type of image processing. The DMS product actually has two distinctive parts.
One part is the DMS Server and the other part is represented by the DMS clients.
The DMS Server represents different configurations of what the centralized
database looks like. The DMS clients are the different programs and
methodologies of accessing the image data within the centralized database (ie.
softWoRx, softWoRx Suite, and softWoRx DMS).
Optional Components
You can purchase several optional DeltaVision components. Some options are
available for both DeltaVision Core and personalDV, but many are available only for
DeltaVision Core (as noted below):
„
The Environmental Chamber provides a controlled temperature environment
for live cell imaging. The chamber also supports CO2 injection, which allows
you to control humidity and pH by maintaining a CO2 flow over the sample.
„
Additional Filter Modules can be loaded with custom filters or with the Live
Cell filter set to provide greater flexibility for your lab.
„
Quantifiable Laser Module components (available for DeltaVision Core only)
allow you to use lasers to perform FRAP and other photokinetic experiments.
„
The TIRF (Total Internal Reflection Fluorescence) module (available for
DeltaVision Core only) includes the Olympus TIRF module and special adapters
that allow you to install it on DeltaVision.
„
The EM CCD Camera (available for DeltaVision Core only) is an optional
camera that provides high signal-to-noise ratios for Low-light Fluorescence,
TIRF, or single molecule fluorescence.
„
Analysis Workstations include the softWoRx Linux workstation and the
softWoRx Suite Windows workstation.
„
Software includes two optional modules for either the DeltaVision Core or
personalDV: the softWoRx Explorer and the softWoRx Suite advanced option.
softWoRx Suite for Windows is also available as an option for personalDV.
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Chapter 7: Facility Requirements and Components
„
A selection of Differential Interference Contrast (DIC) components are
supported by DeltaVision Core and personalDV.
„
The optional Multiplexed Wavelength module for DeltaVision includes a
second shuttered xenon light source and provides nearly simultaneous twochannel imaging.
„
The following optional objectives are tested by Applied Precision to ensure
that they meet our rigid quality standards.
Optional Objectives
U-APO 40X Oil, 0.65-1.35NA, 0.10mm WD
U-PLAN S-APO 100X Oil, 1.4NA, 0.12 WD
U-PLAN APO 60X W/PSF Water, 1.20 NA, 0.25mm WD
The following subsections describe each of the optional DeltaVision components.
The Environmental Chamber
Environmental control is an essential factor in live-cell microscopy or any other
type of experiments requiring system stability. Focus instability can be difficult to
resolve for critical experiments. Small changes in ambient temperature can lead to
thermal expansion or contraction in the microscope stand, stage, and objective
changing the plane of focus. The Environmental Chamber is designed to precisely
control temperature and CO2 levels making it ideal for long term time-lapse
applications and high-resolution imaging. The Environmental Chamber includes a
temperature controller, a CO2 humidifier, and a CO2 chamber.
For instructions that show how to install, remove, and operate the environmental
chamber, see the Environmental Chamber User's Manual (included with your
system).
Additional Filter Modules
You can purchase additional DeltaVision Filter modules and use them to quickly
change the filter sets to meet different imaging requirements.
Each filter module holds up to six filters. You can purchase a Live Cell Filter
Module that is preloaded with four filter sets that are commonly used for live cell
imaging. You can also purchase empty filter modules and insert customized filter
sets into those modules.
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The Live Cell Filter Module
The Live Cell Filter Module includes the following filters:
Filter Name
Excitation CWL/BP
Emission/Eyepiece CWL/BP
CFP
430/24
YFP
510/20
535/30
mCherry
572/35
632/60
EGFP
470/40
525/50
470/24
Quantifiable Laser Module Components
The Quantifiable Laser Module (QLM) adds a laser beam into the back aperture of
the microscope objective to provide a focused illumination spot in the center of the
optical field. The lasers are mounted in a closed system in the cabinet. Light is
directed from the lasers to the light path through the Laser Optic module that
mounts on the back of the Fiber Optic module.
Note The QLM is not an available option for personalDV.
If your system has the QLM hardware module, you can use softWoRx to analyze
Photokinetic (photo-bleaching and photo-activation) experiments.
QLM Laser
Optics
Module
QLM Laser
Module
The QLM module provides software to control the lasers and to analyze the data
obtained from these experiments. For more about QLM, see the DeltaVision QLM
Getting Started Guide.
Total Internal Reflection Fluorescence (TIRF) Module
The TIRF module includes the Olympus TIRF component and special adapters
required to install it on a DeltaVision system. (TIRF requires a system with a QLM
hardware module.)
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Note TIRF is not an available option for personalDV.
TIRF Component
Note TIRF is an optical sectioning technique that limits fluorescence imaging to a thin area
at the surface of a specimen, typically 100 nm or less, resulting in an enhanced signal-tonoise ratio and higher imaging contrast.
EM CCD Camera
The EM CCD Camera is an optional component.
Note The EM CCD Camera is not an available option for personalDV.
The addition of the Cascade II with Electron Multiplying CCD (EM-CCD)
technology further improves the signal-to-noise-ratio (SNR) of the DeltaVision
system.
Unlike conventional CCD cameras, an Electron Multiplying CCD (EMCCD) is not
limited by the readout noise of the output amplifier, even when operated at high
readout speeds. A solid state Electron Multiplying (EM) register allows weak
signals to be multiplied before any readout noise is added by the output amplifier.
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The EM CCD camera can be operated either as a high-performance EMCCD
camera for unparalleled low-light-level sensitivity, or as a traditional, nonelectron-multiplying CCD camera.
Applications for the EM CCD camera include:
•
Low-light Fluorescence
•
Total Internal Reflection Fluorescence Microscopy (TIRF)
•
Single-molecule Fluorescence
Analysis Workstations
You can purchase two types of Analysis workstations: the softWoRx Linux
workstation and the softWoRx Suite Windows workstation.
The softWoRx Linux workstation includes all of the softWoRx Analysis modules,
including 3D Visualization tools, Colocalization, FRET Analysis, FRAP Analysis,
Intensity and Distance Measurement, and Modeling.
The softWoRx Suite Windows workstation includes the softWoRx Browser,
softWoRx Explorer, Modeling and 3D Visualization (powered by Bitplane), and
Deconvolution tools.
Multiplexed Wavelength Module
The optional Multiplexed Wavelength module for the DeltaVision systems allows
you to perform nearly simultaneous two-channel imaging without the drawbacks
associated with true simultaneous two-channel imaging. This option uses two
shuttered illumination sources and a dual-band emission filter to eliminate filter
wheel movement between channels, and therefore greatly reduce the time
required for the DeltaVision system to acquire a set of two-channel images. The
combined light path ensures no registration artifacts are introduced and
independent excitation of probes helps to ensure minimal crosstalk.
Software
softWoRx Explorer
®
softWoRx Explorer is a cross-platform image viewer that is available for many
commonly used operating systems.
softWoRx Explorer allows you to view and explore DeltaVision images and images
from other sources that contain spatial, temporal, and spectral ranges. In addition
to displaying data in the X and Y plane, you can scroll through Z sections and
time-lapse data. Individual spectra (i.e., channels or fluorescent wavelengths) can
be hidden or displayed in a variety of colors.
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softWoRx Suite for Windows Option (for personalDV)
softWoRx Suite is a Windows-based ensemble of software developed by Applied
Precision in collaboration with Bitplane AG. It provides sophisticated multidimensional data visualization, analysis, image restoration, image correction, and
image viewing management, all within an easy-to-use streamlined browser
interface.
softWoRx Suite Advanced Option
The Advanced Option for softWoRx Suite includes two sophisticated analysis
features: 4-D Particle Tracking and ImarisColoc. softWoRx Suite Advanced Option is
offered by Applied Precision in collaboration with Bitplane, AG.
4-D Particle Tracking allows users to observe temporal changes of objects. This
tracking module offers a choice of methods for both detection and tracking and
allows analysis and measurement of various object properties.
The Colocalization feature enables users to easily isolate, visualize, and quantify
regional overlap in 3D and 4D images. Results can be presented in two ways—as a
new 3D or 4D channel or as a statistical report.
softWoRx Data Management Solution (DMS) Support
softWoRx DMS provides a functional infrastructure for the storage of biological
images and their associated metadata in a centralized database. softWoRx DMS is a
standard component included with DeltaVision Core, but for personalDV this
software is optional.
Consumable Parts
Common to 100-120 V and 220-240 V Systems
Component
Fuse for Component
API Part Number
IC/MIC
6.3A 250V UL High Break
Capacity
19-170045-000
Microscope
T5AH
Used in some older
DeltaVision systems
Bulbs
Component
API Part Number
250W xenon Arc Lamp Bulb
34-100390-000
LED Transmitted Light (must replace entire assembly)
52-851243-000
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British Power Cord
Component
Fuse for Component
API Part Number
Power cord with Bussman
TDC180-10A built into plug.
(Must meet British Standard
BS1362.)
10A
19-210046-000
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8
8
Changing Cameras and Filters
This chapter provides the following instructions for cameras and filters:
„
Changing Cameras
„
Using Live Cell or Custom Filter Wheel Modules
„
Changing Filter Wheel Modules
„
Calibrating Filter Wheels
Changing Cameras
DeltaVision Core includes a High-speed Camera as a standard component. If you
have the optional EM CCD camera, you can change cameras to better meet your
imaging needs.
These instructions show how to change the camera and how to select the new
camera in Resolve3D.
Contamination from fingerprints or dust on the camera window
orCAUTION:
inside the Emission filter wheel will degrade image quality.
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To change the camera:
1. Remove the camera cover by lifting it up and sliding it away from the
microscope.
2. Remove the camera by pushing in and then pulling up on the camera end. You
do not need to remove cables from the camera.
3. Remove the other camera from the spare camera tray and place the camera
that you just removed on the camera tray.
Always place the camera in the camera tray when it is not
CAUTION:
secured to the system. Dropping the camera will cause severe damage.
4. Install the new camera by sliding it into place and then pushing in and
pressing down on the end of the camera.
5. Replace the camera cover.
To select the camera:
1. In the Resolve3D window, click Settings to open the Resolve3D Settings dialog
box.
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Chapter 8: Changing Cameras and Filters
2. On the Imaging tab, click the Camera list and select the camera that is
currently installed.
Notes
#1 The EM CCD camera is listed twice in the Camera list:
CASCADE2_512 Conv./… sets the camera in Conventional mode.
Cascade2_512 EMCCD/… sets the camera in electronmultiplication mode.
#2 When the camera is changed, the binning number is reset to 1.
#3 If the camera cables loosen or are accidentally disconnected, you may
need to restart the IC software. To reseat the cables, consult the camera
documentation.
Using Live Cell or Custom Filter Wheel Modules
A filter wheel module includes an Excitation filter wheel, an Emission filter wheel,
and an Eyepiece filter wheel.
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DeltaVision Filter Wheel Module
Excitation (10-position) filter wheel
Emission filter wheel
Eyepiece filter wheel
A filter wheel module includes the Excitation, Emission, and Eyepiece filter wheels
and can support up to six filter pairs.
If your system has an alternate filter wheel module, you can swap modules to
meet your imaging needs.
You can purchase a module of Live Cell filter wheels that includes factoryinstalled filters. You can also purchase a module of empty filter wheels and
customize it with your own filters.
Changing Filter Wheel Modules
Changing the filter wheel modules for your system includes changing the
Eyepiece and Emission filter wheels and, depending on how many filter pairs are
supported, may also include changing the Excitation filter wheel.
If you purchased a filter wheel module as a separate component after you
purchased your system, you must configure the instrument controller for the new
module the first time that you use it (see the instructions included with your filter
wheel module).
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To change the Eyepiece filter wheel:
1. Disconnect the Position Sensor cable that connects the filter wheel to the
IC/MIC. DeltaVision displays a message that indicates the cable is removed. Do
not close this message.
Position Sensor Cable
2. Holding the oculars in one hand, use a 3 mm hex key (Olympus provides one
with the microscope) to loosen the set screw that holds the oculars to the
Eyepiece filter wheel and set the oculars on the table.
set screw
Loosening the set screw and removing the oculars
3. Loosen the set screw that holds the Eyepiece filter wheel to the beveled mount
on the stand and remove the Eyepiece filter wheel.
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set screw
Removing the Eyepiece filter wheel
4. Place the new Eyepiece filter wheel on the beveled mount on the stand and
tighten the set screw that holds it in place.
5. Connect the Position Sensor cable to the new Eyepiece filter wheel. The
IC/MIC will automatically start to re-initialize the EM and EX filter wheels
even though the current EX and EM filter wheels have not yet been replaced.
You can ignore this re-initialization.
6. Place the oculars on the microscope and tighten the set screw that holds them
in place.
7. If the IC/MIC is ON, click OK on the message box that indicates the cable is
removed.
If the IC/MIC is OFF, you will need to manually update the filter set in the
Resolve3D | Settings | Misc tab window.
WARNING: Before you unplug any motor cables, make sure that the xenon lamp
and the IC/MIC are turned OFF.
To change the Emission filter wheel:
1. Turn off the IC/MIC.
2. Remove the camera cover. Then remove the camera by pushing in and pulling
up on the camera end as shown below.
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3. On the Emission filter wheel motor, disconnect the cable that connects the
motor to the IC/MIC. Use a 3 mm hex key to loosen the set screw that holds the
Emission filter wheel to the microscope.
Cable to
IC/MIC
Set Screw
4. Remove the Emission filter wheel.
IC/MIC Cable
Connector
5. Install the alternate Emission filter wheel. Tighten the Emission filter wheel set
screw and connect the cable from the IC/MIC to the new Emission filter wheel.
6. Reinstall the camera by sliding it into place and then pushing in and pressing
down on the camera.
Note If you still need to change the EP or EX filter wheels, do so now before
continuing with this procedure.
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7. When you are finished changing filter wheels, restart the IC/MIC.
8. If Resolve3D does not automatically display the correct filter module, there are
two ways to expressly let the system know you have a new filter set:
•
From the Resolve3D main menu, select Settings, change to the Misc tab,
and select the current filter set.
•
Unplug and then re-plug in the Eyepiece filter wheel position sensor cable
(see below). The system will automatically recognize which filter set the
Eyepiece filter wheel belongs to and update accordingly.
Note that it is not necessary to remove the oculars before plugging or
unplugging the Eyepiece filter wheel.
To change the Excitation Filter Wheel (if necessary):
1. Ensure that the IC/MIC is turned off, and make sure that the xenon arc lamp
has been off and allowed it to cool.
2. Loosen the two silver thumb screws at the top and bottom of the filter wheel
housing.
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Thumb screws should
loosen without the
use of tools, but can
also be removed
with a flat-blade
screwdriver if
necessary.
Loosening top thumb screw from filter wheel housing
Loosening bottom thumb screw from filter wheel housing
3. Loosen the Focusing Lens housing thumb screw to disengage it from the
beveled support on the Excitation filter wheel housing and gently slide the
filter wheel housing outward from its mounted position.
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Removing the Excitation Filter Wheel housing from its mounted position
4. Disconnect the cable to the IC/MIC from the Excitation filter wheel and connect
it to the alternate Excitation filter wheel.
Disconnecting the IC/MIC cable from the Excitation Filter Wheel
5. Slide the alternate Excitation filter wheel housing back into its mounting
position by pulling back on the Focusing Lens assembly as shown.
Pulling back the Focusing Lens housing and re-engaging the filter wheel
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Chapter 8: Changing Cameras and Filters
6. Re-engage the Focusing Lens housing into the beveled support on the new
Excitation filter wheel housing and align and tighten the two thumb screws at
the top and bottom of the Excitation filter wheel housing.
7. Tighten the thumb screw on the Focusing Lens housing.
Note If you still need to change the EP or EM filter wheels, do so now before
continuing with this procedure.
8. When you are finished changing filter wheels, restart the IC/MIC.
9. If Resolve3D does not automatically display the correct filter module, there are
two ways to expressly let the system know you have a new filter set:
•
From the Resolve3D main menu, select Settings, change to the Misc tab,
and select the current filter set.
•
Unplug and then re-plug in the Eyepiece filter wheel position sensor cable
(see Step 8 in the previous procedure). The system will automatically
recognize which filter set the Eyepiece filter wheel belongs to and update
accordingly.
Calibrating the Filter Wheels
Calibration initializes the filter positions and ensures that the filters are centered in
the filter wheel openings.
If you notice poor light transmittance or poor image quality, one possible cause is
a misaligned filter wheel. The filter wheel calibration re-establishes the zero
position of the filter wheel for the Instrument Controller.
To calibrate the Neutral Density, Excitation, and Emission filter wheels:
1. On the Resolve3D window, click Settings to open the Resolve3D Settings
dialog box. Then click the Misc tab.
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2. Verify that the Excitation, Emission, and Eyepiece filter wheel fields all
display the same filter wheel module name (if any of the names are different,
you may need to select or replace one or more of the filter wheels).
3. Press Save Settings and close Resolve3D.
4. Click on the Instrument Controller icon on the workstation desktop as shown.
5. Log on to the Instrument Controller with the user name worx and password
4delta if prompted.
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Chapter 8: Changing Cameras and Filters
6. Remove the filter wheel to be calibrated (Excitation, Emission, or Neutral
Density) from the system so the opening is visible.
7. Press Alt + H. The filter wheels will rotate as they are being initialized. Hold
the EX or ND filter wheels to stabilize them and help ensure that motor torque
does not cause them to fall off of the work surface.
You are then prompted with, “Calibrate the EX filter wheel (y/n).”
8. If you are calibrating the Excitation Filter Wheel, press y. Otherwise press n to
move to the next filter wheel (ND or EM).
When you press y, the following message is displayed:
Pressing the up or down arrow keys advances the filter wheel one complete
position. The left and right arrow keys move the filter wheel in small
increments.
9. Find the Home filter position on the filter wheel. The Home filter position is
marked on each of the filter wheels as follows:
•
EX = 1
•
ND = 1
•
EM = 0
The position is stamped on the surface of the filter wheel and may be viewed
through the filter wheel hole.
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Filter position not centered
Filter position centered
10. Once the filter wheel is centered, press Enter. After all necessary filter wheels
have been centered you are prompted with, “Save the new filter
offsets (y/n).”
11. If you made a mistake during calibration you can press n and start the process
over, otherwise press y to save your offsets.
12. Re-install the filter wheels in the system.
13. To shut down the Instrument Controller, go to the Start tab and select
Windows Security. Then select the Shutdown option (as shown).
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Chapter 8: Changing Cameras and Filters
The IC/MIC will power down.
14. Power the IC/MIC back on and boot the DeltaVision as normal.
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9
9
Maintenance
This chapter provides the following instructions for the basic maintenance of the
system:
„
Shutting Down and Starting the System
„
Replacing the Xenon Bulb
„
Replacing the Transmitted Light
„
Aligning the Illumination Path
„
Replacing IC/MIC Fuses
„
Cleaning
„
Moving the System
Shutting Down and Starting the System
Use the following instructions for shutting the system down during a power
outage or other occasions that require total shutdowns.
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DeltaVision Power Switches
The main DeltaVision power switches are shown below. (The Master Switch on the
isolation transformer that plugs into the wall is not shown.)
Monitor Switch
IC/MIC Switch
Workstation Switch
Main Components
Switch
QLM Switch
(optional)
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Guidelines for Using Switches
Main Components
Use this switch to turn power on and off for the DeltaVision Workstation,
Instrument Controller and Microscope Interface Chassis (IC/MIC), the Fast
Camera Power Supply, and the DeltaVision Microscope.
Workstation, IC/MIC, and Monitor
Leave these switches on except on rare occasions (such as power outages) when
you need to shut down the entire system.
Shutting Down the System
In some situations, such as power outages, you will need to shut down the entire
DeltaVision system.
To shut down the DeltaVision system:
1. Save all data on the workstation.
2. Turn off the xenon light source using the bulb
icon on the Resolve3D
main menu. Clicking the icon will switch it to the off
state.
3. On the softWoRx menu bar, choose File | Exit. Then exit all other workstation
applications.
4. From the main menu button, choose Logout and then Shut Down. Wait until
the monitor displays Power Down.
5. Press the power button on the IC/MIC once to shutdown.
6. Turn off the monitor.
7. Turn off the power strip bar switch.
Note For personalDV, there is no power strip bar. For personalDV, skip this step.
8. Clean the objective (see Turning DeltaVision Off on Page 30).
9. Lower the objective.
Starting the System
Use the following instructions to start the system after a total shut down.
To start the DeltaVision System:
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1. Turn on the power strip bar.
Note For personalDV, there is no power strip bar. Begin the power-up process with
Step 2.
2. Turn on the IC/MIC.
3. Turn on the workstation.
4. Turn on the monitor.
5. Follow the instructions for turning on DeltaVision on Page 19.
Replacing the Xenon Bulb
WARNING: Ensure the xenon lamp is off and has had plenty of time to cool
before starting this procedure. Refer to the “Xenon Lamp Safety” section in
Chapter 2 of this manual for details on safety issues and proper disposal of the
lamp.
Follow these steps to replace the xenon bulb on DeltaVision:
1. If the system is on, exit Resolve3D and ensure that the IC/MIC is off prior to
proceeding. The fan on the lamp housing must be off before you begin this
procedure.
2. Loosen the three hex screws in the flange of the xenon lamp housing.
hex screws
3. Gently slide the lamp housing away from the flange to remove it from the
DeltaVision excitation module.
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Chapter 9: Maintenance
4. Loosen the two thumb screws on the opposite end of the xenon lamp housing.
5. Gently slide the internal lamp mechanism from the lamp housing.
6. Remove the center clip from the internal lamp mechanism as shown.
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7. Lift the bulb assembly (small black box) from the two supporting pins in the
lamp mechanism.
8. Replace the xenon bulb assembly with a new one (Part #34-100390-000).
9. Replace the clip around the internal lamp assembly and gently slide the
internal lamp mechanism into place within the lamp housing.
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10. Tighten the two thumb screws on the end of the lamp housing.
11. Place the open end of the lamp housing over the flange on the DeltaVision and
tighten the three hex screws as shown.
hex screws
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12. Turn the DeltaVision on as usual and start Resolve3D.
13. Before resetting the bulb age, write down the age of the bulb you just replaced.
This will help you to keep track of when you may need to replace the next one.
14. Open the Imaging tab on the Settings window and reset the bulb age.
Bulb age
Reset bulb
age
60
Note For DeltaVision systems with the Multiplexed Wavelength option installed, the
procedure for changing the xenon bulb in the secondary lamp housing is identical
to the procedure described above.
Replacing the Transmitted Light
To replace the LED transmitted light assembly:
1. If the DeltaVision system is on, exit Resolve3D and ensure that the IC/MIC is off
prior to proceeding.
2. Loosen the two hex screws on the top and right sides of the transmitted light
housing as shown.
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hex screws
3. Carefully remove the transmitted light assembly from the housing.
4. Disconnect the other end of the attached cable from the Transmitted
Illumination Source connection on the back of the IC/MIC (upper left-hand
corner when facing the back of the unit; shown below).
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Transmitted Illumination Source Connection (back of IC/MIC)
5. Re-insert the new transmitted light assembly (Part #52-851243-000) into the
housing and tighten the two hex screws.
6. Re-connect the new transmitted light assembly cable to the Transmitted
Illumination Source connection on the back of the IC/MIC (shown above).
Aligning the Illumination Path
To get the best images with DeltaVision, make sure that the light path is aligned in
both Köhler and Critical positions. Use the instructions in this section to test and
adjust alignment. This section contains the following topics:
„
The Fiber Optic Module describes the settings and adjustments on this key
component. You’ll use the Fiber Optic Module to switch from Köhler to
Critical Illumination. You’ll also use the Fiber Optic Module to align the
illumination path with the microscope.
„
Before You Check or Adjust the Illumination Path Alignment lists the settings and
filters that you’ll need to select before you check or adjust alignment.
„
Checking Illumination Path Alignment shows how to check the alignment in both
Köhler and Critical positions. To ensure optimal performance, check the
alignment every two weeks. If acquired images seem dim, you should also
check alignment to verify that the light is reaching the sample.
„
Path Alignment shows how to align the illumination path in both Köhler and
Critical positions.
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The Fiber Optic Module
Use the Fiber Optic Module to align the light path from the fiber optic cable to the
Fluorescence Illuminator. This module allows you to adjust the tilt, horizontal, and
vertical orientation of the light path.
Figure 7: The Fiber Optic Module
Photo sensor Port
Tilt Screws
Critical Spring
Köhler Spring
Locking Knob
Y Adjustment Screw
X Adjustment Screw
Adjustment or Lock
Use to
Tilt Screws
Adjust the tilt of the Fiber Optic Module body.
Photo-sensor Port
Connect the fiber optic cable to the photo-sensor detector
in the IC/MIC.
Köhler Spring
Set the Köhler illumination position. Two locking screws hold
it in position.
Critical Spring
Set the Critical illumination position. Two locking screws
hold it in position.
Locking Knob
Temporarily lock the position of the Focus Control.
Y Adjustment Screw
Adjust the vertical position of the cable.
X Adjustment Screw
Adjust the horizontal position of the cable.
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Before You Check or Adjust Illumination Path
Alignment
Before you check or adjust alignment, remove all sources of blockage to the light
path. Then verify the following settings to make sure that the various lenses and
controls are in the correct positions (you can feel the controls snap into place when
they are set in position).
„
The Fluorescence Illuminator Slider on the back of the microscope is in an
open position.
„
The standard (Sedat) color set polychroic filter (usually labeled) is selected and
in position on the Filter Cube Turret. (Alignment may vary between polychroic
mirrors. Factory alignment is performed on the standard color set.)
„
The shutter on the Filter Cube Turret is open (O).
„
The Field Stop Aperture lever is pulled out all the way to close the aperture.
„
The Magnification Changer is completely in (the 1X position).
„
The 60X objective is in its lowest position.
„
The Beam Selector is on the
„
The Eyepiece Lens Selection Wheel is in the open (O) position.
(eye) position.
Eyepiece Focus
Eyepiece Lens
Selection Wheel
Centering
Telescope
Focus
Eyepiece Filter Wheel
Beam Selector
Z Focus
60X Objective
Fluorescence
Illuminator Slider (back
view)
Field Stop Aperture
And Adjustment Screws
Filter Cube Turret
Auxiliary Magnification
Changer
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Checking Illumination Path Alignment
To provide an optimum light source, the tilt and position of the Fiber Optic
Module must be aligned with the microscope. This procedure shows how to
perform an “inside-outside” focus test to make sure that the module is properly
aligned. It also shows how to perform “in focus” tests to make sure that the
position of the module (distance from the microscope) is correct for Köhler
Illumination, and if required, for Critical Illumination.
To check alignment:
1. Move the Beam Selector to direct the light to the eyepiece and make sure that
the 60X objective is lowered and in position as shown previously in
Before You Check or Adjust Illumination Path Alignment.
2. Use the Eyepiece Filter Wheel to select the FITC eyepiece filter.
3. In the Resolve3D window, select the following filters:
In this Field
Select
Excitation
TRITC
Emission
FITC
%T
0.1%
4. Apply immersion oil to the objective and mount the mirror slide.
5. On the keypad, press EX SHUTTER to open the Excitation shutter.
6. Use the Eyepiece Focus on the oculars to focus on scratches on the mirror slide.
7. Fully close the Field Stop Aperture and slowly rotate the fine Z Focus knob in
and out of focus in both directions. The light should evenly expand around the
aperture. If it flares in one direction, go to Step One: Center the Field Stop
Aperture on Page 144.
8. Check for alignment in Köhler Illumination as follows:
a. Open the Field Stop Aperture. Then loosen the Locking knob and slide
the focus end of the Fiber Optic Module into the Köhler Illumination
position (see Page 84). Then tighten the Locking Knob.
b. Switch the Eyepiece Selection Wheel to the Centering Telescope
position (CT). Use the Centering Telescope Focus control to focus on
the objective back aperture (the outside edge of the middle circle), as
shown below.
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Middle Circle
Inside
Circle
Focus on the outside edge of
the middle circle
After you focus on the outside edge of the middle circle, the outside edge of
the inner circle should also be in focus and the centers of the inside and middle
circles should be aligned.
c. If the image is not if focus, go to Step Three: Set the Köhler Spring Position
on Page 147.
9. If you are using Critical Illumination, check for alignment by moving the
eyepiece to the Open position (O), loosening the Locking knob, and sliding the
Focus Control on the Fiber Optic Module out to the Critical Illumination
position. Then tighten the Locking Knob. Both the scratches and the edge of
the circle should be roughly centered and in focus, as shown below. If not, go
to Step Four: Set the Critical Illumination Position on Page 148.
10. If you observe any alignment problems in Steps 7, 8, or 9, use the instructions
in the next section to align the illumination path.
Path Alignment
This procedure shows how to align the Fiber Optic Module and the fiber optic
cable with the light path. It also shows how to set the positions of the two springs
that allow you to switch between Köhler and Critical Illumination.
Step One: Center the Field Stop Aperture
1. Apply immersion oil to both the objective and the mirror slide and mount the
mirror slide.
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2. Use the Eyepiece filter wheel to select the FITC eyepiece filter. In the
Resolve3D window, set the other filters for alignment as follows:
In this Field
Select
Excitation
TRITC
Emission
FITC
%T
0.1%
3. Press EX SHUTTER on the keypad to open the EX shutter.
4. Use the Z focus knob to focus on the scratches on the mirror slide.
5. Pull out the Field Stop lever all the way to close the Field Stop aperture.
6. Use the Olympus 3mm hex key to adjust the two Field Stop Centering Screws
until the Field Stop Aperture is centered on the reticle.
Field Stop lever with
Centering Screws
The reticle should appear in the center of the octagon as shown below.
7. Fully open the Field Stop aperture.
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Step Two: Adjust the Tilt of the Fiber Optic Module Body
1. Make sure that the Beam Selector on the microscope base is set to direct the
light to the eyepiece and verify that the 60X objective is set in its lowest
position. The Z focus knob should be turned until it stops (with the objective
fully lowered).
2. Remove the oil from the objective (See Cleaning on Page 151) and remove the
Repeatable Slide Holder.
3. Place a piece of paper on the stage with a piece of glass on top to keep it flat.
4. Press EX SHUTTER on the keypad to open the EX shutter.
5. In Resolve3D, set the neutral density filter to 100% light transmittance.
6. Pull the Field Stop lever all the way out to close it. The bright area on the paper
should be circular and centered in the dim field.
7. Use a 3 mm hex key to loosen the Tilt screws and adjust the screws so that the
bright spot on the paper is circular and is centered in the dim field as shown in
the previous step.
Tilt
Screws
8. Tighten the Tilt screws.
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Step Three: Set the Köhler Spring Position
1. Move the Eyepiece Selection Wheel from the open (O) to the Centering
Telescope (CT) lens.
2. Use the Centering Telescope Focus on the ocular to focus on the objective back
aperture (the outside edge of the middle circle).
Middle Circle
Inside
Circle
Focus on the outside edge of
the middle circle
3. Loosen the Locking Knob and use a 2.5 mm hex key to loosen the two Köhler
Spring screws. Then move the Focus Control back and forth until the edge of
the inner circle is in focus. After you focus the edge of the inner circle, the
edges of both the inner and middle circles should be in focus.
Middle Circle
Inside
Circle
4.
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focus
Holding the Focus Control still, tighten the Locking Knob.
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5.
Slide the Köhler Illumination spring so that it is in the groove on the Focus
Control and tighten the Köhler spring locking screws to set the Köhler
position.
Step Four: Set the Critical Illumination Position
1. Move the Eyepiece Selection Wheel to Open (O) and fully open the field
aperture.
2. Apply immersion oil to both the objective and the mirror slide and mount the
mirror slide.
3. Loosen the Locking Knob. Then move the Focus Control on the Fiber Optic
Module out approximately 5 mm and then slide it back and forth until the
edge of the circle snaps into a sharp focus. You should see both the scratches
and the edge of the circle in focus as shown below.
Note Critical Illumination is typically 5mm out from Köhler Illumination.
4. Tighten the Locking Knob.
5. Check the focus. If the edges on the circle are not crisp, loosen the locking
knob, re-adjust the Focus Control, and tighten the Locking Knob again.
6. Slide the Critical Illumination spring into the groove on the Focus Control and
tighten the Critical Illumination spring screws with a 2.5 mm hex key.
Step Five: Adjust and Test the Alignment
1. Fully close the Field Aperture and slowly rotate the microscope Z focus knob
in and out of focus. The light should evenly expand around the aperture.
„
a. If the light does not expand evenly around the aperture, make sure that:
•
•
•
•
„
The shutter is open.
The Eyepiece Selection Wheel is set to 0.
The mirror slide is mounted in immersion oil and in focus.
The Fiber Optic Module is in Critical position (see Step 3).
b. Use a 1.5 mm hex key to adjust the X and Y adjustment screws on the top
and left sides of the Fiber Optic Module until the image is centered in the
reticle as shown below:
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2. Adjust the center alignment in the Köhler position as follows:
„
a. Slide the Focus Control to the Köhler position, set the eyepiece to CT, and
use the Centering Telescope Focus to focus on the objective back aperture.
You should see the area between the back aperture of the objective lens (the
darker area) and the fiber tip (bright green).
„
b. Use a 1.5 mm hex key to adjust the X and Y adjustment screws on the top
and left sides of the Fiber Optic Module until the fiber tip image is at a point
that is midway between its original center and the actual center (the point
where the centers of the two circles are aligned).
3. Adjust the tilt of the Fiber Optic Module as follows:
„
a. Make sure that the viewing knob on the microscope base is set to direct the
light to the eyepiece and verify that the 60X objective is in detent and in its
lowest position.
„
b. Remove the oil from the objective.
„
c. Place a piece of paper on the stage with a piece of glass on top to keep it flat.
„
d. Press EX SHUTTER on the keypad to open the EX shutter and set the neutral
density filter to 100% light transmittance (in Resolve3D).
„
e. Use a 3 mm hex key to adjust the X and Y tilt screws so that the bright spot
on the paper is circular and is centered in the dim field.
4. Repeat steps 1-3 in this procedure until the image is centered in both the
Critical and Köhler positions.
Note Aligning for Critical and Köhler illumination is an iterative process. The
alignment will improve with each iteration.
5. When the image is centered, tighten the clamping screws on the Fiber
Alignment Disk.
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6. Fully close the Field Aperture and slowly rotate the microscope Z focus knob
in and out of focus. The light should evenly expand around the aperture. If the
light tends in one direction, call the API Customer Service Hotline (1-800-8625166).
7. Move the Fiber Optic Module between the Köhler and Critical position several
times and make sure that the positions are repeatable (within about 1%). If the
Köhler and Critical positions are not repeatable, call the API Customer Service
Hotline (1-800-862-5166).
Replacing IC/MIC Fuses
Follow these instructions to replace a fuse in the Microscope Interface Chassis. To
replace fuses for other components, follow the instructions in the manuals that are
provided for those components.
CAUTION: Installation of improperly rated fuses can cause damage to the
system.
To replace a fuse:
1. Shut down the system.
2. Unplug the power cord on the back of the IC/MIC.
3. Remove the fuse holder.
4. Test the fuses with a continuity meter.
5. Replace any bad fuses with 5X20mm 6.3A 250V UL high break capacity fuses
(API P/N 19-170045-000).
6. Install the fuse holder.
7. Plug in the power cord.
AppliedPrecision
Chapter 9: Maintenance
Cleaning
Most system surfaces are best cleaned with a lint-free cloth or lint-free swabs and
spectroscopy-grade isopropyl alcohol or chloroform. Avoid contaminating the
cleaning solution by never reusing the cleaning cloth or swabs. Operators should
be trained in the handling of flammable liquids such as alcohol. Material Safety
Data Sheets (MSDS) should be maintained for the cleaning solutions, as with any
hazardous material.
The exceptions to this cleaning practice are the polychroic mirror and the optical
filters. These components should be cleaned with low-pressure air. For example,
use a bulb designed for cleaning camera lenses, which blows air across the surface.
Do not use high pressure. Do not use canned air, as this often leaves a fluorescent
residue.
To clean the microscope, follow the instructions in the manufacturers’ manuals
that are provided for these components.
Improper cleaning of the polychroic mirror and the optical
CAUTION:
filters will result in damage.
Moving the System
If you need to move your DeltaVision system, call Applied Precision for
instructions.
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A
Appendix A: The Immersion Oil Kit
The immersion oil kit is a collection of oils with refractive indexes that range from
1.500 to 1.534. Use of the correct immersion oil decreases the spherical aberration
in the image data.
•
For DeltaVision Core, the immersion oil kit includes eighteen oils that range
from 1.500 to 1.534, in increments of 0.002.
•
For personalDV, the immersion oil kit includes six oils that range from 1.512
to 1.522, in increments of 0.002.
Many factors influence the optimum refractive index of the immersion oil,
including specimen preparation, temperature, humidity, and atmospheric
pressure.
The Oil Calculator
In order to calculate the desired refractive index, softWoRx is equipped with the
Lens Information function. This function is located in the Utilities menu in
softWoRx. It can also be accessed from Resolve 3D by clicking the Info button. The
following parameters are explained here to help you enter the appropriate
information and use the resulting calculations.
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Distance from Coverslip to Specimen (microns)
Establishes the distance from the surface of the coverslip to the desired focal plane.
Temperature
Defines the temperature of the specimen and the immersion medium.
Specimen Refractive Index
Defines the refractive index of the specimen, which is usually that of the mounting
medium. In some cases, the specimen itself contributes significant refraction.
Recommended Refractive Index
Displays the resulting optimal refractive index of the immersion oil. Actually
experimenting with oils with refractive indexes very close to this value is the best
way to select the optimal oil.
Resolution Ratio
Displays the ratio between the Z resolution and the XY ratio. This serves as a
reference to the degree of Z elongation.
Maximum XY Pixel Size
Displays the maximum recommended XY pixel size for deconvolution.
Recommended Z Step
Displays the smallest possible Z step for this objective. Choosing a smaller Z step
will add to the size of the image file, but will not improve image quality.
AppliedPrecision
B
Appendix B: Troubleshooting
This appendix was designed to help you diagnose and correct the most common
problems encountered on the DeltaVision system. It covers two types of
troubleshooting tasks:
„
Diagnosing System Problems
„
Analyzing Reasons for Poor Image Quality
If you are unable to correct a problem, fill out the DeltaVision Problem Report
Form at the end of this appendix and either e-mail it to [email protected] or fax it to
425-557-1055, attn: Bio Service Hotline.
Diagnosing System Problems
Troubleshooting the Controller
The following table shows the most common Instrument Controller problems and
their resolutions.
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Table B-1: Controller Troubleshooting Chart
Indication
Cause
Correction
Encoder Error when
initializing stage.
Poor cable
connection.
Power down system, including IC/MIC.
Reseat X, Y, and Z motor cables.
Reseat motor cables on excitation
module.
Power up system.
Stalled sequence of
images.
Computer
misallocated
memory.
Exit the software and restart IC/MIC.
Troubleshooting the Workstation
Other system troubles are indicated by messages or readings in the software. The
Resolve 3D message window displays Resolve 3D activity. Observe the messages
in this window when troubleshooting. This table shows possible problems and
corrective actions.
Table B-2: Workstation Troubleshooting Chart
Indication
Cause
Correction
"File system full"
message when
trying to save
images.
There is no more
storage space for
image data.
Delete unwanted files.
"Camera not found"
message.
Power up sequence
was incorrect.
Shut down the system and then restart
it using the steps described in
Chapter 9: Maintenance.
Resolve 3D settings
are not updating
changes made
using the keypad or
joystick (for
example, the filter
selection, or Z
position).
Lack of
communication
between the
Instrument
Controller and
Workstation.
Shut down the system and then restart
it using the steps described in Chapter
9: Maintenance.
At login, user name
not recognized.
User has not been
added.
Add user. See the softWoRx Imaging
Workstation User's Guide.
Save image files to CD or DVD or LAN.
See softWoRx Imaging Workstation
User's Guide for more information.
Analyzing Reasons for Poor Image Quality
The following table documents the most common acquisition difficulties and
abnormalities in image data.
Table B-3: Image Quality Troubleshooting Chart
Indication
Cause
Correction
Dim images or long
exposure times.
Poor illumination.
Fully open field aperture.
Align xenon lamp and fiber optic cable
as shown in Replacing the Xenon Bulb
AppliedPrecision
Appendix B: Troubleshooting
Indication
157
Cause
Correction
on Page 134.
Ensure that filter cube turret is locked in
position on rail mount.
Ensure shutter on filter cube is open.
Ensure slider behind microscope is
seated in an open position.
Ensure proper filter cube is in position
and seated in detent.
Dim illumination.
When fiber optic
cable and focusing
lens are removed,
the projected light
does not form a
circle.
Filter wheel(s) out
of alignment.
Dark, out of focus
spots on image.
Dust interference.
Clean polychroic filter, emission filter,
and camera window using low-pressure
air. Do not use canned air. See Page 151
for further recommendations regarding
cleaning system components.
Image is distorted
around edges or
throughout.
Occlusion seems to
creep in toward
center.
Condensation on
camera window,
possibly due to
improper camera
temperature.
Clean camera window using lowpressure air. Do not use canned air. See
Page 151 for further recommendations
regarding cleaning system components.
Brightness of Z
section images
varies greatly
A broken Photo
sensor cable
Disconnect the Photo sensor cable and
the EX module cable.
Shut down and start up as described on
Pages 19 and 30 or unplug and plug in
the Eyepiece filter wheel. This will reset
the home position of the filter wheels
and align filter wheel position.
Calibrate filter wheels following
instructions on Page Error! Bookmark not
defined..
Check camera temperature in
Resolve3D. Consult camera
documentation for proper setting.
Connect the Photo sensor cable to the
EX module.
Set the Excitation filter to FITC or some
other visible light.
Open the EX shutter.
Bend the cable and examine it for light
leaks. If you observe a light leak, replace
the cable.
Direct the light to a wall. If you observe
inconsistencies in the light output as you
bend the cable, replace the
Photosensor cable.
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Table B-3: Image Quality Troubleshooting Chart Cont’d
Indication
Cause
Correction
Image has a traveling
light or bubble.
Air bubble in
immersion oil.
Clean front and back surfaces of
objective and coverslip.
Reapply immersion oil and restart
experiment.
Interference in image
data.
Possibly dirt, dust,
oil, or air bubble.
Clean front and back surfaces of
objective and coverslip.
Very bright image or
camera saturation
message.
Camera saturation.
Use lower exposure time and/or
higher neutral density filter.
Z series shows uneven
or off center
illumination.
Poorly aligned
illumination.
Align xenon lamp and fiber optic
cable.
No image when
Acquire is pressed.
Knob at base of
microscope is
directing light to the
eyepiece.
Move knob to direct light to
camera.
Z series out of focus
and incomplete.
Stage was not
centered within
sample at start of
experiment.
Position stage in center of the
sample and run experiment again.
See Chapter 9, Maintenance.
AppliedPrecision
Appendix B: Troubleshooting
159
DeltaVision Problem Report Form
Research Facility:
Contact Person:
Phone:
System Serial Number:
softWoRx version: (use Help → Software Versions
to display)
E-mail:
Date:
Problem Encountered: Please write a detailed description, answering as many of the following questions
as possible.
Questions:
1
2
3
4
5
6
7
8
9
10
When did this first occur?
Was there any recent change or update to the system prior to the problem occurring?
What sequence of operations produces the problem?
What other programs were running when you encountered the problem?
What error messages, if any, were shown?
Is the failure the same each time or does it show different symptoms?
Does it occur consistently or is it random?
Does it go away after the workstation is re-booted?
Does it go away after the instrument controller is re-booted?
How often do you re-boot the workstation and instrument controller?
Additional Comments:
(See Page 2 for additional clarification issues)
Please supply the following log files:
Workstation: (/home/userName/softworx-logs/softworxlog.txt)
Instrument Controller: (c:\ic530_dv\log\IC_530.log or c:\ic525_dv\log\IC_525.log or
c:\ic540_dv\log\IC_540.log)
Please e-mail this form to [email protected] or Fax to: 425-557-1055, attn: Bio Service Hotline
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C
Appendix C: Acquiring a PSF
This appendix shows how to acquire a Point Spread Function (PSF) and convert it
to an Optical Transfer Function (OTF).
„
Acquiring a PSF shows how to measure a Point Spread Function.
„
Converting PSF to OTF shows how to convert the Point Spread Function to the
Optical Transfer Function that is required to process images.
Before You Start
Before you attempt to measure a PSF, check the OTF library included with
softWoRx to find out if the library provides an OTF for your objective.
Acquiring a PSF
To measure the point spread function (PSF), you need to optically section a
fluorescent bead. Since the properties of the objective lens are the most important
elements of determining a PSF, it is necessary to measure the PSF whenever new
lenses are added to your microscope. The deconvolution software adapts to the
PSF (actually the OTF) wavelength, so it is unnecessary to measure the PSF at
more than one wavelength.
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Note If you do not have the tools necessary to acquire a PSF, softWoRx includes a
utility that allows you to calculate a theoretical OTF based on the numerical
aperture of the camera lens, index of refraction, and emission wavelength. If the
aperture of the lens is lower than 0.75 N.A., the calculated OTF may work as well, or
even better, than a measured OTF. However, if the aperture is greater than 0.75
N.A., a measured OTF will generally give you better results. For information about
calculating an OTF, see the softWoRx online Help.
A well-measured PSF is a key to successful deconvolution. For this reason, make
sure that you:
„
Thoroughly check all imaging conditions.
„
Take the time you need to get a good signal-to-noise ratio in the image.
„
Completely scan the bead.
Tools
This procedure requires the following tools:
„
A clean and aligned 3-D microscopy system
„
A bead slide with 0.1μm, or smaller, fluorescent beads
„
A grid slide or other microscopic ruler
„
An immersion oil set, if appropriate (see Selecting the Correct Immersion Oil
on page 165.)
The following steps describe how to calculate pixel size, measure the PSF, and
obtain the corresponding OTF.
To calculate pixel size:
1. Place the new objective in the Objective Turret and set the Max image size, best
camera speed, and magnification slider at 1X.
2. Use the Eyepiece Filter Wheel to select the FITC eyepiece filter.
3. In the Resolve3D window, select the following filters:
In this Field
Select
Excitation
TRITC
Emission
FITC
%T
0.1%
4. Place the silicon target grid on the stage and focus it (9.995 um/square). Align
the grid image to the vertical and horizontal axis and maximize the image.
AppliedPrecision
Appendix C: Acquiring a PSF
163
5. Switch the beam selector to SP or SPL. Then adjust the exposure time and %T
filter and click Acquire. Leave Data Collection window 21 open.
6. In the Image window, choose Tools | Measure Distance. Then set the Units to
Pixels in the Measure Distance dialog box.
7. Draw a line across the image from a point on the top left square to a point in
the same relative position on the top right square.
8. If the vertical delta is more than four pixels, re-align the slide. Repeat this
process at the middle and bottom. Then count and record the number of grid
elements and record the distance in pixels.
9. Repeat Steps 7 and 8 in the vertical direction.
10. Calculate the pixel size for each of the six measurements (top, middle, bottom,
left, center, and right) as follows:
Pixel Size(um) =
9.995 um/box
Sum of (Measured pixels)/(Number of grids per measurement)
Your calculation should be accurate to four decimal places.
11. Average the six pixel sizes to obtain the correct pixel size.
To acquire a PSF:
1. Obtain the objective lens ID number (see the softWoRx online Help for
information about lens identification numbers).
2. From the softWoRx main menu, choose Utilities | Revise Microscope
Configuration. Then enter the root password to open the RESOLVE3D.SYS
file.
3. In the RESOLVE3D.SYS file, under the Microscope Specifications section:
a. Increase the number of lenses next to MS_Number_Lenses: by 1.
b. Add the name of the objective to MS_Lens_Names: (e.g., 100Xoil,
60Xwater).
c. Add the lens ID to MS_Lens_ID_Numbers:
d. Enter the pixel size for the new lens.
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For example:
If the desired lens is 40X/1.35 with ID=10403 (the third lens in the list),
then the pixel size is 0.1656.
MS_Lens_Names: 10X 20X 40X 60X 100X
MS_Pixel_Sizes: 0.6680 0.3313 0.1656 0.1103 0.06631
MS_Lens_ID_Numbers: 10105 10205 10403 10602 10002
4. Mount a bead slide on the microscope and focus on the beads to obtain the
maximum intensity. Find a bead that is located by itself. (Refer to “Finding
Beads” immediately following this procedure.)
5. Use the Center Object tool to center a single bead in the X and Y directions. (It
is helpful to collect large images, such as 1024×1024.)
6. Adjust the CCD exposure time so that the maximum intensity at the plane of
best focus is at least 2000 counts. Make sure that the camera does not saturate
at the plane of best focus.
7. Now use a 256×256 image.
8. Ensure there is only one bead in the field of view and that, as you go out of
focus, no rings from other nearby beads enter the image.
9. Verify that your microscope and software are accurately configured for lens
and auxiliary magnification.
10. Execute the Standard PSF Measurement Macro described in the online Help to
measure the standard point spread function (or run a Z series through the
bead consisting of 128 sections acquired in 0.1 μm Z increments).
11. Run the softWoRx PSF to OTF program that converts the optical sections into
an OTF. (Refer to Converting PSF to OTF later in this chapter.)
Finding Beads
It is easiest to find beads in a very dark room. Bead slides from Applied Precision
include 1μm beads that fluoresce brightly at 617 nm and 0.1μm beads. Coarsely
focus on the slide by positioning the lens near the slide. Scan the slide while
looking for fluorescent haze from the 1μm beads. When you focus on the
fluorescence haze from the 1μm beads you should also find the 0.1μm beads.
Note Although a replacement bead slide is included in the Slide kit, bead slides
have a limited shelf life. To purchase bead slides from Applied Precision, contact us
at the appropriate number or address listed in Chapter 1: Getting Started.
AppliedPrecision
Appendix C: Acquiring a PSF
Selecting the Correct Immersion Oil
Accurate PSF measurements depend on the selection of the correct immersion oil.
Our experience has shown that the oils recommended by microscope
manufacturers are often not ideal for 3-D microscopy. We recommend that PSFs
are measured with a minimal amount of spherical aberration. Inappropriate
immersion oils yield asymmetric PSF measurements as a result of spherical
aberration. In the case of Olympus and Zeiss microscopes, an index of refraction
equal to 1.518 is ideal for measuring beads that are mounted in glycerol using 1½
coverslips. Nikon microscopes use an index of refraction equal to 1.512. There are
many variables that can affect the selection of the correct immersion oil. The
softWoRx Lens Information program can help you select the proper oil.
Note An oil kit is included with your DeltaVision system. To purchase replacement
oil, please contact Applied Precision.
To confirm that you are selecting the correct immersion oil:
1. Collect the image data and generate a 3-D maximum intensity volume
projection.
2. Rotate the 3-D image to get a view of the XZ or YZ plane. For example, rotate
the image 90 degrees about the X axis or 90 degrees about the Y axis. To better
see the shape of the PSF, it is helpful to do an exponential scaling—an
exponent of .5 usually works well.
Note If you are using softWoRx, use the Volume Viewer to generate a 3-D
rendering of the bead scan.
3. Look for symmetric flare in the resulting image. Symmetry indicates that the
oil is correct, and in virtually all situations, the most symmetric PSF along the
Z axis is also the smallest and has the highest intensity. In other words,
symmetry corresponds with the highest resolution.
4. Repeat the process with different oils until you determine the optimal
immersion oil.
The following figure demonstrates how image flare can be affected by the use of
different immersion oils.
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Figure C-1: Flare from Immersion Oils (3-D Maximum Intensity Projections)
X
Z
Correct Immersion Oil
Immersion Oil Index Too Low
Immersion Oil Index Too High
Converting PSF to OTF
The PSF to OTF program converts a measured point spread (PSF) to an optical
transfer function (OTF). Essentially, the OTF is the Fourier transform of the PSF.
The pixel size of the resulting image is given in cycles/μm. To reduce problems
associated with measurement noise, the PSF is radially averaged during the
conversion and, as a result, the 3D PSF image becomes a 2D OTF image.
The horizontal axis of the OTF represents axial (Z) frequency and the vertical axis
represents radial (XY) frequency. The brightness of the OTF image elements, on a
scale of 0 to 1, represents the frequency response of the microscope system at the
corresponding radial and axial frequencies.
AppliedPrecision
Appendix C: Acquiring a PSF
167
Radial Resolution (XY)
Figure C-2: Sample OTF Image
Axial Resolution
Figure C-3: PSF to OTF Conversion
Each option in PSF to OTF Conversion is described briefly below. For additional
information regarding these options, refer to the online Help.
PSF File
Defines the name of the PSF image file to be converted to an OTF.
OTF File (sym)
Displays the name of the resulting axially symmetric OTF. (For your convenience,
the OTF filename is created by appending “_otf” to the PSF filename.)
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X Range
Defines the start and end pixel numbers in X.
Y Range
Defines the start and end pixel numbers in Y.
Z Range
Defines the start and end pixel numbers in Z.
T Range
This field is not used for PSF to OTF conversion.
Wavelengths
Determined by PSF wavelength.
Lens ID
Specifies the lens identification number (e.g., 12004).
Sub-Image: Center
Specifies the central XYZ coordinates of the point spread.
Sub-Image: Size
Specifies the XYZ image dimensions about the central coordinates. (The standard
softWoRx point spread measurement is 256×256×128.)
Additional Parameters: Border Rolloff (voxels)
Specifies the number of voxels to roll off at the edge of the image. This reduces
edge effects resulting from the Fourier Transform used in the PSF to OTF
conversion.
The procedure for converting a PSF to an OTF is very simple. After the PSF file has
been identified in PSF to OTF Conversion, softWoRx assigns default settings to the
rest of the options in the dialog box. In almost every instance, these settings will be
appropriate to use for the conversion.
To convert a PSF to an OTF:
1. Click Conversions on the main menu bar of softWoRx.
2. Click Convert PSF to OTF in the Conversions menu. PSF to OTF Conversions
will appear.
3. Use one of the following options to enter the PSF file to convert.
•
Drag the appropriate PSF file from the File Manager into the PSF File text
box.
•
Click PSF File to display a small version of the File Manager and then
choose the PSF file that you wish to convert.
AppliedPrecision
Appendix C: Acquiring a PSF
•
Type the desired path and filename into the PSF File text box.
4. Click Do It.
To place OTF into OTF Library
If the objective used is in addition to those already present, you’ll need to modify
softWoRx to use the new objective by adding the file to either
/usr/local/softWoRx or dv2.10/config/system.dvrc as follows:
1. Log in to Linux as root.
2. Navigate to /usr/local/softWoRx/config/system.dvrc
3. Find the section labeled, “Lens-to-OTF matching” and follow the instructions
provided for the OTF file.
The following is an example of this section of the file:
# Lens-to-OTF matching. These are of the form
LENS_<lensIDNumber>_OTF
# and are defined to be the file name in the OTF directory
of the OTF that is
# to be used for this lens ID.
LENS_12_OTF
60X140_sample.otf
LENS_10602_OTF
60X140.otf
LENS_10003_OTF
100X135.otf
LENS_10403_OTF
40X135_sample.otf
LENS_10603_OTF
60Xw_120.otf
LENS_10205_OTF
20X0.75c.otf
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D
Appendix D: Reference Information
The appendix includes the following topics:
„
Standard Filename Extensions lists the filename conventions used by
DeltaVision.
„
Standard Fluorescence Filters shows the excitation and emission peaks of the
standard filters included with DeltaVision.
„
Live Cell Filter Sets shows the excitation and emission peaks of the filters
included in the optional Live Cell filter wheel module.
„
Reference List includes references for microscopy, Linux, image processing,
optics, microscopy, and sample preparation.
Standard Filename Extensions
The following is a list of filename conventions used by DeltaVision.
Filename
Extension
Type of File
*.dv
Standard DeltaVision image
*.otf
Optical Transfer Function
*_R3D.dv
Resolve3D image
*_D3D.dv
Deconvolved image
*_VOL.dv
Volume Rendered image
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Standard Fluorescence Filters
DeltaVision provides a four color filter set, a polarizer, and multiple sets of optional
filters. These filters are designed to be used with many common fluorescent
probes. If you are using fluorescent probes that are not well matched with the
standard DeltaVision filters, contact Applied Precision for assistance.
The excitation and emission peaks of the DeltaVision filters2 are provided in the
following table.
Filter
Name
Excitation
Emission
Appropriate
Probes
DAPI
UV, 350nm
Blue, 455nm
DAPI, Hoechst,
Coumarin
FITC
Blue Green, 490nm
Green, 525nm
Fluorescein,
GFP, CY3
TRITC
Green, 555nm
Orange, 605nm
Rhodamine,
Texas Red,
Phycoerythrin,
CY-5®
Red, 645nm
Infrared, 705nm
CY-5
CFP
(optional)
Deep Blue, 436 nm
Blue Green, 470nm
CFP
YFP
(optional)
Blue Green, 500 nm
Yellow Green, 535nm
YFP
Live Cell Filter Sets
The optional Live Cell filter wheel module includes four of the most common sets
of filters used for live cell imaging.
2
Filter
Name
Excitation
Emission
Appropriate
Probes
CFP
Deep Blue, 430nm
Blue, 470nm
Cyan GFP (CFP)
YFP
Blue Green, 500nm
Yellow Green, 535nm
Yellow GFP
mCherry
Yellow, 572nm
Red, 632nm
DsRed Express,
DsRed2
EGFP
Blue, 470nm
Green, 525nm
EGFP, sgGFP
Data for filters provided by Chroma Technology Corp.
AppliedPrecision
Appendix D: Reference Information
Reference List
Selected references are provided on the following pages. Contact Applied
Precision for the most recent list. If you notice omissions from the list, please
inform Applied Precision.
Microscopy
Agard, D. A., Sedat J. W. (1983) Three-dimensional architecture of a polytene nucleus.
Nature 302: 676-681.
Agard, D. A. (1984) Optical Sectioning Microscopy: Cellular Architecture in Three
Dimensions. Ann. Rev. Biophys. Bioeng. 13: 191-219.
Agard D.A., Hiraoka Y., Sedat J.W. (1988) Three-dimensional light microscopy of diploid
Drosophila chromosomes. Cell Motility & Cytoskeleton 10:18-27.
Agard D.A., Hiraoka Y., Shaw P.J., Sedat J.W. (1989) Fluorescence microscopy in three
dimensions. Methods in Cell Biology 30:353-377.
Aikens R.S., Agard D.A., Sedat J.W. (1989) Solid-state imagers for microscopy. Methods in
Cell Biology 29:219-313.
Anderson J.T., Paddy M.R., Swanson M.S. (1993) PUB1 is a major nuclear and cytoplasmic
polyadenylated RNA-binding protein in Saccharomyces cerevisiae. Molecular &
Cell Biology 13:6102-6113.
Asada T., Kuriyama R., Shibaoka H. (1997) TKRP125, a kinesin-related polypeptide
involved in the centrosome-independent organization of the cytokinetic apparatus
of tobacco BY-2 cells. Journal of Cell Science 110, in press.
Babcock D.F., Herrington J., Goodwin P.C., Park Y.B., Hille B. (1997) Mitochondrial
Participation in the Intracellular Ca2+ Network. Journal of Cell Biology, in press
12/96.
Bass H.W., Marshall, W.F., Sedat J.W., Agard D.A., Cande W.Z. (1997) Telomeres cluster de
novo before the initiation of synapsis: A three-dimensional spatial analysis of
telomere positions before and during meiotic prophase. Journal of Cell Biology 137
(1):5-18.
Belmont A.S., Braunfeld M.B., Sedat J.W., Agard D.A. (1989) Large-scale chromatin
domains within mitotic and interphase chromosomes in vivo and in vitro.
Chromosoma 98:129-143.
Belmont A.S., Sedat J.W., Agard D.A. (1987) A three-dimensional approach to mitotic
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Cell Biology 105:77-92.
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Belmont A.S. and K. Bruce. (1994) Visualization of G1 chromosomes: a folded, twisted,
supercoiled chromonema model of interphase chromatid structure. Journal of Cell
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Charlton C.A., Mohler W.A., Radice G.L., Hynes R.O., Blau H.M. (1997) Fusion
Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture.
Journal of Cell Biology 138: 331-336.
Chen H., Sedat J.W., Agard D.A. (1989) Manipulation, display, and analysis of threedimensional biological images, in Handbook of Biological Confocal Microscopy
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Chen H., Hughes D.D., Chan T.-A., Sedat J.W., Agard D.A. (1996) IVE (Image Visulization
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Chikashige Y., Ding D.Q., Funabuki H., Haraguchi T., Mashiko S., Yanagida M., Hiraoka
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Csink A.K., Henikoff S. (1996) Genetic modification of heterochromatic association and
nuclear organization of Drosophila. Nature 381: 529-531.
Dawe R.K., Sedat J.W., Agard D.A., Cande, W.Z. (1994). Meiotic chromosome pairing in
maize is associated with a novel chromatin organization. Cell 76: 901-912.
Dawe R.K., Cande W.Z. (1996) Induction of centromeric activity in maize by suppressor of
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Dernburg A.F., Daily D.R., Yook K.J., Corbin J.A., Sedat J.W., Sullivan W. (1996) Selective
loss of sperm bearing a compound chromosome in the Drosophila female. Genetics
143: 1629-1642.
Dernburg A.F., Sedat J.W., Hawley R.S. (1996) Direct evidence of a role for
heterochromatin in meiotic chromosome segregation. Cell 86: 135-146.
Dernburg A.F., Broman K.W., Func J.C., Marshall W.F., Agard D.A., Sedat J.W. (1996)
Perturbation of nuclear architecture by long-distance chromosome interactions.
Cell 85: 745-759.
Gertler F.B, Niebuhr K., Reinhard M., Wehland J., Soriano P. (1996) Mena, a Relative of
VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament
Dynamics. Cell 87: 227-239.
Goodwin P.C. (1996) Wide-Field Deconvolution vs. Confocal Microscopy of Living Cells.
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Appendix D: Reference Information
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during prometaphase in Drosophila melanogaster embryos. Journal of Cell Biology
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Hiraoka Y., Dernburg A.F., Parmalee S.J., Rykowski M.C., Agard D.A., Sedat J.W. (1991)
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Hiraoka Y., Haraguchi T. (1996) Fluorescence imaging of mammalian living cells.
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Hiraoka Y., Minden J.S., Swedlow J.R., Sedat J.W., Agard D.A. (1989) Focal points for
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Hiraoka Y., Rykowski M.R., Lefstin J.A., Agard D.A., Sedat J.W. (1990) Three-dimensional
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Hiraoka Y., Sedat J.W., Agard D.A. (1987) The use of a charge coupled device for
quantitative optical microscopy of biological structures. Science 238:36-41.
Hiraoka Y., Sedat J.W., Agard D.A. (1990) Determination of three-dimensional imaging
properties of a light microscope system: partial confocal behavior in
epifluorescence microscopy. Biophysical Journal 57:325-333.
Hiraoka Y., Swedlow J.R., Paddy M.R., Agard D.A., Sedat J.W. (1991) Three-dimensional
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Kam Z., Agard D.A., Sedat J.W. (1997) Three-dimensional microscopy in thick biological
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Kam Z., Chen H., Sedat J.W., Agard D.A. (1991) Analysis of three-dimensional image data:
display and feature tracking, in Electron Tomography (Frank J, ed.) Plenum Press,
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Kam Z., Jones M.O., Chen H., Agard D.A., Sedat J.W. (1993) Design and construction of an
optimal illumination system for quantitative wide-field multidimensional
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Kam Z., Minden J.S., Agard D.A., Sedat J.W., Leptin M. (1991) Drosophila gastrulation:
analysis of cell shape changes in living embryos by three-dimensional fluorescence
microscopy. Development 112:365-70.
Kam Z., Volberg T., Geiger B. (1995) Mapping of adherens junction components using
microscopic resonance energy transfer imaging. Journal of Cell Science 108:10511062.
Kaplan K.B., Swedlow J.S., Varmus H.E., Morgan D.O. (1992) Association of p60c-src with
endosomal membranes in mammalian fibroblasts. Journal of Cell Biology 118:321-33.
(cover article)
Kaplan K.B., Bibbins K.B., Swedlow J.R., Arnaud M., Morgan D.O., Varmus H.E. (1994)
Association of the amino terminal half of c-Src with focal adhesions alters their
properties and is regulated by phosphorylation of tyrosine 527. EMBO Journal
13:4745-4756.
Kaplan K.B., Swedlow J.R., Morgan D.O., Varmus H.E. (1995) c-Src enhances the spreading
of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Devel
9:1505-1517.
Kirk K.E., Harmon B.P., Reichardt I.K., Sedat J.W., Blackburn E.H. (1997) Block in
Anaphase Chromosome Separation Caused by Telomerase Template Mutation.
Science 275: 1478-1481.
Lauer S. A., P. K. Rathod, N. Ghori, K. Haldar (1997) A Membrane Network for Nutrient
Import in Red Cells Infected with the Malaria Parasite. Science 276.
Lieb J. D., Capowski E. E., Meneely P., Meyer B. J. (1996) DP-Y, a Link Between Dosage
Compensation and Meiotic Chromosome Segregation in the Nematode. Science
274: 1732-1736.
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Appendix D: Reference Information
Ma X, Ehrhardt D. W., Margolin W. (1996) Colocalization of cell division proteins FtsZ and
FtsA to cytoskeletal structures in living Escherichia coli cells by using green
fluorescent protein. PNAS USA, 93: 12998-13003.
Marshall W.F., Dernburg A.F., Harmon B., Agard D.A., Sedat J.W. (1996) Specific
interactions of chromatin with the nuclear envelope: Positional determination
within the nucleus in Drosophila melanogaster. Molecular Biology of the Cell, 7: 825842.
Marshall W.F., Fung J.C., Sedat-J-W. (1997) Deconstructing the nucleus: Global architecture
from local interactions. Current Opinion in Genetics & Development, 7: 259-263.
Marshall W.F., Straight A., Marko J.F., Swedlow J.R., Dernburg A., Belmont A., Murray
A.W., Agard D.A., Sedat J.W. (1997) Interphase chromosomes undergo constrained
diffusional motion in living cells. Curr. Biol. 7:930-939.
Mathog D., Sedat J.W. (1989) The three-dimensional organization of polytene nuclei in
male Drosophila melanogaster with compound XY or ring X chromosomes. Genetics
121:293-311.
Minden J.S., Agard D.A., Sedat J.W., Alberts B.M. (1989) Direct cell lineage analysis in
Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of
living embryos. Journal of Cell Biology 109:505-516. (cover article)
Moritz M., Braunfeld M.B., Sedat J.W., Alberts B., Agard D.A. (1995) Microtubule
nucleation by gamma-tubulin-containing rings in the centrosome. Nature 378
(6557): 638-640.
Moritz M., Braunfeld M.B., Fung J.C., Sedat J.W., Alberts B.M, Agard D.A. (1995) Threedimensional structural characterization of centrosomes from early Drosophila
embryos. Journal of Cell Biology 130: 1149-1159.
Näthke I.S., Hinck L., Swedlow J.R., Papkoff J., Nelson W.J. (1994) Defining interactions
and distributions of cadherin and catenin complexes in polarized epithelial cells.
Journal of Cell Biology 125:1341-1352.
Näthke I.S., Adams C.L., Polakis P., Sellin J.H., Nelson W.J. (1996) The adenomatous
polyposis coli tumor suppressor protein localizes to plasma membrane sites
involved in active cell migration. Journal of Cell Biology 134:165-179. (cover article)
Neugebauer K.M., Roth M.B. (1997) Distribution of pre-mRNA splicing factors at sites of
RNA polymerase II transcription. Genes and Development 11:1148-1159.
Paddy M.R. (1998) Determining Nuclear Structure Using the Fluorescence Light
Microscope. Methods in Cell Biology 53: 49-77.
Paddy M.R., Agard D.A., Sedat J.W. (1992) An extended view of nuclear lamin structure,
function, and dynamics. Seminars in Cell Biology 3:255-266.
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Paddy M.R., Belmont A.S., Saumweber H., Agard D.A., Sedat J.W. (1990) Interphase
nuclear envelope lamins form a discontinuous network that interacts with only a
fraction of the chromatin in the nuclear periphery. Cell 62:89-106.
Paddy M.R., Chelsky D. (1991) Spoke: a 120-kD protein associated with a novel
filamentous structure on or near kinetochore microtubules in the mitotic spindle.
Journal of Cell Biology 113:161-171.
Paddy M.R., Saumweber H., Agard D.A., Sedat J.W. (1996) Time-resolved, in-vivo studies
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deconvolution in multi-wavelength fluorescence images. Journal of Microscopy 182:
50-60.
Schwartz K., Richards K, Botstein D. (1997) BIM1 encodes a a microtubule-binding protein
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transduction pathways by Helicobacter pylori. PNAS USA, 94: 7595-7599.
Shaw P. (1994) Deconvolution in 3-D optical microscopy. Histochemical Journal 26: 687-694.
Shaw P.J., Agard D.A., Hiraoka Y., Sedat J.W. (1988) Tilted view reconstruction in optical
microscopy: Three-dimensional reconstruction of Drosophila melanogaster embryo
nuclei. Biophysical Journal 55:101-110.
Shimanuki M., Miki F., Ding D.-Q., Chikashige Y., Hiraoka Y., Horio T., Niwa O. (1997) A
novel fission yeast gene, kms1+, is required for the formation of meiotic prophasespecific nuclear architecture. Molecular and General Genetics 254: 238-249.
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American Laboratory April 1996.
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Appendix D: Reference Information
Straight, A.F, Marshall W.F., Sedat J.W., Murray A.W. (1997) Mitosis in living budding
yeast: Anaphase A but no metaphase plate. Science 277:574-578.
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Current Opinions in Cell Biology 5:412-416.
Swedlow J.R., Hirano T. (1996) Chromosome dynamics: Fuzzy sequences, specific
attachments? Current Biology 6: 544-547.
Swedlow J.R., Sedat J.W., Agard D.A. (1993) Multiple chromosomal populations of
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Swedlow J.R., Sedat J.W., Agard D.A. (1997) Deconvolution in optical microscopy.
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E
Appendix E: Resolve3D and Keypad
Options
This chapter describes the following Resolve3D windows and dialog boxes:
„
The Resolve3D Window is the main window for data acquisition.
„
The Design/Run Experiment Dialog provides tools to select, design, edit, and
execute experiment macros.
„
The Experiment Designer is used to generate experiment command macros.
„
The Settings Dialog used to control how images are displayed, select camera
settings, and specify file output.
„
Keypad/Joystick Operation is a reference for the buttons on the keypad. Many of
the Resolve3D functions are also available on the keypad and joystick.
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The Resolve3D Window
The Resolve3D window is the main data acquisition window. In addition to
providing many of the acquisition options and controls, it provides access to the
other dialogs and windows that are used for data acquisition.
To open Resolve3D:
X From the softWoRx menu, choose File | Acquire (Resolve3D).
Figure E-1: Resolve3D Window
The Resolve3D Menu includes
commands for acquiring
images, controlling marked
points, specifying image and
display settings, and calibration
(see Page 183).
The Resolve3D Toolbar allows
you to acquire images, open
the Design/Run Experiment
dialog to set up and run
experiments, and open the
Settings dialog to control
display, image, and file options
(see Page 186).
Image Control Fields allow you
to choose filters, exposure time,
lens type, image size, and
binning settings (see Page 187).
Stage Position Control Fields and
Buttons can be used to move
the stage and mark points of
interest (see Page 189).
Blue box and bar can be
dragged to move the stage in
X, Y, and Z (see Page 193).
Image Intensity and Scale
Values display image properties
in numerical and graphical
formats (see Page 193).
The Message Pane reports the
status of various Resolve3D
activities (see Page 193).
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Appendix E: Resolve3D and Keypad Options
The Resolve3D Menu
The Resolve3D menu has the following menu items:
„
The File menu includes commands to acquire images and to open the key
dialog boxes for setting up and running experiments.
„
The View menu includes commands to manage marked points and to create a
blank screen.
„
The Options menu includes commands to open the Settings dialog box
(where you can set display and image options) and to save settings.
„
The Calibration menu opens the Calibration tool.
„
The Help menu provides options to show or hide ToolTips and to get Help.
The File Menu
Use the following Resolve3D File menu commands to acquire images, create
scratch files, and open the Design/Run Experiment dialog box.
Figure E-2: The Resolve3D File Menu
Acquire Image
Collects and displays an image from the microscope. This image is only displayed
in the Data Collection window. It is not saved to a disk file for later use.
Continuous Acquire
Opens the Continuous Acquire dialog box that you can use to collect and display
images continuously. These images are only displayed; they are not saved to a
disk file.
Snapshot
Launches a tool to let you collect a single 2-D, multi-wavelength "snapshot" image.
If you need to collect a Z series, time-lapse image, or other complex scheme, you
will need to design an experiment with the Resolve3D Experiment Designer.
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Scratch File
Creates a "scratch file" to which you can save individual image frames for later
use. After a file is opened, clicking Save Current Image saves the most recently
collected image frame. Clicking Close Scratch File or Done closes the file. Note
that the image size cannot be changed while a scratch file is open.
Experiment
Opens the Design/Run Experiment dialog box. You can use Design/Run
Experiment to select a previously created experiment, open the Experiment
Designer window to design a new experiment, or open the Experiment Macro
Editor to create or edit an experiment macro.
Quit
Closes the Resolve3D window.
The Resolve3D View Menu
Use the View menu to manage points that you have marked, to clear the history of
the path that you took while exploring your sample, or to create a black screen for
light-sensitive conditions.
Figure E-3: The Resolve3D View Menu
Point List
Opens the Points List dialog box. This dialog box helps you manage a list of points
(with X, Y, and Z coordinates) that you want the system to remember. These
points can be interactively "visited" at any time and they can be used in
experiments.
Clear Stage Trails
Clears the Stage Trails history. (The system maintains a history of the paths of
motion that you take while exploring your sample. These paths are displayed as
"Stage Trails" on the Stage View.)
Clear Stage Thumbnails
Clears all of the thumbnail images currently displayed on the stage view.
Blank Screen
Turns the computer screen blank. This is useful when you are imaging under very
light-sensitive conditions. Clicking anywhere on the screen restores it.
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Appendix E: Resolve3D and Keypad Options
Command Line Interface
Opens the Command Line dialog box that provides advanced users with the
ability to issue individual Resolve3D commands to the system.
CAUTION: The Command Line Interface should be used carefully because
it can put the system in an unstable state.
The Resolve3D Options Menu
Use the Options menu to open the Settings dialog box (where you can set display
and image options) and to save configuration settings and state information.
Figure E-4: The Resolve3D Options Menu
Settings
Opens the Settings dialog box which allows you to control display, imaging, and
file output options.
Save Settings
Saves the configuration settings and information (current filters, image size, etc.)
to be used the next time Resolve3D is opened.
The Resolve3D Calibration Menu
Use the Calibration menu to make calibration tables, read calibration tables, and
designate which calibration tools are active.
Figure E-5: The Resolve3D Calibration Menu
Make
Opens the Calibration tool used to create flat-field calibration tables and optional
tables of "bad" pixels. These tables may be applied to images, either when they are
acquired or at a later time. (To apply the tables to images after they are acquired,
use the Calibrate utility available from the Process item on the main softWoRx
menu.)
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Read
Opens the Read Calibration Files dialog box that you can use to read calibration
tables.
Manage
Opens the Manage tool that you can use to designate which calibration tables are
active and to remove tables from the system's session memory.
The Resolve3D Help Menu
Use this menu to turn ToolTips on or off, get help on the Resolve3D window, or
find out which version of softWoRx you are using.
Figure E-6: The Resolve3D Help Menu
Turn ToolTips On/Off
Turns tool tips on or off. Tool tips display “pop-up” information about buttons on
the interface. They open when the mouse pointer is held over a button for a few
seconds.
On Window
Opens Help for the Resolve3D window.
Version
Displays version numbers for the softWoRx components.
The Resolve3D Toolbar
Use the buttons on the Resolve3D toolbar to acquire images, open the Experiment
Designer/Run dialog box that allows you to set up and run experiments, and open
the Display Settings dialog box that allows you to control display, imaging, and
file output options.
Figure E-7: The Resolve3D Toolbar
Acquire
Collects and displays an image from the microscope with the current settings. This
image is only displayed in the Data Collection window. It is not saved in a file.
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Appendix E: Resolve3D and Keypad Options
Experiment
Opens the Design/Run Experiment dialog box that you can use to select a
previously created experiment, open the Experiment Designer window to design a
new experiment, or open the Experiment Macro Editor to create or edit an
experiment macro.
Settings
Opens the Settings dialog box that allows you to control display, imaging, and file
output options.
Image Control Fields
Use the Resolve3D Image Control fields to:
„
Select the Excitation, Emission, and Neutral Density filters.
„
Select exposure time.
„
Determine whether to calibrate the image.
„
Select the desired shutter.
„
Select the image size.
„
Select the lens and get lens information.
„
Select whether to use auxiliary magnification.
„
Determine binning parameters.
„
Get pixel size.
Figure E-8: The Resolve3D Image Control Fields
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Excitation
Specifies an excitation filter. When this filter is selected, a corresponding emission
filter is automatically selected and the following parameters are set to the last
values that were used for that excitation filter.
„
Exposure time
„
Neutral Density filter
„
Active illumination shutters
„
Number of frames to average
„
Target intensity
„
Camera gain
You can choose Save Settings from the Options menu to store the configuration.
The wavelength/bandwidth of the selected filter is indicated to the right of the
filter choice box.
Emission
Specifies an emission filter. The wavelength/bandwidth of the selected filter is
indicated to the right of the filter choice box.
%T
Specifies a Neutral Density filter. The relative illumination intensity is indicated in
the menu. (This value indicates the amount of light passing through the filter. A
value of 100% indicates the light is unfiltered.)
Exposure
Specifies camera exposure time (in seconds). The minimum and maximum
exposure times allowed for this field depend upon the camera type.
Find
Finds the exposure time necessary to reach the target intensity currently entered in
the Target Intensity dialog box. (This dialog box opens when you click the Find
button.) For dim samples, 300-900 counts are acceptable. For bright samples, 30003600 counts provide a high dynamic range without saturation.
Calibrate
Calibrates images when they are collected from the camera. If you use this option,
you must load and activate a calibration table that fits the current imaging
parameters (wavelength, image size, bin choice) before you acquire images.
Shutter Control
Selects the desired excitation shutter.
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Image Size
Specifies image size (pixels or CCD detector elements) for acquired images. The
pull-down list contains predefined sizes for convenience. You can enter special
sizes by choosing Other... from the pull-down list. (Image size must be a multiple
of four.)
Lens
Specifies the objective lens name. The pull-down list contains the lenses that are
known to be part of the microscope system.
Info
Opens the Lens Information dialog box which displays information about the
current objective lens.
Bin
Specifies the number of CCD detector elements to add together to form one image
element. Binning is applied in both the X and Y directions. It increases intensity,
but it decreases resolution. Pixel size is a function of binning.
Aux. Mag.
Specifies to use the microscope's 1.6x manual auxiliary magnification. (On IX70
stands, the manual auxiliary magnification is 1.5x. On IX71 stands that have a
Cascade II camera, the auxiliary magnification is 2.0x)
Pixel size
Displays pixel dimensions in microns/pixel. The value is calculated from the Lens,
Bin, and Auxiliary magnification settings.
Stage Position Control Fields and Buttons
The Resolve3D Stage Position Control area allows you to control the microscope's
X, Y, and Z stage positions. The Center, Mark, and Visit tools and the Stage Motion
Controls are shown below.
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Figure E-9: The Resolve3D Stage Position Control
Stage Control and Display Tools
Center Object
Centers the stage on an object selected in an Image Window. The pixel size must
be correct in order for object centering to work properly, which means that the
correct lens and auxiliary magnification setting must be selected. An image will be
acquired after the object is centered.
Mark Point
Marks the current X, Y, and Z stage coordinates as points to be visited later.
Mark Top of Sample
Marks the current stage Z position as the "top" of your sample. Because the
scanning process always moves the stage toward the objective, this position also
represents the point where the stage is closest to the objective (the most negative Z
value). This also represents the focal plane that is the closest to the slide side of a
sample. Use this along with the Mark Bottom of Sample button to establish the
thickness of the sample. You can use these marked positions to aid with the Z
sectioning setup.
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Note All scans that are set up using the Experiment Designer scan in Z, using relative
coordinates. The Mark Top of Sample and Mark Bottom of Sample buttons are most
helpful in determining the thickness of the sample to be scanned. When an
experiment is started, the scan region is determined by the current focus point and
the thickness of the sample. So, for example, if you have marked three points to visit
in an experiment and they all have different middle-Z positions, the experiment will
calculate the scan based on these different Z positions and the fixed thickness.
Visit Top
Moves the stage to the position marked as the top of the sample.
Mark Bottom of Sample (end of scan)
Marks the current stage Z position as the "bottom" of your sample for a potential
scan. This corresponds to the positive stage Z coordinate value or the coverslip
side of the sample.
Visit Bottom
Moves the stage to the position marked as the bottom of the sample.
Visit Middle
Visits the point in the middle of the defined scan region.
Autofocus
Automatically focuses.
Acquire Image
Collects and displays an image from the microscope. The current settings are used
to collect the image. This image is only displayed in the Data Collection window.
It is not saved in a file for later use.
XY Stage Controls
Moves the stage in the X and Y axis. The left and right arrows move the stage in
the X axis in the increment set in the dX field and the up and down arrows move it
in the Y direction in the increment set in the dY field.
Z stage Motion Controls
Moves the stage in the Z axis in the increment set in the dZ field.
Pan
Moves the stage view. Note that this tool is "sticky." To disable it, click the Pan tool
again.
Clear Stage Trails
Clears all of the stage trail lines from the stage view.
Clear Thumbnails
Clears all of the thumbnail images currently displayed on the stage view.
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Marked Points list
Opens the Point List dialog box to manage the list of marked points.
Zoom Tool
Controls zoom. Drag the thumbwheel down to zoom in; up to zoom out. The
button under the thumbwheel returns the zoom to 1:1.
Z Slider
Moves the stage up or down.
dX
Specifies the X step size, in microns.
dY
Specifies the Y step size, in microns.
Stage Trails Window
The blue box represents the current stage location. Drag the box to move the stage
in X and Y.
dZ
Specifies the Z step size, in microns.
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Image Intensity and Scale Values
The Resolve3D Intensity values show the statistics for the intensity values of the
image in numerical and graphical formats. The scale of the image is also shown.
Figure E-10: The Resolve3D Image and Intensity and Scale Values
Min, Max, Mean
Displays the minimum, maximum, and mean intensity values of the most recently
acquired image. For a 12 bit CCD camera, these values range between 0 and 4095.
A value of 4095 indicates camera saturation, unless image calibration is in effect.
(If you are using 0.5X Gain, the saturation is less than 4095 counts.)
Histogram
Shows the intensity distribution for the most recently acquired image. The vertical
blue bars indicate the Scale min and Scale max and can be dragged interactively
with the mouse to change how the display is scaled. The X-axis represents
intensity and the Y-axis represents the number of pixels.
Scale min/Scale max
Specifies the settings for the minimum and maximum display values. These
numbers can be changed manually or by moving the histogram threshold bars.
Io
Indicates valid (or invalid) photo sensor values or saturation. The indicator is
green if the photo sensor value is valid. It is red before the first image is acquired.
After the first scan, a red color indicates that the most recently acquired image has
an invalid photo sensor value. A red indicator can also signify unreasonable
saturation or an improperly functioning photo sensor device.
The Message Pane
The Resolve3D Message pane reports the status of various Resolve3D activities.
Use the scroll-bar to view messages that have scrolled off the top of the pane.
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Resolve3D Shortcuts
You can right-click anywhere in the Resolve3D window to open a shortcut menu
that allows you to acquire images, mark points, and create a blank screen (for
imaging under very light-sensitive conditions).
Figure E-11: Resolve3D Shortcut Menu
Acquire
Collects and displays an image from the microscope. The current settings are used
to collect the image. This image is only displayed in the Data Collection window.
It is not saved in a file for later use.
Snapshot
Launches a tool to let you collect a single 2-D, multi-wavelength "Snapshot" image.
(The current settings are used to collect the image.)
Mark point
Marks the current X, Y, and Z stage coordinates as a point to be visited later.
Blank Screen
Turns the computer screen black for imaging under very light-sensitive
conditions. Clicking anywhere on the screen restores it.
The Design Experiment Tab
The Design Experiment tab is used to generate experiment command macros.
To open the Design Experiment tab:
X From the Resolve3D window choose Experiment. The Design/Run Experiment
window opens with the Design Experiment tab selected.
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Figure E-12: Design Experiment
Experiment name
Specifies the name of the experiment macro.
Enable Fast Acquisition
Enables fast acquisition experiments (see Page 196).
Sectioning
Specifies sectioning for 3D images (see Page 196).
Channels
Specifies channels (filters) and exposure time (see Page 198).
Time-lapse
Specifies criteria for time-lapse experiments (see Page 199).
Point Visiting
Specifies a list of marked points (see Page 200).
Panels
Allows you to set up panel collections that you can use to review a large area of a
slide or to stitch together to form a single, large image (see Page 201).
Actions
Lets you designate laser events, Autofocus, pause, wait, time-lapse, ratio imaging,
and other specific actions to occur during the experiment.
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Experiment name and Enable Fast Acquisition
Experiment name
Specifies the name of the file that is generated by the Experiment Designer. A file
extension of .exp will be added to the name.
Enable Fast Acquisition
Enables fast image acquisition of 2D images. (This is only available when you are
using an interline camera.)
This data acquisition mode should be used carefully. Fast Acquisition uses a single
command to set up a data stream to the Instrument Controller. Because no
checking for user input occurs once the command is executed, you cannot stop an
acquisition sequence once it is started.
There is an upper memory limit for data collection that is based on the size of
RAM memory for the Instrument Controller (typically about 350-400 MB). If you
run into this limitation, turning Fast Acquisition off allows you to collect larger
data sets.
If the system is unstable after collecting a large data set with Fast Acquisition,
restart the workstation or stop using Fast Acquisition unless it is strictly necessary.
Sometimes Fast Acquisition isn’t much faster than standard acquisition, and more
is risked than gained by using it.
Note You can set Fast Image Acquisition options to control the scan sequence, the
shutter open mode, the camera readout mode, and the starting Z location for the
scan. (See the online Help for more information.)
Sectioning Setup
Once your microscope is focused near the middle of the vertical zone of interest of
your sample, you can use the parameters in the Design Experiment Sectioning tab
to control the Optical Sectioning procedure.
Note The standard scan direction moves the objective lens towards the specimen.
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197
Figure E-13: Design Experiment Sectioning Setup Options
Focus point when scan starts
Specifies the Z location of the sample when the experiment starts. The
recommended option is Middle of Sample.
Optical section spacing
Specifies the spacing (in microns) between each optical section. The focal point
will be changed by this value after each image is collected.
Number of optical sections
Specifies the number of sections to collect (for each wavelength) for the
experiment. softWoRx automatically calculates and displays this value if the
Sample thickness and Optical section spacing are entered. If you specify this
value, the Sample thickness value is changed.
Sample thickness
Displays the Number of optical sections multiplied by the Optical section
spacing.
Get Thickness
Retrieves the Sample thickness, based on the locations defined with the
buttons in the Resolve3D window.
and
OAI
Acquires a 3D Z projection of the interval defined by the marked top and bottom
of the sample. The optical section spacing and the number of optical sections are
determined automatically, based on the depth-of-field of the objective and the
exposure time.
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Channels Setup
Use the Design Experiment Channels tab to select wavelengths (filters) and to
specify an exposure time for each filter.
Figure E-14: Design Experiment Channels Options
Refresh exposure conditions
Updates the filter and exposure settings to those last used in the main Resolve3D
window.
Active Wavelength Toggle Buttons
Enable the exposure time, filters, and display settings for specific wavelengths.
Select the buttons that activate the wavelengths that you want to collect. (You
must select one button for each wavelength.) If no wavelengths are selected, the
exposure and filters that are set in the Resolve3D window are used.
Exp
Specifies the exposure time (in seconds) to be used when acquiring an image for
the selected wavelength. If left blank, the value specified in Resolve3D will be
used when the experiment is run.
EX Filter
Specifies the excitation filter to use for this experiment or image. When it is
changed, the currently "paired" emission filter is automatically selected.
EM Filter
Specifies the emission filter to use. This filter may be selected independently of the
EX Filter setting.
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%T Filter
Specifies the Neutral Density filter to use. The % value indicates light
transmittance. A value of 100 % indicates that no light is blocked (or no filtering).
Ex Shutter
Specifies the excitation (illumination) shutter to use for each channel of the image.
Reference Image
Specifies to use an alternate filter or the transmitted light to acquire a reference
image that can be combined with other images. This option is useful for
Differential Interference Contrast (DIC) analysis. It can also be useful for other
types of reference images. Only one reference image per Z stack is collected.
Time-lapse Setup
Use the Design Experiment Time-lapse tab to specify the number of time points,
the time periods, and the total time for a time-lapse experiment.
Figure E-15: Experiment Designer Time-lapse Setup Options
Time-lapse option
Specifies running a time-lapse experiment.
Time-lapse
Specifies the hours, minutes, and seconds for each time period.
Total Time
Displays the total time of the experiment, which can also be calculated as follows:
Total time = (The number of time points - 1) x time-lapse
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Time Points
Specifies the number of time samples to collect in a time-lapse experiment.
Enable Cell Tracking
Moves the stage laterally to follow cells as they move during a time-lapse
experiment. With the Enable Cell Tracking option selected, DeltaVision
automatically keeps cells in the field of view.
Cell Tracking Options
Opens the Cell Tracking Options dialog box that allows you to set the parameters
for cell tracking. For more information on using this dialog box, see Tracking Cells
on Page 67.
Autofocus before imaging
Automatically focus the camera before each time point. For point visiting
experiments, the camera is automatically focused every time that a point is visited.
Point Visiting Setup
Use the Design Experiment Point Visiting tab to specify a list of marked points to
visit during the experiment.
Tip Before you specify these points, make sure that the point list is open in the Point
List dialog. You cannot specify the points when the point list is closed.
Figure E-16: Experiment Designer Point Visiting Setup Options
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Visit Point List
Specifies the list of points, described by number, to visit during the experiment.
All sectioning and wavelength procedures are repeated at each of the listed points.
A point list can be entered as a series of numbers separated by commas or dashes.
Separating two numbers with a dash '-' (as shown in the following example)
indicates that all point numbers in between should also be visited.
For example:
1,2,5,7-10
Note When you use Point Visiting with Z sectioning, the microscope uses the Z value
of the current focus of each point as the initial reference for that point. Values
specified for Z sectioning are incremented relative to that point.
Autofocus before imaging
Automatically focus the camera at each visited point before acquiring an image.
Panel Collection Setup
Use the Design Experiment Panels tab to create panel collection macros. These
macros are useful when you want to scan a large area with a relatively high
magnification lens. You can use the panels as a means of reviewing a large area of
a slide, or as data that you want to stitch together to form a single, large image.
Panel collection macros are sensitive to microscope settings such as image size and
magnification. Because the number of panels required depends upon many
factors, it is usually not a good idea to reuse panel collection macros.
Figure E-17: Experiment Designer Panel Collection Setup Options
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Collect Panels
Specifies to use a panel collection macro.
Overlap (pixels)
Specifies the amount of overlap (in pixels) between adjacent panels.
Start Coordinates
Specifies the XYZ coordinates at which to start collecting panels. Use the Get Start
button to obtain the current XYZ stage coordinates.
End Coordinates
Specifies the XYZ coordinates at which to finish collecting panels. Use the Get End
button to obtain the current XYZ stage coordinates.
The Design/Run Experiment Dialog Box
The Design/Run Experiment dialog box provides tools to select, design, edit, and
execute experiment macros. (Experiment macros are "scripts" of commands that
guide the DeltaVision system to collect images.)
To open the Design/Run Experiment dialog box:
X From the Resolve3D window, click Experiment and click the Run Experiment
tab.
Figure E-18: The Design/Run Experiment dialog box
Note The Design PK Experiment tab is available only for systems that have the
QLM module.
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Image file name
Specifies the file name to use for the image in this text field. If you do not provide
a file name, you will be asked to provide one when the experiment starts running.
If you have the Auto-increment file names setting (in the Settings dialog box)
turned on, you will only need to provide a name once for each session. The names
will have incrementing numbers appended to them automatically.
Image title
Specifies text to save in the header of the image file created by the experiment. The
title can be viewed later using the Header Labels button of the Image Window's
Image Information dialog box.
Add note to log
Inserts a note in the experiment's log file at any time before or while an experiment
is running. (You will need to click Do It to insert the note.)
Change time lapse
Changes the time lapse value while an experiment is executing. (You will need to
type the desired time, in seconds, in the text field and click the Do It button to
change the value.) This value only affects experiment macros that contain a
TLAPSE command. It does not permanently change the macro.
Show PK Progress graph
See the QLM Getting Started Guide.
Show images during acquisition
Displays images in a Data Collection window as they are collected. In cases where
acquisition speed is important, deselecting this option may increase performance.
Launch viewer after experiment
Launches an Image window when the experiment is finished. Note that if more
than two files are created in a point-visiting experiment, the Image windows will
not launch even if this option is selected.
Images acquired/requested:
Displays the current number of images acquired, compared to the total number
requested. (During fast acquisition, the reported number of acquired images may
not be updated regularly.)
Disk space required
Displays the estimated size of the experiment (i.e., the size of the image file).
Resolve3D checks the disk space to make sure that enough space is available
before it runs the experiment.
Elapsed time:
Displays the elapsed time of the experiment, in seconds.
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Estimated Finish
Displays the estimated clock time in which a running time-lapse experiment will
finish.
Current command:
Reports each macro command as it is executed.
Start Scan
Starts the selected macro to run an experiment.
Cancel Scan
Terminates a scan while the experiment is running. (The images that are collected
before the experiment is cancelled are automatically saved.)
Help
Opens the Help for the Design/Run Experiment dialog box.
The Settings Dialog Box
Use the Resolve3D Settings dialog box to control how images are displayed, select
camera settings, and specify file output.
To open the Settings dialog box:
X From the Resolve3D Window, click the Settings button.
Figure E-19: The Settings Dialog Box Display Options
Note The QLM tab is available only for systems that have the QLM module.
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Settings Dialog Box Display Options
The Settings dialog box Display tab options allow you to control how the images
are displayed.
Image Display Mode
Specifies which window and wave are used for image display. Five modes are
currently available:
Model
Description
None
Displays images in the current window and wave.
Scratch
Displays all images in the specified window, Wave 1.
Auto Grayscale
Displays images in a separate window for each Emission (EM) filter.
Auto Color
Displays images in a different wave for each Emission (EM) filter. This
option should be used only when you are running an experiment.
Point Track
Opens a separate window for each visited point in a point-visiting
experiment.
Window
Specifies the number of the active display window. Select a new window to
change where the next image will be displayed. (Temporary windows are
numbered 21 or higher.)
Wave
Specifies the number of the active display wave (channel) as it is listed on the left
side of the Data Collection window. Select a new wave to change which display
window channel to use.
Calculate statistics
Calculates image intensity statistics. The typical reason for disabling this feature is
to improve readout speed.
Calculate histogram
Calculates and displays the image intensity histogram in the Resolve3D window
during image collection. (This option does not set scaling.)
Auto histogram range
Provides automatic scaling of the histogram width for each image that is analyzed.
This option provides automatic histogram ranging from the minimum to the
maximum intensity. When off, the histogram range remains at its current settings.
Display images
Displays images when the Acquire button is clicked or an experiment is running.
Deselecting this option can increase performance.
Deconvolve preview images
Displays instantly processed 2D image previews that closely resemble images
processed with advanced 3D image restoration techniques.
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Auto intensity scale
Provides automatic scaling of the image intensity between the minimum and
maximum brightness. (This switch applies only to the appearance of the image,
not the actual data.)
Acquire after point visit
Automatically acquires an image when the Visit Point option is selected.
Settings Dialog Box Imaging Options
The Settings dialog box Imaging options allow you to select camera settings.
Figure E-20: The Settings Dialog Box Imaging Options
Camera
Specifies which camera to use. For the EM CCD camera, this list also specifies
whether to use Conventional or Electron Multiplication mode.
Frames to average
Specifies the number of successive camera images to average into a result that is
displayed in the window. (This is similar to the AVG macro command.)
Gain
Specifies a gain value for the selected camera.
Transfer speed
Specifies a transfer rate for the selected camera (in kHz). Higher speeds may boost
performance at the expense of image noise.
Target temperature
Displays the cooled CCD camera's target temperature. This can be changed only
by modifying the appropriate configuration file on the Instrument Controller
computer.
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Current temperature
Displays the current temperature. Resolve3D monitors the temperature and
updates this value occasionally. If you want to force a request for the current
temperature, click the Refresh button.
Xe Lamp bulb age (hours)
Displays a count for the current number of hours on the xenon bulb.
Use photosensor
Enables use of the Photo sensor.
Settings Dialog Box Files Options
Use the Settings dialog box File options to set the image output directory, set the
directory where experiment macros are stored, and select auto incrementing for
file names.
Figure E-21: The Settings Dialog Box Files Options
Data Folder
Specifies the destination directory for Resolve3D output images. If you supply a
directory that you do not have permissions to use, you will be warned. If you
provide a directory name that does not exist, you will be presented with the option
of creating the directory. (You can also change the destination directory by
changing the global softWoRx Image Data Directory in the User Parameters tool.)
Experiment macros folder
Specifies the directory where your experiment macros are stored.
Data folder is temporary
Specifies that any directory entered in the Data Folder field only applies to the
current Resolve3D session. In subsequent Resolve3D sessions, the Data folder
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value reverts back to the global softWoRx Data Directory as specified in User
Parameters.
Use this option in environments where several users are running the microscope
and using a single system login. Data folders can be created for each user under a
common parent folder. As users start Resolve3D, they can choose to use their own
folders.
In most cases, you should not use this option if each user has their own system
login in your environment. With the option deselected, the Data folder assignment
modifies the global softWoRx Data Directory definition and the system remembers
the directory used in the last Resolve3D session.
Auto-increment file names
Creates a new file name by appending a serialized number to the base file name
each time you run an experiment.
Convert to 2 byte signed integer
If toggled On, Resolve3D saves all images as signed 16-bit (the pre-3.5.0 default).
This setting is designed to increase compatibility with older software. Note that
this setting cannot be On for EMCCD cameras.
Settings Dialog Box Autofocus Options
Use the options on the Resolve3D Autofocus tab to set up Autofocus parameters.
Automatically determine parameters
Autofocus parameters are determined from the depth of field of the objective lens,
which is calculated from the lens’ numerical aperture. You must select the proper
objective lens in order to get the appropriate Autofocus settings.
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Channel for Autofocus
This setting indicates the wavelength to use for Autofocus.
Contrast calculation method
This setting determines the polarity of the contrast calculation. There are three
choices for image contrast calculation methods:
•
Auto – The instrument controller usually can determine which contrast
calculation method to use, but not always.
•
Brightfield – for dark objects on a light background
•
Fluorescence – for light objects on a dark background
Autofocus Z test step (μm)
This option sets the step size used for Autofocus within the maximum Z range.
Maximum Z test range (μm)
The setting indicates the maximum range that the Autofocus will search.
Post-autofocus Z offset (μm)
This setting can provide a constant offset after the Autofocus position determines
the best plane of focus. Often times Autofocus will find a plane that is consistently
different from the desired plane.
Settings Dialog Box Misc Options
Use the options on the Resolve3D Misc tab to select or clear Lost Motion
Compensation or to select a filter wheel configuration (if your system is
configured to use alternate filter wheels).
Figure E-22: The Settings Dialog Box Misc Options
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Allow Lost Motion Compensation (LMC)
Enable Lost Motion Compensation to remove the effect of hysteresis in the stage.
This option should be selected for most applications. When it is turned off, the Z
series range may shift. Clear this option if you want to improve speed at the
expense of position repeatability.
Show stage trails
Display the path of stage movement on the Stage View.
Show stage thumbnails
Display a thumbnail image of each image on the Stage View as the image is
acquired.
Show point numbers
Displays the number of each point in a point list on the Stage View.
Excitation filter wheel
Allows you to select the name of the Excitation filter wheel that you are using.
This option list appears only if you have configured your system for alternate filter
wheels.
Emission filter wheel
Allows you to select the name of the Emission filter wheel that you are using. This
option list appears only if you have configured your system for alternate filter
wheels.
Eyepiece filter wheel
Allows you to select the name of the Eyepiece filter wheel to use. This option list
appears only if you have configured your system for alternate filter wheels.
Reinitialize Filter Wheels
Reinitializes the filter wheels.
Action Buttons
Done
Closes the Resolve3D Settings dialog box.
Save Settings
Preserves the current options for your next Resolve3D session. In addition to
options in the Resolve3D Settings dialog box, current state information such as
current filters and exposure time is saved.
Help
Opens the Help for the Settings dialog box.
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Appendix E: Resolve3D and Keypad Options
Keypad/Joystick Operation
Many of the functions accessible through Resolve3D are also available on the
keypad/joystick. This section describes each key on the keypad/joystick.
Note Some buttons on your keypad may not be active.
Figure E-23: Keypad/Joystick
RESET
Clears communication buffers, closes shutters, stops all motors, and clears encoder
errors. Use the RESET button when you suspect that the workstation and controller
are not synchronized.
CONTROL MODE
Toggles the controller mode between local mode and remote mode. Local mode
enables control by the keypad and joystick. The remote mode disables the joystick
and the keypad buttons (except for CONTROL MODE and a few other buttons)
and shifts control to other internal components.
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LMC RESET ON/OFF
Executes a Lost Motion Compensation (LMC) move.
Tip You can disable LMC on the Resolve3D Settings dialog. (For more information,
see the online Help.)
REMOVE TRAIL
Clears the stage movement history from memory.
KEYLIGHT ON/OFF
Turns the keypad backlight on and off.
QUIT
Quits the controller program when pressed twice. (To restart the Instrument
Controller program, double-click the Instrument Controller icon on the desktop.)
BLANK SCREEN
Suspends (or activates) the monitor's light display (BLANK SCREEN is a toggle
button). Use this feature when viewing dim samples or performing light sensitive
experiments.
SLOW/MEDIUM/FAST
Adjusts the joystick stage movement speed.
DISABLE MOTION KEYS
Disables the eight keys below the joystick. This prevents accidental input from the
keys when using the joystick.
YES/NO
Allows response to questions prompted on the screen. For example, during
initialization, the computer will ask if you want to continue with initialization. The
Yes and No buttons are an easy way to respond.
ACQUIRE MODE ARROWS
Changes the acquisition mode. (This includes the Excitation filter, exposure time,
shutter configuration, and many of the options defined in the Settings dialog box.)
JOYSTICK
Moves the stage in X and Y.
POINT ARROWS
Scrolls through the list of marked points. Press ACQUIRE IMAGE to view the
image for a selected marked point.
ND ARROWS
Changes the neutral density filter to one of a lower () or higher () value. When
the neutral density filter is at the minimum value and you push the decrease
arrow (), the wheel moves to engage the maximum value filter. When the
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Appendix E: Resolve3D and Keypad Options
213
Neutral Density filter is at the maximum value and you push the increase arrow
(), the wheel moves to engage the minimum value filter.
RETRACE ARROWS
Retrace () moves the stage one position back in the stage movement history
buffer. Retrace () moves the stage one position forward in the stage movement
history buffer.
EX ARROWS
Changes the excitation filter to the previous () or next () filter on the filter
wheel. This button changes the excitation filter but not any other settings, such as
exposure time and shutter configuration.
PANEL 1/2 MARK
Defines the area of the specimen to be stitched, as shown in the following diagram.
Figure E-24: Panels Diagram
Panel 2
Panel 1
OR
Panel 2
Panel 1
ACQUIRE IMAGE
Commands the system to collect image data. This key function is identical to
clicking Acquire in Resolve3D. Use this key when you are scanning through your
sample and using the eyepiece to find a region of interest, or when you want to get
a quick look at the specimen on the monitor.
SAVE IMAGE
Saves the last acquired image to the currently open file.
STEP DECREASE/INCREASE
Changes the step size of the movement controlled by the arrow keys located
beneath the joystick. Pressing either button many times will change the step size to
the minimum/maximum. There are 8 possible step sizes: 50nm, 100nm, 200nm,
400nm, 800nm, 2250nm, 4500nm, and 9000nm.
X AND Y ARROW KEYS
Move the stage in the direction shown.
Z ARROW KEYS
Move the stage in the direction shown.
Z1 MARK/Z2 MARK
Define the boundaries of the Z series.
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DeltaVision Core and personalDV User’s Manual
EX SHUTTER
Toggles the excitation shutter between open and closed.
TRANS SHUTTER
Toggles the shutter for the transmitted light source between open and closed.
POINT MARK
Adds the stage position to the marked points list.
AppliedPrecision
Index
%T Filter field, 212
3D images, 4, 38–44
3D Z projections, 78–80
Acquire Image button, 19, 228
Acquire Image setting, 204
Acquire images, 22–27, 28–30
Acquire Mode arrows, 227
Active Wavelength buttons, 211
Agard, Dr. David A., 2
Altitude requirements, 103
Ambient illumination, 104
Applied Precision, LLC, contacting, xii, 7
Aquiring a PSF, 173–78
Auto histogram range, 219
Auto intensity scale, 219
Autofocus, 5
before imaging, 213, 214
options, 222–23
setting in Resolve3D, 204
within experiment macros, 69–71
Autofocus button, 52
Autofocus Z test step option, 223
Automated Optical Sectioning, 4
Axial frequency, 179
Bead slides, 177
Beads
finding for PSF measurement, 176
measuring in immersion oil, 177–78
Bibliography, 15
Bin setting, 202
Blank Screen button, 227
Border rolloff, 180
determining, 56
Bottom of sample, 41
Bottom of Sample setting, 204
Brightfield, 3
Bulb icon, 32, 143
Bulbs, 124
Cabinet components, 112–14
Calculating OTF. See Optical transfer
function
Calculating pixel size, 174–75
Camera
changing, 125–27
damage prevention, 11
EM CCD, 87, 121–22
high-speed, 87, 107–8
power supply, 108
saturation, 87, 170
setting in Resolve3D, 220
supported for DeltaVision, 107–8
Camera not found message, 167
CCD camera, 107–8
damage prevention, 11
Cell mitosis, 47
Cell tracking, 71–77
Center Object setting, 203
Center of geometry, 74
Center of intensity, 74
Channel for Autofocus setting, 223
Channels tab, 211–12
Choosing filters, 17–19
Cleaning DeltaVision, 162
Coarse focus, 17, 23, 26
Collect Panels setting, 57, 215
Collecting data, 84–98
Collecting panel images, 55–57
Command line interface, 197
Components, 99–124
cabinet, 112–14
camera, 107–8
consumable, 123–24
desktop, 110–12
EM CCD camera, 121–22
enviromental chamber, 119
fiber optic module, 116
fluorescense microscope, 106
instument controller, 113
optical, 105–10
optional, 118–23
optional software, 123
photo sensor, 110
repeatable slide holder, 114–16
software, 117
softWoRx Workstation, 114
TIRF module, 121
tool kit, 116
Xenon lamp, 109
Consumable parts, 123–24
Contact Applied Precision, xii
Continuous Acquire, 196
Contrast calculation polarity, 223
Control Mode button, 226
Controlling the stage, 202–6
Conventions, document, x
Converting PSF to OTF, 178–81
Counter space, for system, 101
Country-specific requirements, 101
Critical illumination, 5, 89–91, 150, 155
position, 159
Critical illumination spring, 89
Critical spring, 151
Current rating, of electric supply, 100
Current temperature display, 220
Customer Service Hotline, xii
Customer support, 7
Damage prevention, 11
Data
collection techniques, 84–98
monitoring acquisition of, 91–96
saving, 30–32
Data Collection window, 93–95
Deconvolve preview images, 219
DeltaVision
capabilities, 3, 4
components, 99–124
damage prevention, 11
prerequisites, 15
turning off \r, 32
Depth of field, 42
Design Experiment tab, 36–38, 40, 207–8
Design/Run Experiment dialog box, 215–17
Desktop components, 110–12
DIC, 80–82
DIC imaging, 4
Dichroic filter wheel, 24
Differential Interference Contrast. See DIC
Digital Microscopy, 4
Dim illumination problems, 168
Dim image problem, 168
Disable Motion keys, 227
Display modes, 93–95
Displaying histogram, 96
Document conventions, x
Editing a point list, 53
Editing macros, 96–98
Electrical requirements, 100–101
EM CCD camera, 121–22
EM Filter field, 212
Emission filters, 18, 26, 47
changing, 129–32
specifications, 184
Enable fast acquisition, 209
Encoder error, 167
End Coordinates setting, 215
End of scan, 204
Enviromental chamber, 119
Environmental requirements, 101–4, 102
EX arrows, 228
EX Filter field, 211
EX Shutter button, 19, 229
Ex Shutter field, 212
Exact method for Z scan settings, 44
Excitation filters, 18, 26, 46
specifications, 184
Excitation shutter, 19
safety, 27
Exp field, 211
Experiment Macro Editor, 96–98
Experiment macros, 36–38
Experiments
2D image in 3 wavelengths, 17–19
3D, 38–44
and autofocus macros, 69–71
and focal planes, 41
and macros, 36–38
and marking points, 50–52
and optical section spacing, 41–44
and panel collection, 55–57
and sample thickness, 43–44
Design tab, 207–8
editing macros for, 96–98
guidelines for 3D, 41
macros folders, 221
photokinetic, 120
point visiting, 49–55
running, 35–57
setting up, 35–57
time-lapse, 44–49, 71–77
Explorer, 123
Exposure time, 87–89
AppliedPrecision
Appendix E: Resolve3D and Keypad Options
Eyepiece
filter wheel, 26, 129–30
filter wheel and safety, 9
filters, 18
focus, 17
Fast button, 227
Fiber Optic Cable, 11
Fiber optic module, 116, 151
adjust tilt of, 156–57
checking alignment \r, 153
Field stop aperture, 155–56
File system full error, 167
Filename extensions, 183
Filter sets, 17–19
Filters
activating multiplexed sets, 58–62
and changing wheels, 128–35
and safety, 9
and wheel modules, 128–35
changing emission wheel, 130
choosing, 17–19, 17–19
cleaning, 162
emission, 18
excitation, 18
excitation and emission peaks, 184
eyepiece, 18
eyepiece wheel change, 129–30
fluorescense, 184
live cell sets, 184
neutral density, 18
optional modules for, 119
Sedat polychroic, 152
sets of, 17–19
specifications, 184
standard optical, 106
usuable life span, 106
wheel calibration, 135–39
Find exposure, 201
Finding a sample, 23, 27–28, 84–87
Finding exposure time, 87–89
Fine focus, 17
Flare, 178
Flare from immersion oil, 178
Flat-panel monitor, 111
Fluorescence, 3
Fluorescence microscope, 106
Fluorochromes, 17–19
Focal planes, 41, 42
Focus, 17
Focus knob, 86
Focus lock, 17
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Focus point, 40, 41
Fourier transform, 178
Frames to average setting, 220
Frequency, operating, 100
Fuses, 12, 100
Gain setting, 220
Get Thickness setting, 210
Goodwin, Paul, 3
Guidelines for 3D experiments, 41
Hi Res Camera, 122
Histogram of intensity values, 96
Histograms, 206
History, 2–3
Humidity requirements, 103
IC/MIC
replacing fuses, 161
view of back, 150
IC/MIC switch, 142
IC/MIC switches, 143
Illumination
adjust fiber optic module tilt, 156–57
aligning the path, 150–61
ambient, 104
center field stop aperture, 155–56
checking alignment, 153–55
critical position adjustment, 158
exposure time, 87–89
Kohler sping position, 157–58
switching between methods, 89–91
testing path alignment, 159
types of, 89–91
Image acquisition, 22–27, 28–30
2D in 3 wavelengths, 17–19
and exposure time, 87–89
and live specimens, 68–82
collecting panels for, 55–57
Exposure time, 88
marking points for, 50–52
monitoring, 91–96
point visiting, 49–55
troubleshooting, 168–70
with continuous Z sweep, 78–80
Image control fields, 200–202
Image Display and Analysis, 4
Image Fusion tool, 82
Image intensity, 206
Images
2D in 3 wavelengths, 17–19
3D, 38–44
acquiring, 22–27, 28–30
and optical section spacing, 41–44
calibrate, 201
collecting panels for, 55–57
display mode, 218
distortion, 168
intensity values, 206
live cell, 5, 68–82
modes for viewing, 93–95
reference, 80–82
running experiments for, 35–57
saving, 30–32
scale values, 206
setting size, 202
symmetric flare, 177
viewing previews, 93
Immersion oil
flare from, 178
selecting, 177–78
Immersion oil kit, 85, 164–65
Purchasing from Applied Precision, 177
Instrument controller, 113
troubleshooting, 166–67
Intensity values, 87
Internet Address, Applied Precision, xii
Joystick, 19–20, 111, 225–29, 227
Keylight button, 227
Keypad, 19–20, 111, 225–29
Kohler illumination, 5, 89–91, 150, 155
Kohler spring, 151
position, 157–58
LCD monitor, 111
Lens
ID, 180
setting with Resolve3D, 202
Lens information window, 25
Light path, 16
Line requirements, 100
Listing points, 49–55
Live cell filter module, 119
Live cell filter sets, 184
Live cell imaging, 5, 68–82
Live specimens
tracking, 71–77
LMC Reset button, 226
lo setting, 206
Loading a point list, 53
Locating a sample, 84–87
Locking knob, 151
Long exposure times, 168
Macros, 36–38
editing, 96–98
Maintenance
adjust fiber optic module tilt, 156–57
centering field stop aperture, 155–56
changing MIC fuses, 161
checking fiber optic cable alignment, 153–
55
cleaning DeltaVision, 162
critical illumination position, 158
filter wheel calibration, 135–39
illumination path alignment, 150–61
Kohler spring adjustment, 157–58
moving DeltaVision, 162
system shutdown, 143
testing illumination path alignment, 159
Mark Point button, 229
Mark Point setting, 203
Marked Points List button, 205
Marking points, 50–52, 52
Replacing points in a list, 52
saving a point list, 53
Visiting marked points, 52
Master switch, 142
Material Safety Data Sheets (MSDS), 162
Maximum intensity, 206
Maximum Z test range setting, 223
Mean intensity, 206
Medium button, 227
Message pane, 207
Microscope interface chassis, 143
replacing fuses for, 161
Microscope Interface Chassis, 113
Middle of sample, 41
Middle of the sample option, 40
Minimum intensity, 206
Monitor switch, 142
Monitoring data acquisition, 91–96
Monitoring point visiting, 54
Move threshold, 75
Moving DeltaVision, 162
Multiplexed wavelength
experiment design, 35–66
filter set activation, 58–62
option, 35–66, 122
overview, 58
ND arrows, 227
Network connection, 103
Neutral density filters, 18
No button, 227
Number of optical sections field, 41
OAI. See Optical axis integration
OAI setting, 210
Objective
AppliedPrecision
Appendix E: Resolve3D and Keypad Options
cleaning, 32
damage prevention, 11
Oil calculator, 164–65
Oil, immersion. See Immersion oil
Optical axis integration, 68, 78–80
Optical components, 105–10
Optical filters, 106
damage prevention, 11
specifications, 184
Optical section spacing, 210
Optical sections, 41–44
Optical transfer function, 173–81
conversion from PSF, 178–81
file, 180
sample image, 179
Optional components, 118–23
Optional software, 123
OTF. See Optical transfer function
Overlap setting, 215
Overscan method for Z scan settings, 44
Pan tool, 204
Panel 1 and 2 Mark buttons, 228
Panel collection, 5, 55–57, 214–15
Personal data folder, 30–32
Phase contrast, 3
Photo sensor, 110
Photo sensor port, 151
Photo-bleaching, 87
Pixel size, 174–75
setting, 202
Point arrows, 227
Point list, 49–55, 197
dialog box, 52
editing, 53
Loading, 53
Saving, 53
Point Mark key, 51
Point marking, 50–52
Point spread function, 173–81
acquiring, 173–78
conversion to OTF, 178–81
file, 180
finding beads, 176
tools needed, 174
wavelength, 180
Point visiting, 4, 5, 41, 49–55
monitoring, 54
Points tab, 213–14
Polychroic mirror, 162
Post-autofocus Z offset setting, 223
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Power switches, 142
Prerequisites, 15
Previous experience method for Z scan
settings, 44
Primary light path, 58
Probes, 17–19
PSF. See Point spread function
QLM switch, 142
Quantifiable laser module, 26
Quantitative Processing, 4
Quit button, 227
Radial frequency, 179
Recommended Z step, 165
Recommended Z Step Size field, 40
Reference books, 185–92
Reference channel, 74–75
Reference images, 80–82, 212
Refraction, index of. See Refractive Index
Refractive index, 164–65
Nikon, 177
Olympus and Zeiss, 177
Refresh exposure conditions, 47
Refresh exposure conditions button, 211
Remove Trail button, 226
Repeatable slide holder, 25, 50, 85, 114–16
Replacing MIC fuses, 161
Replacing the tran light, 149–50
Requirements
altitude, 103
country-specific, 101
electrical, 100–101
environmental, 101–4
humidity, 103
line, 100
power cord, 100
temperature, 103
Reset button, 226
Resolution ratio, 165
Resolve3D
%T setting, 201
Acquire Image, 196
Autofocus options, 222–23
Aux Mag setting, 202
Bin setting, 202
bulb icon, 32, 143
calibrate images, 201
Calibration menu, 198–99
cell tracking options, 213
Channels tab, 211–12
Clear Stage Thumbnails, 197
Clear Stage Trails, 197
Command Line Interface, 197
Continuous Acquire, 196
Design Experiment tab, 207–8
Design/Run Experiment dialog box, 215–
17
Emission setting, 201
enable fast acquisition, 209
Excitation setting, 201
Experiment, 197
Exposure setting, 201
Exposure time, 88
Find exposure, 201
Help menu, 199
image control fields, 200–202
Image Size setting, 202
Info setting, 202
Lens setting, 202
menus, 195–200
message pane, 207
Options menu, 198
Panels tab, 214–15
Pixel Size setting, 202
Point List, 197
Points tab, 213–14
Scratch File, 196
Sectioning tab, 209–10
selecting cameras, 125–27
Settings dialog box, 217–25
shortcuts, 207
Snapshot, 196
Time-lapse tab, 212–13
toolbar, 199–200
window, 194
Retrace arrows, 228
Reviewing large areas, 55–57
ROI percent, 76
Run Experiment tab, 36–38
Running experiments, 35–57
Sadat, Dr. John W., 2
Safety, 8–12
Bright Light Exposure, 9
Danger of Shock, 9
using proper filters, 9
UV Exposure, 9
Warning Labels, 12
Sample thickness, 43–44
Sample thickness setting, 210
Saturation, 87–89, 88, 170
Save Image button, 228
Saving images, 30–32
Scale settings, 206
Scale values, 206
Scaletter, Beth, 3
Secondary light path, 58
Sectioning specimens, 38–44
Sectioning tab, 209–10
Sedat polychroic filter, 152
Settings dialog box, 217–25
action buttons, 225
Autofocus tab, 222–23
Display tab, 218–19
Files tab, 221–22
Imaging tab, 219–20
Misc tab, 223–25
Seubert, Ron, 2
Shutting down DeltaVision, 32–33, 143
Slide holder, 114–16
adapter, 115
Slow button, 227
Snapshot, 196
softWoRx, 1
softWoRx Explorer, 123
softWoRx software, 117
softWoRx Suite, 118
advanced option, 123
softWoRx Workstation, 114
Troubleshooting, 167
Space requirement, of system, 101
Spherical aberration, minimizing, 177
Stage controls, 202–6
Stage speed, 86
Standard filename extensions, 183
Standard filter sets, 107
Standard microscope slide, 85
Start Coordinates setting, 215
Statistics, 96
Step Decrease and Increase buttons, 228
Stopping DeltaVision, 32–33
Sub-image, 180
Symmetric flare, 177
Target temperature display, 220
Technical support, xii, 103
Temperature requirements, 103
Theoretical OTF. See Optical transfer function
Thickness of sample, 43–44
Tilt screws, 151
Time-lapse, 4, 5
setting up experiments, 44–49
Time-lapse tab, 48, 212–13
TIRF module, 121
Tool kit, 116
Toolbar, Resolve3D, 199–200
AppliedPrecision
Appendix E: Resolve3D and Keypad Options
Top of sample, 41
Top of Sample setting, 203
Total Internal Reflection Fluoresence
Module. See TIRF module
Tracking cells, 71–77
Tracking methods, 74
Trans Shutter button, 19, 229
Transfer speed setting, 220
Transmitted light source, 19
plugging in, 150
replacing, 149–50
Troubleshooting, 166–70
encoder error, 167
image quality, 135, 168–70
instrument controller, 166–67
light transmittance, 135
softWoRx Workstation, 167
stalled image sequence, 167
Turning off DeltaVision, 32–33
Ultraviolet light, 9
University of California, San Francisco, 2
URL, Applied Precision, xii
Use photosensor setting, 221
Viewing image previews, 93
Viewing modes, 93–95
Viewing statitics, 96
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Visit Bottom setting, 204
Visit Middle setting, 204
Visit Top setting, 204
Visiting points. See Point visiting
Voxels, 56, 180
Warning labels, 12
Workstation switch, 142
X adjustment screw, 151
X and Y arrows keys, 228
X step size setting, 205
Xenon bulb replacement, 144–48
Xenon light source, 109
and bulb replacement, 144–48
Y adjustment screw, 151
Y step size setting, 205
Yes button, 227
Z arrow keys, 228
Z focus, 17
Z sectioning, 38–44, 43
methods for, 44
Z slider, 205
Z step size setting, 205
Z sweep acquisition, 68, 78–80
Z1 Mark and Z2 Mark buttons, 228
Zoom tool, 205
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