Olympus xcellence User Manual [ru]

Imaging Software

Life Science Microscopy

User Manual

Imaging Software for Life Science Microscopy

Software Manual for Imaging Stations

Version 1.1

Imaging Excellence

We at Olympus Soft Imaging Solutions GmbH have tried to make the information in this manual as accurate and reliable as possible. Nevertheless, Olympus Soft Imaging Solutions GmbH disclaims any warranty of any kind, whether expressed or implied, as to any matter whatsoever relating to this manual, including without limitation the merchantability or fitness for any particular purpose. Olympus Soft Imaging Solutions GmbH will from time to time revise the software described in this manual and reserves the right to make such changes without obligation to notify the purchaser. In no event shall Olympus Soft Imaging Solutions GmbH be liable for any indirect, special, incidental, or consequential damages arising out of purchase or use of this manual or the information contained therein.

No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the prior permission of Olympus Soft Imaging Solutions GmbH.

© 2003 – 2010 by Olympus Soft Imaging Solutions GmbH. All rights reserved.

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Tel: +49 89 - 89 55 805 660

Fax +49 89 - 89 55 805 6606 Email: [e-mail]

www.olympus-sis.com

LICENSE AGREEMENT between END USER and OLYMPUS SOFT IMAGING SOLUTIONS regarding the OSIS SOFTWARE PRODUCT.

IMPORTANT-READ CAREFULLY: Below you will find the contractual agreements governing the use of the OSIS SOFTWARE PRODUCT. These conditions apply to you, the user, and to OLYMPUS SOFT IMAGING SOLUTIONS. With any of the following actions you explicitly agree to be bound by the conditions of this contract: purchasing the software, opening the package, breaking of one of the seals or using the software.

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§ 2. User rights

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(1)OLYMPUS SOFT IMAGING SOLUTIONS guarantees for the period of 12 months after the date of purchase, that the software works in all major aspects according to the descriptions in the manuals. OLYMPUS SOFT IMAGING SOLUTIONS, as the producer of the software, provides this warranty. It does not replace or restrict other warranties or liabilities provided to the User by local or other sales people or organizations. OLYMPUS SOFT IMAGING SOLUTIONS does not guarantee that the software is defect free; that the software fulfills the specific requirements of the User, or that the OBS SOFTWARE PRODUCT works with other software provided by the User.

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Software Manual

Contents

Contents

Chapter 1

Imaging stations for life science experiments

Chapter 2

A system chart and a list of all components

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

Chapter 5

Beyond snapshots: setting up simple and complex experiments in an intuitive, graphical way

1	Introduction ..................................................................................	1
2	System Overview..........................................................................	3
2.1	System Chart....................................................................................	4
2.2	Hardware ..........................................................................................	5
2.2.1	Motorized Microscope Modules.......................................................	5
2.3	Software ...........................................................................................	6
3	Brief Introduction to the Software and First Steps...................	7
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
4	Image Acquisition and Hardware Control ..............................	19
4.1	Simple Image Acquisition ...............................................................	20
4.1.1	Snapshot and Live View .................................................................	20
4.1.2	AVI Recorder ..................................................................................	21
4.2	Camera Control ..............................................................................	21
4.3	Illumination Control.........................................................................	25
4.4	Microscope Control........................................................................	26
4.5	Motorized Stage Control ................................................................	29
4.5.1	Defining a Positions List .................................................................	29
4.5.2	Correcting a Positions List .............................................................	30
4.5.3	Calibrating the Motorized Stage.....................................................	32
4.6	Autofocus .......................................................................................	34

4.6.1Autofocus options…....................................................................... 35

4.6.2	Executing an Autofocus Scan ........................................................	36
5	Experiment Manager..................................................................	37
5.1	A Graphical Tool .............................................................................	38
5.2	Concept of Usage ..........................................................................	39
5.2.1	Experiment Manager Components ................................................	39
5.2.2	Arrangement and Customization....................................................	40
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

Contents

Chapter 6

Touching up the image display by brightness, contrast and color adjustment and navigation through multidimensional image sets

Chapter 7

Basic processing routine like size calibration, overlay drawings, extraction and conversion of image sets

5.3.3	Types of Experiments .....................................................................	61
5.4	Conducting Experiments / Data Acquisition ..................................	70
5.4.1	Opening a Database .......................................................................	70
5.4.2	Executing an Experiment ................................................................	71
5.4.3	Data Storage and Preferences........................................................	75
6	Image Display and Navigation...................................................	77
6.1	The Viewport ..................................................................................	78
6.2	Image Display.................................................................................	80
6.2.1	General............................................................................................	80

6.2.2Adjust Display…..............................................................................80

6.2.3	Auto Adjust......................................................................................	84
6.2.4	White Balance .................................................................................	84
6.2.5	Black Balance .................................................................................	85
6.2.6	Gray Scale.......................................................................................	85
6.2.7	Fluorescence Color .........................................................................	86
6.2.8	Edit Fluorescence Color… ..............................................................	86
6.2.9	False-Color… ..................................................................................	87
6.2.10	Edit False-Color…...........................................................................89
6.3	Image Navigation ...........................................................................	95
6.3.1	General............................................................................................	95
6.3.2	Multi-Color Images..........................................................................	95
6.3.3	Displaying Different Color Bands in the Tile View Mode.................	96
6.3.4	Time-Lapse Sequences ..................................................................	96
6.3.5	Z-Stacks..........................................................................................	98
6.3.6	Multi-dimensional Sequences.........................................................	98
6.3.7	Parallel Navigation in Multiple Viewports........................................	99
6.4	Projections and Extended Focal Imaging ......................................	99
6.4.1	Projections Along the Z and Time Axes..........................................	99
6.4.2	EFI – Extended Focal Imaging ......................................................	100
6.5	Fluorescence and Transmission Image Overlay ..........................	101
6.6	Intensity Modulated Display.........................................................	102
7	Image Data Handling................................................................	103
7.1	Calibrate Images ..........................................................................	104
7.1.1	Why Calibrate Images? .................................................................	104
7.1.2	Calibrating the Camera Channel...................................................	104
7.1.3	XY-Calibration ...............................................................................	106
7.1.4	Z-Calibration .................................................................................	108
7.2	Scale Bar......................................................................................	109
7.2.1	General..........................................................................................	109
7.2.2	Setting the Scale Bar Properties...................................................	109
7.2.3	Show in Viewport ..........................................................................	110
7.2.4	Draw into Overlay..........................................................................	110
7.3	Show Markers, Time and Z Information.......................................	111

7.4Grid…........................................................................................... 111

7.5

Overlays .......................................................................................

113

Software Manual

Contents

Chapter 8

Data changing tools to improve the image quality, for example, by increasing the contrast or reducing the noise

7.5.1	General .........................................................................................	113
7.5.2	Activating the Overlay Toolbar .....................................................	113
7.5.3	Creating and Editing Overlays......................................................	114
7.6	Separate .......................................................................................	118

7.7Extract….......................................................................................118

7.8Combine…....................................................................................119

7.8.1	General .........................................................................................	119
7.8.2	Combining Data Sets ...................................................................	120
7.9	Convert Image ..............................................................................	121
7.9.1	General .........................................................................................	121
7.9.2	To 8-Bit.........................................................................................	121
7.9.3	To 16-Bit.......................................................................................	122
7.9.4	To RGB (3x8-Bit) ..........................................................................	122
7.9.5	Invert.............................................................................................	122
7.10	Image Information.........................................................................	123
7.10.1	The General Tab ...........................................................................	124
7.10.2	The Dimensions and Markers Tabs..............................................	125
7.11	Image Statistics ............................................................................	126
8	Image Processing ....................................................................	127
8.1	Shading Correction.......................................................................	129
8.1.1	General .........................................................................................	129
8.1.2	Define Shading Correction ...........................................................	129
8.1.3	Executing a Shading Correction...................................................	132
8.2	Bleaching Correction....................................................................	133
8.3	Thresholds and Binarization .........................................................	135
8.3.1	Set Thresholds..............................................................................	135
8.3.2	Set Color Thresholds....................................................................	140
8.3.3	Binarize.........................................................................................	144
8.4	Filters ............................................................................................	145
8.4.1	General .........................................................................................	145
8.4.2	Sharpen I ......................................................................................	148
8.4.3	Sharpen II .....................................................................................	149
8.4.4	Differentiate X ...............................................................................	149
8.4.5	Differentiate Y ...............................................................................	149
8.4.6	Laplace I .......................................................................................	150
8.4.7	Laplace II ......................................................................................	150
8.4.8	Mean.............................................................................................	151
8.4.9	Median..........................................................................................	151
8.4.10	Pseudo Filter.................................................................................	152
8.4.11	Sobel ............................................................................................	153
8.4.12	Roberts .........................................................................................	153
8.4.13	Reimer ..........................................................................................	154
8.4.14	User Filter .....................................................................................	154
8.4.15	NxN...............................................................................................	156
8.4.16	Lowpass .......................................................................................	157
8.4.17	Edge Enhance ..............................................................................	157

Contents

Chapter 9

The software contains a host of measuring tools in a special measurement environment

8.4.18	Rank ..............................................................................................	158
8.4.19	Sigma ............................................................................................	159
8.4.20	DCE – Differential Contrast Enhancement ....................................	160
8.4.21	Separator ......................................................................................	161
8.5	Morphological Filters....................................................................	164
8.5.1	Define Morphological Filter... ........................................................	165
8.5.2	Erosion ..........................................................................................	167
8.5.3	Dilation ..........................................................................................	168
8.5.4	Morph. Open.................................................................................	169
8.5.5	Morph. Close.................................................................................	169
8.5.6	Gradient ........................................................................................	170
8.5.7	Top Hat Bright...............................................................................	170
8.5.8	Top Hat Dark.................................................................................	171
8.5.9	Distance Bright..............................................................................	171
8.5.10	Distance Dark................................................................................	172
8.5.11	Ultimate Erode Bright....................................................................	172
8.5.12	Ultimate Erode Dark......................................................................	173
8.5.13	Skeleton ........................................................................................	173
8.5.14	Separate Particles.........................................................................	174

8.6Arithmetic Operations…............................................................... 175

8.7	Image Geometry ..........................................................................	177
8.7.1	Resize............................................................................................	177
8.7.2	Rotate............................................................................................	178
8.7.3	Mirror.............................................................................................	180
8.7.4	Align ..............................................................................................	180
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..................................................................................	184
8.8.4	Nearest Neighbors ........................................................................	185
8.8.5	Wiener Filter ..................................................................................	186
8.8.6	3-D AMLE Deconvolution: Advanced Maximum Likelihood .........	187
9	Measurements ..........................................................................	189
9.1	Measurements Toolbar ................................................................	190
9.2	Drawing Tools for Length and Area Measurements ....................	191
9.2.1	Point ..............................................................................................	191
9.2.2	Touch Count .................................................................................	191
9.2.3	Length ...........................................................................................	192
9.2.4	Angle .............................................................................................	194
9.2.5	Area...............................................................................................	195
9.3	Magic Wand .................................................................................	200
9.3.1	Magic Wand Options ....................................................................	201
9.4	Results .........................................................................................	202
9.4.1	Move Origin...................................................................................	202

Software Manual		Contents
9.4.2	Create Measurement Sheet..........................................................	203
9.4.3	Deleting Measurement Results.....................................................	204
9.4.4	Image Link ....................................................................................	205
9.4.5	Show/Hide Statistic ......................................................................	205
9.4.6	Select Measurements...................................................................	205
9.4.7	Define Statistics............................................................................	207
9.4.8	The Preferences Measure Tab...................................................	208
9.4.9	Measurement Sheets: Statistics...................................................	208

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...................................................................	214
10.3.2		Vertical Line Profile.......................................................................	215
10.3.3		Arbitrary Line Profile .....................................................................	215
10.3.4		Average Intensity of Neighboring Pixels.......................................	216
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) ..............................................................	220
10.5		Background Subtraction... ...........................................................	221
10.5.1		General .........................................................................................	221
10.5.2		Subtracting the Image Background .............................................	221
10.6		Intensity Kinetics in Time and Z ...................................................	223
10.7		DeltaF / F (∆F/F) Analysis .............................................................	224
10.7.1		General .........................................................................................	224
10.7.2		Generating a (∆F/F) sequence ......................................................	224
10.8		Ratio Analysis ...............................................................................	226
10.8.1		General .........................................................................................	226
10.8.2		Generating a Ratio Sequence ......................................................	227
10.9		Spectral Unmixing ........................................................................	228
10.9.1		Application....................................................................................	228
10.9.2		The Problem .................................................................................	229
10.9.3		The Solution..................................................................................	230
10.9.4		How Does it Work?.......................................................................	231
10.9.5		Spectral Unmixing with ..............................................	232
10.9.6		Calibration ....................................................................................	233
10.9.7		Unmixing.......................................................................................	234
10.9.8		Unmixing of Color Camera Images ..............................................	236
10.10		Phase Color Coding and Analysis ................................................	237
10.10.1		Phase Color Coding .....................................................................	237
10.10.2		Phase Analysis .............................................................................	238
10.11		Colocalization ...............................................................................	238
10.12		The FRET Software Module .........................................................	240
10.12.1		Image Acquisition .........................................................................	240
10.12.2		FRET Image Correction Factors ...................................................	242

Contents

	10.12.3 FRET Analysis ...............................................................................		245
	10.13	Kymogram....................................................................................	251
Chapter 11	11	Graph Display and Graph Analysis .........................................	253
Analyses of intensity	11.1	Graph Documents........................................................................	254
kinetics of fluorescence	11.2	The Graph Window ......................................................................	255
image time series	11.2.1	The Cursor: Changing the XY Scaling in the Diagram ..................	255
	11.2.2	The Cursor: Measuring Individual Graph Points ...........................	256
	11.2.3	The Graphs Button Bar .................................................................	256
	11.3	The Graph Menu ..........................................................................	260
	11.3.1	Markers and Labels.......................................................................	260
	11.3.2	Protecting and Deleting a Graph ..................................................	263
	11.3.3	Graph Information... ......................................................................	263
	11.3.4	Sheet .............................................................................................	264

Chapter 12

Images generated during an experiment are automatically stored in structured databases – analytical data can be added

12	Database ...................................................................................	269
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..................................................................	274
12.4.3	Adjusting the Database Window...................................................	275
12.5	Working with the Database..........................................................	278
12.5.1	Loading Documents......................................................................	278
12.5.2	Inserting Documents .....................................................................	278
12.5.3	Query.............................................................................................	280
12.5.4	Administration: Defining Organizational and Database Fields......	281

Chapter 13

Setting personal preferences of the software user interface

13	The Special Menu and the Window Menu .............................	283
13.1	Macros .........................................................................................	284
13.1.1	General..........................................................................................	284
13.1.2	Record Macro ...............................................................................	284
13.1.3	Executing Macros .........................................................................	285
13.1.4	Stop Macro Recorder ...................................................................	287
13.1.5	Run Macro.....................................................................................	287
13.1.6	Single Step....................................................................................	287
13.1.7	Reset Interpreter ...........................................................................	288
13.1.8	Set as Default-Macro ....................................................................	289
13.1.9	Define Macros...............................................................................	289
13.2	Add-In Manager... ........................................................................	291

13.3Define Menu Bar…....................................................................... 292

13.4	GUI Configuration ........................................................................	295
13.4.1	Reset .............................................................................................	296
13.4.2	Load... ...........................................................................................	296
13.4.3	Save... ...........................................................................................	297
13.5	Preferences... ...............................................................................	298

Software Manual

Contents

Chapter 14

A complete, integrated development environment for macros based on the programming language Imaging C

13.5.1	The Preferences	Image Tab.......................................................	298
13.5.2	The Preferences	View Tab .........................................................	300
13.5.3	The Preferences	File Tab ...........................................................	302
13.5.4	The Preferences	Measure Tab...................................................	307
13.5.5	The Preferences	Module Tab.....................................................	309
13.5.6	The Preferences	Graph Tab.......................................................	311
13.5.7	The Preferences	Database Tab .................................................	312
13.6	Window.........................................................................................		313
13.6.1	Minimize All...................................................................................		313
13.6.2	Close All........................................................................................		313
13.6.3	Document Manager… ..................................................................		313
13.6.4	Viewport Manager	........................................................................	315
13.6.5	Image Manager.............................................................................		315
13.6.6	Status Bar.....................................................................................		316
13.6.7	Command Window.......................................................................		316
14	Imaging C..................................................................................		319
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 .........................			336
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

Chapter 15

Telling the software which hardware modules are available and what the current installation is, for example, what filters are loaded

15	Configuration...........................................................	347
15.1	The Illumination System MT20 / MT10.........................................	348
15.1.1	Configuring the Excitation Filters .................................................	348
15.1.2	Burner Configuration ....................................................................	350
15.1.3	Using MT20 / MT10 without the Imaging Software......................	351
15.2	Configuring the Microscope .........................................................	351
15.2.1	General Configuration ..................................................................	351
15.2.2	Z-Drive Configuration ...................................................................	353
15.2.3	Configuration of the Objectives....................................................	354
15.2.4	Configuration of the Fluorescence Filter Turret............................	355
15.2.5	Configuration of the Transmission Contrast Inserts.....................	356

Contents

	15.2.6	Configuration of the Filters of a Filter Wheel.................................	357
	15.3	Definition of Image Types ............................................................	358
	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 ...................................................	366
	15.9.2	Configuring the Image Types........................................................	367
Chapter 16	16	Installing the Software ...........................................	369
The details to take care	16.1	Updating the Software...............................................	370
of when installing a new	16.2	Updating the Hardware Control ................................	372
software version	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 Imaging Station and for your 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.

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. 1.1 is the immediate 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 attenuation to meet the experimental requirements for fast real-time acquisition by highly sensitive digital cameras.

•The Real-Time Controller of real-time systems, a hyper-precision control board to 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 professional systems, a control board to synchronize the 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 imaging software is a powerful all-embracing platform that features 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 in-

cluded, 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.

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 imaging station. 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.

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Chapter 2 – System Overview 3

2 System Overview

The following chapter gives you a short overview of the basic system components (without additional peripherals). The system consists of different hardware and software devices, which are full, integrated.

4 Chapter 2 – System Overview

2.1System Chart

The system chart shown exemplifies the different components of a standard system and how they are interconnected.

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Chapter 2 – System Overview 5

2.2Hardware

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.).

2.2.1Motorized 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

6Chapter 2 – System Overview

2.3 Software

•ObsConfig Configuration Software to configure the Illumination System MT10 / MT20.

• Imaging Software integrating the Experiment Manager for planning and executing 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.

• software updates only: Update Software for the System Coordinator / Real-time Controller and the MT10 / MT20 electronics

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Chapter 3 – Brief Introduction and First Steps 7

3Brief 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, Configuration, of this manual carefully as well as 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

8 Chapter 3 – Brief Introduction and First Steps

3.1The User Interface

Viewport
		Viewport
Manager

Operands Box

Image

Manager

Image

Buffer Box

Document

Area

Double-click the icon on your desktop to start the software or open it via Start Programs . The screenshot below shows the program's graphical user interface (GUI). It is composed of:

aMenu bar: access to pull-down menus with assorted commands.

bTool bar: direct access to the most important commands for image acquisition, processing and analysis.

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Chapter 3 – Brief Introduction and First Steps 9

cStatus bar: shows the connected CCD camera and displays information depending on the active functions.

dViewport 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.

eImage 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.

fDocuments Area which always contains:

gViewport: displays the active image or a selection of currently loaded images.

hGraph document: the currently active graph (if a corresponding analysis has been carried out). It is minimized when is started.

iAdditionally the Documents Area may contain:

jDatabase Documents

kData Sheets

lText Documents and Macros

3.1.1The 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	multi-color image
Z-stack	multi-color Z-stack
single-color time-lapse	multi-color time-lapse
single-color Z-stacks in 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 window shows a red 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.

3.1.3The 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 Viewports 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.

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Chapter 3 – Brief Introduction and First Steps 11

12 Chapter 3 – Brief Introduction and First Steps

3.2Simple 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:

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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.

3.3Saving Images – The Database

To save a snapshot in the most basic way select File Save 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 Database Insert Experiment… (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.

3.4Loading Images

To load images that are not stored in a database and to display them in the Viewport use the command File Open (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.

3.5Conducting 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 Imaging Software includes the Experiment Manager, an easy-to-use and intuitive tool to plan, configure and execute even the