Olympus xcellence User Manual [ru]
User Manual
Imaging Software
Life Science Microscopy
Imaging Software for
Life Science Microscopy
Imaging Software for
Life Science Microscopy
Software Manual for Imaging Stations
Version 1.1
Imaging Excellence
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Software Manual Contents
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Contents
Chapter 1
Imaging stations for life
science experiments
Chapter 2
A system chart and a list
of all components
1 Introduction ..................................................................................1
2
System Overview..........................................................................
2.1 System Chart....................................................................................4
2.2 Hardware..........................................................................................5
2.2.1 Motorized Microscope Modules....................................................... 5
2.3
Software ...........................................................................................6
Chapter 3
Getting you started – a
quick guide through the
main features of the
imaging stations and how
to use them
Chapter 4
Simple ways to take
images, how to control
the hardware modules
and which parameters to
set
3 Brief Introduction to the Software and First Steps...................
3.1 The User Interface .........................................................8
3.1.1 The Image Manager..........................................................................
3.1.2 The Viewport Manager ...................................................................1
3.1.3 The Viewport................................................................................... 1
3.2 Simple Image Acquisition ............................................................... 12
3.3 Saving Images – The Database ...................................................... 13
3.4 Loading Images .............................................................................. 14
3.5 Conducting Experiments with the Experiment Manager................ 14
3.6 Displaying Multi-Color Images .......................................................16
3.7 Displaying Sequences ....................................................................17
4 Image Acquisition and Hardware Control ..............................1
4.1 Simple Image Acquisition ............................................................... 20
4.1.1 Snapshot and Live View .................................................................2
4.1.2 AVI Recorder .................................................................................. 21
Camera Control ..............................................................................21
4.2
4.3 Illumination Control.........................................................................25
4.4 Microscope Control........................................................................26
4.5 Motorized Stage Control ................................................................29
4.5.1 Defining a Positions List .................................................................2
4.5.2 Correcting a Positions List .............................................................3
4.5.3 Calibrating the Motorized Stage..................................................... 32
4.6
Autofocus .......................................................................................34
4.6.1 Autofocus options…....................................................................... 35
Executing an Autofocus Scan ........................................................3
4.6.2
Chapter 5
Beyond snapshots:
setting up simple and
complex experiments in
an intuitive, graphical
way
5 Experiment Manager.................................................................. 3
5.1 A Graphical Tool ............................................................................. 38
5.2 Concept of Usage ..........................................................................39
5.2.1 Experiment Manager Components ................................................ 3
5.2.2 Arrangement and Customization.................................................... 4
5.3 Setting Up Experiment Plans .........................................................42
5.3.1 Types of Graphical Icons and the General Principles of Usage..... 42
5.3.2
The Command Symbols and Their Properties Pages ....................43
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5.3.3 Types of Experiments .....................................................................61
Conducting Experiments / Data Acquisition .................................. 70
5.4
5.4.1 Opening a Database .......................................................................7
5.4.2 Executing an Experiment ................................................................71
Data Storage and Preferences........................................................75
5.4.3
Chapter 6
Touching up the image
display by brightness,
contrast and color adjustment and navigation
through multidimensional image sets
6 Image Display and Navigation...................................................7
6.1 The Viewport .................................................................................. 78
6.2 Image Display................................................................................. 80
6.2.1 General............................................................................................8
6.2.2 Adjust Display…..............................................................................8
6.2.3 Auto Adjust......................................................................................8
6.2.4 White Balance .................................................................................8
6.2.5 Black Balance .................................................................................85
6.2.6
Gray Scale.......................................................................................85
Fluorescence Color.........................................................................8
6.2.7
6.2.8 Edit Fluorescence Color… ..............................................................8
6.2.9 False-Color… ..................................................................................87
Edit False-Color…...........................................................................8
6.2.10
6.3 Image Navigation ........................................................................... 95
6.3.1 General............................................................................................95
Multi-Color Images..........................................................................95
6.3.2
6.3.3
Displaying Different Color Bands in the Tile View Mode.................9
6.3.4 Time-Lapse Sequences ..................................................................9
6.3.5 Z-Stacks..........................................................................................9
6.3.6 Multi-dimensional Sequences.........................................................9
6.3.7 Parallel Navigation in Multiple Viewports........................................9
6.4 Projections and Extended Focal Imaging ...................................... 99
6.4.1 Projections Along the Z and Time Axes..........................................9
6.4.2 EFI – Extended Focal Imaging ......................................................10
6.5 Fluorescence and Transmission Image Overlay .......................... 101
6.6 Intensity Modulated Display......................................................... 102
Chapter 7
Basic processing routine
like size calibration,
overlay drawings, extraction and conversion of
image sets
7 Image Data Handling................................................................10
7.1 Calibrate Images .......................................................................... 104
7.1.1 Why Calibrate Images?.................................................................10
7.1.2 Calibrating the Camera Channel...................................................10
7.1.3 XY-Calibration...............................................................................10
7.1.4 Z-Calibration .................................................................................10
7.2 Scale Bar...................................................................................... 109
7.2.1 General..........................................................................................10
7.2.2 Setting the Scale Bar Properties...................................................10
7.2.3 Show in Viewport ..........................................................................11
7.2.4 Draw into Overlay..........................................................................11
7.3 Show Markers, Time and Z Information....................................... 111
7.4 Grid…........................................................................................... 111
7.5 Overlays ....................................................................................... 113
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7.5.1 General ......................................................................................... 113
Activating the Overlay Toolbar .....................................................113
7.5.2
7.5.3
Creating and Editing Overlays...................................................... 11
7.6 Separate .......................................................................................118
7.7 Extract…....................................................................................... 118
7.8 Combine…....................................................................................119
7.8.1 General ......................................................................................... 11
7.8.2 Combining Data Sets ................................................................... 12
7.9 Convert Image ..............................................................................121
7.9.1 General ......................................................................................... 121
To 8-Bit......................................................................................... 121
7.9.2
7.9.3
To 16-Bit....................................................................................... 122
7.9.4
To RGB (3x8-Bit) .......................................................................... 122
Invert............................................................................................. 122
7.9.5
7.10
Image Information.........................................................................123
7.10.1 The General Tab ...........................................................................12
7.10.2 The Dimensions and Markers Tabs.............................................. 125
7.11
Image Statistics ............................................................................ 126
Chapter 8
Data changing tools to
improve the image quality, for example, by increasing the contrast or
reducing the noise
8 Image Processing ....................................................................12
8.1 Shading Correction.......................................................................129
8.1.1 General ......................................................................................... 12
8.1.2 Define Shading Correction ...........................................................12
8.1.3 Executing a Shading Correction................................................... 132
Bleaching Correction.................................................................... 133
8.2
8.3 Thresholds and Binarization ......................................................... 135
8.3.1 Set Thresholds.............................................................................. 135
Set Color Thresholds.................................................................... 14
8.3.2
8.3.3 Binarize......................................................................................... 14
8.4 Filters ............................................................................................ 145
8.4.1 General ......................................................................................... 145
8.4.2
Sharpen I ......................................................................................14
8.4.3 Sharpen II .....................................................................................14
8.4.4 Differentiate X ...............................................................................14
8.4.5 Differentiate Y............................................................................... 14
8.4.6 Laplace I .......................................................................................15
8.4.7 Laplace II ...................................................................................... 15
8.4.8 Mean............................................................................................. 151
8.4.9
Median.......................................................................................... 151
Pseudo Filter................................................................................. 152
8.4.10
8.4.11
Sobel ............................................................................................ 153
8.4.12
Roberts ......................................................................................... 153
Reimer ..........................................................................................15
8.4.13
8.4.14 User Filter .....................................................................................15
8.4.15 NxN............................................................................................... 15
8.4.16 Lowpass .......................................................................................157
8.4.17
Edge Enhance ..............................................................................157
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8.4.18 Rank ..............................................................................................15
8.4.19 Sigma ............................................................................................15
8.4.20 DCE – Differential Contrast Enhancement ....................................16
8.4.21 Separator ......................................................................................161
Morphological Filters.................................................................... 164
8.5
8.5.1 Define Morphological Filter... ........................................................165
8.5.2
Erosion ..........................................................................................167
Dilation ..........................................................................................16
8.5.3
8.5.4 Morph. Open.................................................................................16
8.5.5 Morph. Close.................................................................................16
8.5.6 Gradient ........................................................................................17
8.5.7 Top Hat Bright...............................................................................17
8.5.8 Top Hat Dark.................................................................................171
Distance Bright..............................................................................171
8.5.9
8.5.10
Distance Dark................................................................................172
8.5.11
Ultimate Erode Bright....................................................................172
Ultimate Erode Dark......................................................................173
8.5.12
8.5.13
Skeleton ........................................................................................173
8.5.14
Separate Particles.........................................................................17
8.6 Arithmetic Operations…............................................................... 175
8.7 Image Geometry .......................................................................... 177
8.7.1 Resize............................................................................................177
Rotate............................................................................................17
8.7.2
8.7.3 Mirror.............................................................................................18
8.7.4 Align ..............................................................................................18
8.7.5 Auto Align Z...................................................................................181
8.7.6
Shift Correction .............................................................................181
8.8
Deblurring and Deconvolution ..................................................... 182
8.8.1 General..........................................................................................182
8.8.2
Edit Image Parameters..................................................................182
8.8.3
No Neighbor..................................................................................18
8.8.4 Nearest Neighbors ........................................................................185
8.8.5
Wiener Filter ..................................................................................18
8.8.6 3-D AMLE Deconvolution: Advanced Maximum Likelihood .........187
Chapter 9
The software contains a
host of measuring tools
in a special measurement
environment
9 Measurements .......................................................................... 18
9.1 Measurements Toolbar ................................................................ 190
9.2 Drawing Tools for Length and Area Measurements .................... 191
9.2.1 Point..............................................................................................191
Touch Count .................................................................................191
9.2.2
9.2.3
Length ...........................................................................................192
9.2.4
Angle .............................................................................................19
9.2.5 Area...............................................................................................195
9.3
Magic Wand ................................................................................. 200
9.3.1 Magic Wand Options ....................................................................201
Results ......................................................................................... 202
9.4
9.4.1 Move Origin...................................................................................202
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9.4.2 Create Measurement Sheet.......................................................... 203
Deleting Measurement Results..................................................... 20
9.4.3
9.4.4 Image Link ....................................................................................205
9.4.5
Show/Hide Statistic ...................................................................... 205
Select Measurements................................................................... 205
9.4.6
9.4.7
Define Statistics............................................................................ 207
9.4.8
The PreferencesMeasure Tab................................................... 20
9.4.9 Measurement Sheets: Statistics................................................... 20
Chapter 10
Sophisticated analyses of
fluorescence intensities,
mostly for time sequences and Z-stacks
10 Intensity Analyses ....................................................................211
10.1 Pixel Value ....................................................................................212
10.2 Histogram .....................................................................................213
10.3 Line Profiles: Intensity ..................................................................214
10.3.1 Horizontal Line Profile................................................................... 21
10.3.2 Vertical Line Profile....................................................................... 215
Arbitrary Line Profile .....................................................................215
10.3.3
10.3.4
Average Intensity of Neighboring Pixels....................................... 21
10.4 Regions of Interest – ROIs............................................................ 217
10.4.1 General ......................................................................................... 217
10.4.2
Drawing ROIs................................................................................ 217
10.4.3
ROI Measurements (2-D) .............................................................. 22
10.5 Background Subtraction... ...........................................................221
10.5.1 General ......................................................................................... 221
10.5.2
Subtracting the Image Background ............................................. 221
Intensity Kinetics in Time and Z ...................................................223
10.6
10.7 DeltaF / F (∆F/F) Analysis .............................................................224
10.7.1 General ......................................................................................... 22
10.7.2 Generating a (∆F/F) sequence ...................................................... 22
10.8 Ratio Analysis ...............................................................................226
10.8.1 General ......................................................................................... 22
10.8.2 Generating a Ratio Sequence ...................................................... 227
10.9
Spectral Unmixing ........................................................................228
10.9.1 Application.................................................................................... 22
10.9.2 The Problem ................................................................................. 22
10.9.3 The Solution.................................................................................. 23
10.9.4 How Does it Work?....................................................................... 231
Spectral Unmixing with .............................................. 232
10.9.5
10.9.6
Calibration ....................................................................................233
10.9.7
Unmixing....................................................................................... 23
10.9.8 Unmixing of Color Camera Images ..............................................23
10.10 Phase Color Coding and Analysis ................................................ 237
10.10.1 Phase Color Coding .....................................................................237
10.10.2
10.11 Colocalization ...............................................................................238
10.12 The FRET Software Module .........................................................240
10.12.1 Image Acquisition ......................................................................... 24
10.12.2 FRET Image Correction Factors................................................... 242
Phase Analysis ............................................................................. 23
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10.12.3 FRET Analysis ...............................................................................245
Kymogram.................................................................................... 251
10.13
Chapter 11
Analyses of intensity
kinetics of fluorescence
image time series
Chapter 12
Images generated during
an experiment are automatically stored in structured databases –
analytical data can be
added
Chapter 13
Setting personal preferences of the software
user interface
11 Graph Display and Graph Analysis .........................................25
11.1 Graph Documents........................................................................ 254
11.2 The Graph Window ...................................................................... 255
11.2.1 The Cursor: Changing the XY Scaling in the Diagram ..................255
11.2.2
The Cursor: Measuring Individual Graph Points ...........................25
11.2.3 The Graphs Button Bar .................................................................25
11.3 The Graph Menu .......................................................................... 260
11.3.1 Markers and Labels.......................................................................26
11.3.2 Protecting and Deleting a Graph ..................................................263
Graph Information... ......................................................................263
11.3.3
11.3.4
Sheet.............................................................................................26
12 Database ...................................................................................26
12.1 Directories for Data Storage ........................................................ 270
12.2 Open Database... ......................................................................... 271
12.3 New Database.............................................................................. 272
12.4 The Database Features................................................................ 273
12.4.1 General Remarks ..........................................................................273
12.4.2
The Database Window..................................................................27
12.4.3 Adjusting the Database Window...................................................275
Working with the Database.......................................................... 278
12.5
12.5.1 Loading Documents......................................................................27
12.5.2 Inserting Documents.....................................................................27
12.5.3 Query.............................................................................................28
12.5.4 Administration: Defining Organizational and Database Fields......281
13 The Special Menu and the Window Menu .............................28
13.1 Macros ......................................................................................... 284
13.1.1 General..........................................................................................28
13.1.2 Record Macro ...............................................................................28
13.1.3 Executing Macros .........................................................................285
Stop Macro Recorder ...................................................................287
13.1.4
13.1.5
Run Macro.....................................................................................287
13.1.6
Single Step....................................................................................287
Reset Interpreter ...........................................................................28
13.1.7
13.1.8 Set as Default-Macro ....................................................................28
13.1.9 Define Macros...............................................................................28
13.2 Add-In Manager... ........................................................................ 291
13.3 Define Menu Bar…....................................................................... 292
13.4 GUI Configuration ........................................................................ 295
13.4.1 Reset .............................................................................................29
13.4.2 Load... ...........................................................................................29
13.4.3 Save... ...........................................................................................297
Preferences... ............................................................................... 298
13.5
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13.5.1 The PreferencesImage Tab....................................................... 29
13.5.2 The PreferencesView Tab .........................................................30
13.5.3 The PreferencesFile Tab ........................................................... 302
13.5.4
The PreferencesMeasure Tab................................................... 307
The PreferencesModule Tab..................................................... 30
13.5.5
13.5.6 The PreferencesGraph Tab....................................................... 311
13.5.7
The PreferencesDatabase Tab ................................................. 312
Window......................................................................................... 313
13.6
13.6.1 Minimize All................................................................................... 313
13.6.2
Close All........................................................................................ 313
Document Manager… ..................................................................313
13.6.3
13.6.4
Viewport Manager ........................................................................315
13.6.5
Image Manager............................................................................. 315
Status Bar..................................................................................... 31
13.6.6
13.6.7 Command Window....................................................................... 31
Chapter 14
A complete, integrated
development environment for macros based
on the programming
language Imaging C
Chapter 15
Telling the software
which hardware modules
are available and what
the current installation is,
for example, what filters
are loaded
14 Imaging C ..................................................................................31
14.1 General .........................................................................................320
14.2 New Module... ..............................................................................321
14.3 Open Module................................................................................327
14.4 Add to Module..............................................................................328
14.5 Save Module Configuration .......................................................... 329
14.6 Edit Module... ...............................................................................330
14.7 Build Module ................................................................................331
14.8 Close Module ...............................................................................332
14.9 About Module ............................................................................... 332
14.10 Module Manager... .......................................................................333
14.10.1 The Define Search Path for Modules Dialog Box ......................... 33
14.11 Browser... .....................................................................................337
14.12 Find Symbol..................................................................................338
14.13 Goto Definition.............................................................................. 339
14.14 Quick Watch... ..............................................................................340
14.15 Watch Variables............................................................................ 341
14.16 Toggle Breakpoint ........................................................................342
14.17 Edit Breakpoints ...........................................................................344
15 Configuration........................................................... 34
15.1 The Illumination System MT20 / MT10.........................................348
15.1.1 Configuring the Excitation Filters .................................................34
15.1.2 Burner Configuration .................................................................... 35
15.1.3 Using MT20 / MT10 without the Imaging Software...................... 351
Configuring the Microscope ......................................................... 351
15.2
15.2.1 General Configuration .................................................................. 351
15.2.2
Z-Drive Configuration ................................................................... 353
Configuration of the Objectives.................................................... 35
15.2.3
15.2.4 Configuration of the Fluorescence Filter Turret............................ 355
15.2.5
Configuration of the Transmission Contrast Inserts..................... 35
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Configuration of the Filters of a Filter Wheel.................................357
15.2.6
Definition of Image Types ............................................................ 358
15.3
15.4 Configuration of Additional Shutters............................................ 360
15.5 Configuration of the PIFOC.......................................................... 361
15.6 Configuration of the Motorized Stage.......................................... 362
15.7 The UCB Control Box Light Panel ............................................... 363
15.8 Parfocality Correction of Objectives ............................................ 364
15.9 Configuration of the DV2/Dual-View™ Micro-Imager.................. 365
15.9.1 Configuring the Emission Filters ...................................................36
15.9.2 Configuring the Image Types........................................................367
Chapter 16
The details to take care
of when installing a new
software version
16 Installing the Software ...........................................36
16.1 Updating the Software............................................... 370
16.2 Updating the Hardware Control ................................ 372
16.3 Selecting the Camera................................................................... 373
16.4 Single-User Systems (Administrator Users)................................. 374
16.5 Multi-User Systems...................................................................... 375
16.6 PC-to-Controller Network Connection ........................................ 377
Software Manual Chapter 1 – Introduction 1
1 Introduction
Thank you very much for purchasing Olympus' state of art
confidence in our products and service. It is Olympus' main objective to provide you with solutions
able to meet your experimental demands and thus pave the way to your scientific success.
Imaging Station and for your
2 Chapter 1 – Introduction
Introduction
The concept and technology of introduces the next generation of imaging workstations.
is designed as a modular imaging system for a broad range of life science experiments
that supports the Olympus microscopes of the IX and BX series.
successor of the cell^M /cell^R Imaging Software 3.3.
Hallmarks of the systems are:
• The unique all-in-one Illumination System MT10 or MT20 for fast wavelength switch and at-
tenuation to meet the experimental requirements for fast real-time acquisition by highly sensitive
digital cameras.
• The Real-Time Controller of
synchronize the all the hardware devices and modules. This additional independent plug-in CPU
board assures highest accuracy in experiment timing (temporal resolution: 1 ms; precision <
0.01 ms). In practice this ensures that illumination of the specimen can be strongly limited to the
acquisition of the image. As a consequence bleaching and photo damage of the specimen can
be minimized.
• The System Coordinator of
all the hardware devices and modules (temporal resolution: 1 ms). It carries out all the tasks that
the Real-Time Controller does, but lacks its timing precision and ability to run all tasks in parallel.
• The sophisticated
tures an intuitive and user-friendly graphical drag-and-drop interface, the Experiment Manager,
for setting up and executing even the most complex experiments in a convenient and concise
way. A structured database for multi-dimensional data handling (xyz, time, color) is also included, as well as tools for image processing, image analyses, and more complex analysis, like
rationing, ∆F/F, FRET, and spectral unmixing. The integrated Imaging C Module and Macro Recorder give the opportunity for advanced users to customize applications and automate functions.
This manual is the complete documentation necessary for using the Olympus imaging station
correctly and efficiently.
imaging software is a powerful all-embracing platform that fea-
real-time systems, a hyper-precision control board to
professional systems, a control board to synchronize the
1.1 is the immediate
Special care has been undertaken for this manual to guarantee correct and accurate information,
although this is subject to changes due to further development of the
Thus, the manufacturer cannot assume liability for any possible errors. We would appreciate reports of any mistakes as well as suggestions or criticism.
If you find any information missing in this manual or you need additional support, please contact
your local Olympus dealer.
imaging station.
Software Manual Chapter 2 – System Overview 3
2 System Overview
The following chapter gives you a short overview of the basic
out additional peripherals). The system consists of different hardware and software devices, which
are full, integrated.
system components (with-
4 Chapter 2 – System Overview
2.1 System Chart
The system chart shown exemplifies the different components of a standard system and
how they are interconnected.
Software Manual Chapter 2 – System Overview 5
2.2 Hardware
The hardware devices of the imaging station are listed below:
• Olympus microscope of the IX or BX series, according to your specification.
• CCD camera (Olympus XM10T, Hamamatsu ORCA-R2 or other) or EMCCD camera (Hamamatsu
C9100-13, Andor iXon or other)
• Illumination System, typically MT10 / MT20 bearing the arc burner (150W Xe or 150W Xe/Hg),
the motorized filter wheel (8 positions), attenuator and shutter.
• System Coordinator / Real-Time Controller for multi-task acquisition: bears an I/O panel with
seven BNC plugs to trigger peripherals, RS-232 socket to integrate external devices such as
motorized stages, analog OUT for piezo objective or nosepiece movers (PIFOC) and an Olympus
Device Bus (ODB) interface for fast filter wheels, mirror unit turret and transmission shutter.
• Computer: latest generation PC with all standard features, modified for hardware control and
peripherals integration, monitor.
• System-specific accessories (filter sets etc.).
2.2.1 Motorized Microscope Modules
supports the following motorized IX2 and BX2 microscope components:
• z-drive
• fluorescence filter turret
• transmission condenser
• nosepiece
• ocular-to-camera switch
• bottom-to-side port switch
• observation filter wheel
• transmission shutter
6 Chapter 2 – System Overview
2.3 Software
• ObsConfig Configuration Software to configure the Illumination System MT10 / MT20.
•
experiments, Viewport, Viewport Manager, and Image Manager for managing and displaying the
images on the desktop, Image and Graph Analysis tools to analyze the acquired data, and a database for storing and archiving the images.
•
Controller and the MT10 / MT20 electronics
Imaging Software integrating the Experiment Manager for planning and executing
software updates only: Update Software for the System Coordinator / Real-time
Software Manual Chapter 3 – Brief Introduction and First Steps 7
3 Brief Introduction to the
Software and First Steps
The following sections explain the basic features and functions. They also introduce the
most important terms used in the software and in this manual and thus should help you to get
started.
For you to follow the contents of this chapter it is necessary that the system (hardware and software) has been installed and properly configured. For a detailed description of the system installation and configuration read Chapter 15,
the Hardware Manual, especially Chapter 7, System Assembly and Adjustment.
3.1
The User Interface ..........................................................8
3.1.1 The Image Manager.......................................................................... 9
3.1.2 The Viewport Manager ...................................................................10
3.1.3 The Viewport...................................................................................10
3.2 Simple Image Acquisition ............................................................... 12
3.3 Saving Images – The Database ...................................................... 13
3.4 Loading Images .............................................................................. 14
3.5 Conducting Experiments with the Experiment Manager................ 14
3.6 Displaying Multi-Color Images .......................................................16
3.7 Displaying Sequences ....................................................................17
Configuration, of this manual carefully as well as
8 Chapter 3 – Brief Introduction and First Steps
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3.1 The User Interface
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icon on your desktop to start the software or open it via
. The screenshot below shows the program's graphical user inter-
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Double-click the
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face (GUI). It is composed of:
a Menu bar: access to pull-down menus with assorted commands.
b Tool bar: direct access to the most important commands for image acquisition, processing and
analysis.
Software Manual Chapter 3 – Brief Introduction and First Steps 9
c Status bar: shows the connected CCD camera and displays information depending on the ac-
tive functions.
d Viewport Manager (top left below button bars): shows a thumbnail of the active image; a red
rectangle indicates the current zoomed-in sector of the image in the Viewport. The rectangle
can be mouse-dragged across the thumbnail to bring a different area into display.
e Image Manager (underneath the Viewport Manager): consists of two parts:
— the Operand Box, an operational area to determine source and destination buffers for
image processing
— the Image Buffer Box listing the currently loaded images or graphs. Four tabs are
available:
1 The List View that lists the names, the XY size and the bit depth of the images.
2 The Gallery View that shows thumbnails of the images.
3 The Graph View that shows thumbnails of loaded graphs.
4 The Measurement View that lists the results of area and length measurements in
images.
f Documents Area which always contains:
g Viewport: displays the active image or a selection of currently loaded images.
h Graph document: the currently active graph (if a corresponding analysis has been carried out).
It is minimized when
is started.
i Additionally the Documents Area may contain:
j Database Documents
k Data Sheets
l Text Documents and Macros
3.1.1 The Image Manager
In the Image Manager different image types, like single color images, multi-color images, or the
various image sequences (z-stack; time-lapse, etc.), are represented by different symbols.
The highlighted image frame in the Image Manager field is active and displayed in the Viewport
Manager and the Viewport.
single-color image
Z-stack
single-color time-lapse
single-color Z-stacks in time-lapse
multi-color image
multi-color Z-stack
multi-color time-lapse
multi-color Z-stacks in time-lapse
10 Chapter 3 – Brief Introduction and First Steps
3.1.2 The Viewport Manager
The image in the Viewport Manager in the top left corner of the
rectangle. It represents the region of the image currently displayed in the Viewport – if the image is
zoomed to an extent that is larger than the Viewport. The rectangle is interactive: It can be freely
moved within the Viewport Manager to display different areas in the Viewport. It can also be resized
by mouse drag to change the zoom factor in the Viewport display.
window shows a red
3.1.3 The Viewport
The Viewport window allows displaying one image or a number of images at the same time. The
number of Viewports to be displayed and their arrangement can be set using the Arrange View-
ports button in the toolbar of the Image window. Just mark the columns and rows by moving the
mouse cursor over the schematic Viewport, which opens with 4x4 image icons symbolizing independent image areas. The maximum number of images that can be shown at one time is 16 (4x4)
by default.
This setting can be increased to 5x5 as maximum via the Display Properties. Right-click on the
Viewport Manager to open the Display Properties window and change the Viewport limit entry
accordingly.
Software Manual Chapter 3 – Brief Introduction and First Steps 11
12 Chapter 3 – Brief Introduction and First Steps
3.2 Simple Image Acquisition
Live View, Snapshot, Camera Control, Illumination Settings
The most important tools for simple image acquisition are the Live View and Snapshot buttons
and the control boxes that are opened by the Camera Control and Illumination Settings buttons;
all can be found in the Acquisition toolbar.
In the following a typical procedure for the acquisition of a fluorescence image will briefly be described. For detailed explanations, see Chapter 4, Image Acquisition and Illumination Control.
Make sure to select the correct objective and fluorescence filter and place your sample on the
stage. The usage of the microscope will not be explained here.
1. Click Illumination Settings to open the Illumination system MT20 / MT10 control box.
2. Click Main switch / Burner ON to ignite the burner. (For a stable light output, wait about
10 min.)
3. Click on one of the Excitation filter buttons to select an illumination color.
4. Click Camera Control to open the corresponding window. Reasonable starting settings
are:
– Binning 2x2
– Exposure time 50 ms
– Brightness adjustment automatic
These settings have to be adjusted in the course of the working session.
5. Direct the light path of the microscope to the camera. (Of course, focusing can be done
via the ocular, but this will not be explained here.)
6. Click Shutter in the Illumination System MT20 / MT10 control box to illuminate the
specimen.
7. Click Acquire in the Acquisition toolbar or the Camera Control window. The following
happens:
Software Manual Chapter 3 – Brief Introduction and First Steps 13
– The camera starts acquiring images at maximal speed. None is stored; instead, the
newest image overwrites the previous one in the temporary buffer.
– The current image is displayed in the active Viewport.
– A single image icon appears in the Image Buffer Box.
8. Focus your sample with the microscope Z-drive. (Even if the optional PIFOC is available
for focus change, its total range may be too narrow to find the focus at the beginning.
For this reason it is usually better to start with the Z-drive.)
9. If necessary, change the Binning factor in the Camera Control window: 1x1 in order to
get highest spatial resolution, larger factors increase the signal intensity at the cost of
resolution.
10. Adjust the Exposure time for a good signal-to-noise ratio.
11. Click Snapshot in the Acquisition toolbar or the Camera Control window to stop the
Live View. The very last image is being stored as a Snapshot in the Image Buffer Box
and displayed in the Viewport.
12. Click Shutter in the MT20 / MT10 control box to stop the illumination.
13. Save your image as described in the next chapter.
Select an empty buffer in the Image Manager if you want to acquire a new image, otherwise the current image will be overwritten and lost: snapshots are only stored temporarily and need to be saved.
3.3 Saving Images – The Database
To save a snapshot in the most basic way select FileSave from the menu bar or use the short cut
<Ctrl + s>. The snapshot will be stored in a 16-bit tiff format by default. Other data types can be
chosen as usual in the Save Image As window. As with other files, you have to give a name and
select the destination (path) of the storage.
features a database module for the storage of images and entire experiments including
Experiment Plans, data sets, analyses and so on. The module is explained in detail in Chapter 12,
Database.
14 Chapter 3 – Brief Introduction and First Steps
The command Open Database… to load an existing one can be found redundantly in the menus
File, Acquisition and Database. Database files carry the extension *.apl. New databases can be
created with the command New Database…, to be found in the same menus.
In order to be able to store your images in the database, first you have to create an experiment
folder via DatabaseInsertExperiment… (or use an existing one). To store an image in the
database just drag it from the Image Buffer Box into the experiment folder. You will be asked in a
dialog box to give it a name.
Images acquired via the Experiment Manager will be stored automatically in a database.
3.4 Loading Images
To load images that are not stored in a database and to display them in the Viewport use the command FileOpen (short-cut <Ctrl + o>) or click on the Open button in the toolbar. As usually you
have to navigate to the storage folder of the file and select it.
Open the experiment folder in the database to load an image or an image sequence from it (see
previous chapter 3.3). In the Structure Strip on the left hand side of the database window you will
find the Image Icon and in the Gallery Field – the Image Thumbnail. Drag either the icon or the
thumbnail into the Viewport or the Image Manager to load the image set.
3.5 Conducting Experiments with the Experiment
Manager
Experiment Manager
In general, most imaging applications in life science are rather complex and go beyond taking simple snapshots, for example, multi-color imaging, time-lapse imaging, ion imaging with ratiometric
fluorescence dyes, multi-dimensional imaging, etc. The
Experiment Manager, an easy-to-use and intuitive tool to plan, configure and execute even the
Imaging Software includes the
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