OLYMPUS SCAN R User Manual [ru]

®

INSTRUCTIONS

AUTOMATED IMAGE AND DATA

ANALYSIS SOFTWARE

This instruction manual describes the Olympus Automated Image and Data
Analysis Software for Life Science. To ensure safety, obtain optimum performance
and familiarize yourself fully with the use of these products, we recommend that you
study this manual thoroughly before operation. Together with this manual, please also
read the
well as the manuals of all other devices controlled by that software in order to under­stand general operation methods. Retain this manual in an easily accessible place
near a system for future reference.

Automated Image Acquisition Software and Hardware manuals as

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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 product described in this manual and re­serves the right to make such changes without obligation to notify the purchaser. In no event shall
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No part of this document may be reproduced or transmitted in any form or by any means, electronic or
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© 2006 – 2008 by Olympus Soft Imaging Solutions GmbH. All rights reserved.

Manual version: 2.1, December 2008

LICENSE AGREEMENT

Preamble

OLYMPUS SOFT IMAGING SOLUTIONS GMBH
scan^R software and drivers/interfacing software to Olympus Soft
Imaging Solutions manufactured hardware. These components are
referred to as OSIS scan^R SOFTWARE PRODUCT.

LICENSE AGREEMENT between END USER and OLYMPUS SOFT
IMAGING SOLUTIONS regarding the OSIS scan^R SOFTWARE
PRODUCT.
IMPORTANT-READ CAREFULLY: Below you will find the contractual
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PRODUCT. These conditions apply to you, the user, and to OLYMPUS
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explicitly agree to be bound by the conditions of this contract: purchas­ing the software, opening the package, breaking of one of the seals or
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§ 1. Scope
(1) This License agreement explicitly covers only the software diskettes
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§ 2. User rights
(1) OLYMPUS SOFT IMAGING SOLUTIONS permits the User, for the
duration of this contract, to use the software on a single computer and a
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§ 3. Additional user rights
Only if OLYMPUS SOFT IMAGING SOLUTIONS provides the User with
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is manufacturer of the

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Without prior written permission the User may not:
– change, translate, de-compile or de-assemble the software,
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§ 5. Warranty
(1) OLYMPUS SOFT IMAGING SOLUTIONS guarantees for the period of
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OLYMPUS SOFT IMAGING SOLUTIONS does not guarantee, that the
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cannot be held liable for damages or injuries resulting from the use of the
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SOLUTIONS. This applies, without exceptions, also to losses of produc­tivity or profit, interruptions in the flow of business or manufacture, loss
of information, and other financial losses. Without exceptions the possi­ble liability of OLYMPUS SOFT IMAGING SOLUTIONS is limited to the
amount that the User paid for the product. These limitations on the
liability do not influence claims for reasons of product liability.

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Analysis Software Manual

Contents

1 Introduction............................................................................................ 1

1.1 Abstract.............................................................................................. 2

1.2 Technical Support.............................................................................. 2

2 Main User Interface................................................................................ 3

2.1 General.............................................................................................. 4

2.2 General Settings................................................................................ 6

2.3 The scan^R Data Structure................................................................ 7

2.4 Managing Histograms and Scatter Plots............................................ 7

2.4.1 The Histogram Context Menu.......................................................... 9

2.4.2 The Region Context Menu............................................................. 10

2.5 Using the Image Viewer................................................................... 11

2.6 Adjusting the Image Displays........................................................... 12

2.7 Selecting Wells for analysis............................................................. 13

3 Assays.................................................................................................. 15

3.1 General............................................................................................ 16

3.2 Object Finder: Detecting Main Objects............................................ 16

3.3 Sub-object Finder: Detecting Sub-objects ....................................... 18

3.4 Object Finder Modules..................................................................... 20

3.4.1 Entire Image................................................................................... 20

3.4.2 Intensity Threshold......................................................................... 20

3.4.3 Edge Detection............................................................................... 21

3.5 Measurement Parameters ............................................................... 25

3.6 Derived Parameters......................................................................... 26

3.7 Image Processing............................................................................ 27

3.7.1 Background Correction .................................................................. 28

3.7.2 XY Shift.......................................................................................... 29

3.7.3 Inversion......................................................................................... 29

3.7.4 Cut Image....................................................................................... 30

3.8 Virtual Channels............................................................................... 31

3.8.1 Simple Math................................................................................... 31

3.8.2 Spectral Unmixing.......................................................................... 33

3.9 Tracking – Analyzing Time-Lapse Data........................................... 35

3.9.1 Tracking Configuration................................................................... 37

3.9.2 Track Analysis Parameters............................................................ 38

3.9.3 Trace Viewer.................................................................................. 43

4 Analysis Results.................................................................................. 45

4.1 Running an Analysis........................................................................ 46

4.2 Managing Gates............................................................................... 47

4.2.1 Gates and Regions. ....................................................................... 47

4.2.2 The Gate Manager......................................................................... 47

Analysis Software Manual

4.2.3 Color Gating................................................................................... 49

4.3 Well Results..................................................................................... 52

4.3.1 Measurement Results.................................................................... 52

4.3.2 Well Results: Populations.............................................................. 53

4.3.3 Export Definitions........................................................................... 54

4.3.4 Export results of individual objects ................................................ 56

5 Example Assay – Step by Step........................................................... 57

5.1 Setting-up and Executing an Assay................................................. 58

5.2 Analyzing the Data .......................................................................... 61

5.3 Time-Lapse Analysis....................................................................... 64

6 Appendix.............................................................................................. 71

6.1 File Conversion................................................................................ 72

6.1.1 Images List .................................................................................... 72

6.1.2 Image Channel Definition .............................................................. 73

6.1.3 Scan Settings................................................................................. 74

6.1.4 View Conversion Results............................................................... 74

6.2 FCS Export Functionality................................................................. 75

6.3 Libraries........................................................................................... 77

6.3.1 Object Analyzers Library (OAL)..................................................... 77

6.3.2 Object Finders Library (OFL)......................................................... 78

6.3.3 Image Processing Library.............................................................. 78

6.3.4 Virtual Channel Library.................................................................. 79

6.4 Valid functions for derived parameters............................................ 80

Analysis Software Manual Chapter 1 – Introduction 1

1 Introduction

2 Chapter 1 – Introduction

Thank you very much for purchasing Olympus’ Screening Station for Life Science and for your confi­dence in our products and services.
The
the Olympus
tended for the use in biomedical research.

Analysis Software is designed for the automated analysis of images that were acquired by

Screening Station and with the Acquisition Software. The software is in-

The
ponents of the Olympus Screening Station for Life Sciences are for research use
only.

Analysis Software, the Acquisition Software as well as the hardware com-

1.1 Abstract

This user manual will guide you through the usage of the analysis software of the Olympus Screening
Station scan^R. It will assist you in setting up efficient and reliable assays from scratch. This scan^R
module is intended to be used for the analysis, quantification and navigation through your results. The
analysis module of scan^R allows you to run the analysis during acquisition or in “offline mode” after­wards.

Special care has been taken to guarantee correct and accurate information within this documentation,
although this is subject to changes due to further development of the Screening System. Thus, the
manufacturer cannot assume liability for any possible errors. We would appreciate reports of any mis­takes as well as suggestions or criticism.

1.2 Technical Support

If you find any information missing in this manual or you need additional support, please contact
Olympus directly.

Analysis Software Manual Chapter 2 – Main User Interface 3

2 Main User Interface

This chapter explains the features of the image displays and briefly introduces the different menu
points and buttons accessible from the main user interface.

2.1

General.........................................................................................................4

2.2 General Settings...........................................................................................6

2.3 The scan^R Data Structure........................................................................... 7

2.4 Managing Histograms and Scatter Plots....................................................... 7

2.4.1 The Histogram Context Menu.....................................................................9

2.4.2 The Region Context Menu........................................................................10

2.5 Using the Image Viewer..............................................................................11

2.6 Adjusting the Image Displays...................................................................... 12

2.7 Selecting Wells for analysis........................................................................13

4 Chapter 2 – Main User Interface

2.1 General

The scan^R Analysis main graphical user interface contains four histograms and an image viewer win­dow. The functions of these histograms are explained in Chapter 2.4,

The image viewer window shows the image with the object corresponding to a data point selected in a
histogram. A description of the image viewer functions is given in Chapter 2.5,

Navigation through the images of a scan is possible with the tools in the Image box. Color channel
selection is done with respective pull-down shortlists in the Display box.

An assay can be started and followed online with the tools and displays in the field at the lower right of
the main window.

The menu bar at the top contains several pull-down menus and commands. They are listed in the fol­lowing overview:

Managing Histograms

Using the Image Viewer

.

.

AnalysisRun: starts the analysis with the current settings

AnalysisBatch Run: starts multiple analysis with the current settings

Analysis Software Manual Chapter 2 – Main User Interface 5

AnalysisOpen: opens a previous analysis (*.sca-file)

AnalysisSave/Save as: saves the current analysis (*.sca-file)

AnalysisExport Table: exports the values determined for each detected object to a spread sheet. The
values exported depend on the active view. When also sub-objects are detected not only one file is
exported but for every sub-object a separate list is exported. The values that are exported depend on
the active view: population view / trace view (see Chapter4.3.4)

AnalysisEdit Assay: opens the “Assay settings” menu

AnalysisLoad Assay: loads an existing assay (*.say-file). In contrast to the *sca-file the *.say-file con­tains only the analysis, i.e. the operations to perform on a data set but not the results of a specific
analysis.

AnalysisSave Assay: saves the current assay (*.say-file)

AnalysisAssay Gating: opens the gate manager (see Chapter 4.2.2)

AnalysisExit: exits the analysis

ScanOpen: opens a scan and assigns it to the current assay. The file types that can be opened are
the scan^R experiment descriptor files (*.xml-format) and dotslide images (*.wtp-, and *.ets-format)

ScanRelink images: re-links acquired images to an analysis. To do so, navigate to the folder where
the images are stored and select Current Folder.

ScanCustom conversion: converts a third party data set into the scan^R format (see Chapter 6.1)

ScanSelect wells: selects a set of wells for analysis and data navigation; allows also to display a well
overview, i.e. an overview of the images that were acquired in a well. (see Chapter 2.7)

ScanScan Info: displays the path to the image data

ScanSettings: displays the settings to a scan

TrackingTrace View: toggles between the Population and Trace View modes.

TrackingConfigure Tracer: opens the Trace Configuration window to select how the object tracking is
to be performed.

TrackingDefine Parameters: opens the Trace Parameters window to select trace analysis operations.

TrackingShow traces: opens the Trace Viewer that visualizes the trace graphs.

ViewLayout: display properties for the RGB display (affects only the displays).

ViewParameter View: lists all parameters for a selected object, that were determined during analysis

ModulesObject Finders: list and configure available Object Finder Modules (Chapter 6.3, Libraries)

ModulesObject Analysers: list and configure available Object Analyzer Modules (Chapter 6.3, Librar­ies)

ModulesImage Processors: list and configure available Image Processing Modules (Chapter 6.3,
Libraries)

ModulesVC Processors: list and configure available Virtual Channel Modules (Chapter 6.3, Libraries)

RepositioningInteractive: the scan^R screening system moves to the position of the selected object

Settings: Gives access to some preferences including galleries, directories, data export and settings for
repositioning an reclassification.

6 Chapter 2 – Main User Interface

2.2 General Settings

The general preferences can be defined in the Settings menu.

Max Gallery Objects. Sets the number of objects that are displayed in a gallery.

Sort mode. The options are Typical, Random, yx and –yx. When Typical is set, the galleries display the
objects which are closest to the center of gravity of the selected region or histogram in the order of
distance to that center. Random displays randomly selected objects within the selected region. The
options yx and –yx allow to create a gallery that is ordered by one parameter.

Default Data Directory. Enter the directory where the data are read by default (used for “open scan”)

Result Export Directory. Enter the directory where the results are to be stored. When this field is empty
the results will be stored in the scan directory\Population Results. The results of a tracking analysis will
bestored in the scan directory\Trace Results. (Note that in earlier versions the results are stored in the
scan directory\Results folder.

Analysis Software Manual Chapter 2 – Main User Interface 7

Export2Txt. The results can be exported as .txt files or as .fcs files. (For export to .fcs format, see
Chapter 6.2)

Repositioning & Reclassification. Set the Port and the Address for communication with the scan^R
screening system for experiments with repositioning.

2.3 The scan^R Data Structure

scan^R analysis data can be separated into the acquired images and the assay being applied on them.
The acquired images as a whole are called a scan; it includes the individual images and their acquisi­tion settings like color channels, integration time, plate information etc. An assay describes the proc­essing and analysis steps applied to extract data out of the images.

This separation between analysis settings and acquired images allows the reuse of once adapted
analysis settings for different scans.

The images acquired during a scan^R scan are stored as 16-bit *.tif files in a Data subfolder in the ex­periment scan storage folder.

Additionally, the scan settings are stored in an Experiment_descriptor.xml and the stage positions in
the Acquisitionlog.dat file.

The scan^R analysis software serves for the analysis of the scans. The instructions (assays) for these
analyses are stored in the scan^R Analysis/Assays folder as *.say files. These files can be loaded via
AnalysisLoad Assay… to then apply the assay on a scan data set.

Once an assay has been performed on a data set, a *.sca file is generated and can be stored in the
experiment storage folder. These files contain all analysis data including histograms and scatter plots
etc. *.sca files can be loaded via AnalysisOpen… to revisit the analysis results.

To clarify this: if you open a Scan via ScanOpen… the experimental settings of a scan will be loaded
by reading in the Experiment_descriptor.xml file. Thus, you get access to the raw image data. In con­trast to this, if you open an Analysis (*.sca) you will get the results in addition to the images.

2.4 Managing Histograms and Scatter Plots

Histograms are used in scan^R for data representation, classification and navigation. scan^R uses 2-D
and 1-D histograms following the common representation in cytometry.

A 1-D histogram shows the frequency distribution of only one parameter. A 1-D histogram is created
when the same parameter for X and Y is selected. On the X-axis of the histogram the selected parame­ter and on the Y-axis the number of counts is plotted.

A 2-D histogram (scatter plot) is a plot of one parameter of a number of objects against a second pa­rameter. Color-coding is used as "third dimension" to represent the frequency of occurrences.

The data displayed in a histogram are assigned to an object type (main-/ sub-object). The detection of
the objects is defined by the assay. The object type can be chosen from the Object pull down menu

8 Chapter 2 – Main User Interface

underneath the histogram. The X and Y pull down menus are then automatically updated to correspond
to the measured parameters of the chosen object type (see assay definition). The X and Y pull down
menus are used to change the parameters displayed in a histogram. The axes of abscissa and ordinate
are labeled with the chosen parameter.

Two buttons are located in the lower right corner of each histogram. They allow toggling between the
Navigation and the Region-Selection modes.

Navigation

The Navigation button with the pointer symbol allows navigating within the data. Each data point within
a histogram is directly linked to the object from which it is derived. A selected data point is highlighted
by a red circle in all histograms in navigation mode as long as the data point is within the displayed
area. The corresponding object is displayed in the Image Viewer. The X and Y values of the data point
are displayed next to the X and Y pull-down menus. Holding and dragging the mouse using this tool
allows to virtually following the objects changes within the parameter set. The navigation tool also al­lows dragging and modifying existing regions.

Region

The Region tool is used to draw polygons into a 2-D histogram and to set a range in 1-D histograms.
Regions define bi-dimensional intervals within the parameter range and thus subpopulations of data
points. Double-click in order to close a region in a 2-D histogram.

Analysis Software Manual Chapter 2 – Main User Interface 9

2.4.1 The Histogram Context Menu

The histogram can be managed through the context menu accessible via right-click into the histogram.
(Note that you will get the region context menu, if you right-click on a region border as described be­low). The histogram context menu contains the following commands:

• Set Gate. Apply a gate on the selected histogram by choosing a region from the list that appears.

• Show region. Select a region from the list that appears to generate a zoomed-in view of it with the

X/Y parameters that were used to define this region.

• Clone from. Select a histogram from the list that appears to duplicate the histogram.

• Gallery. This command generates an image gallery of objects in the current histogram. The number

of images and the selection criteria is set according to the gallery preferences given in the Settings
menu.

• Zoom out. This command zooms out of the current view by a factor of 2 (in case of linear axis scal-

ing).

• Color Gating. This command causes the population of each gate to be displayed in a different color

in the histogram.

• Settings… This command opens the Histogram Properties dialog with the following settings.

Axis attributes. This field offers different choices to set the axis scaling.

Grid cosmetics. This field offers different choices to set the grid display.

Autoscale. This field offers different choices to set the auto scale of the axes.

BKColor. A click on the colored field opens a window that allows selecting the background color.

10 Chapter 2 – Main User Interface

Selector Color. A click on the colored field opens a window that allows selecting the cross-hair color.

Region Color: A click on the colored field opens a window that allows selecting the color of the region
outline.

Color scheme. This color palette defines the coloring of data points (pixels) in the histogram in depend­ence of the number of counts (events) they represent. For example, if the Color table bin width is set to
three and a data point represents 7, 8 or 9 counts, it will be displayed in the third color from the left.

Color table bin width. It defines the range of counts to be given the same color from the Color scheme.

Binned color table. Click this button to activate the color binning as set in Color table bin width.

Color Gating. This command causes the population of each gate to be displayed in a different color in
the histogram.

Show Legend. This command causes a legend of the colors in the Color Gating mode to be displayed
in the histogram.

2.4.2 The Region Context Menu

The region context menu will open, if you right-click on a region border

• Remove. Deletes the selected region or gate.

• Zoom to. Gives a zoomed view of the selected region.

• Gate. Converts the region into an AND Gate. (See also Chapter 4.4.2,

The Gate Manager.

) When a
gate is applied to a histogram, only the data points within this gate are displayed. To display all data
points open the histogram context menu and go to Set gatenone.

• Region Gallery. This command generates an image gallery of objects in the selected region. The

number of images and the selection criteria is set according to the gallery preferences given in the
Settings menu. (For more information see Chapter 2.2,

General Settings.

)

Analysis Software Manual Chapter 2 – Main User Interface 11

2.5 Using the Image Viewer

Each analysis data point is directly linked to the image object it is generated from. These objects can
be displayed in a close-up view in the image viewer. Browsing in the data set by clicking on data points
with the histograms Navigation tool automatically leads to a corresponding update of the image in the
viewer. Additionally the Well, Position, Time and Well/Name Description fields are updated and give
information about the image origin. The small arrow buttons on the left of these fields can likewise be
used to navigate. Values can be typed in as well.

Display. Multi-color images consist of individual color channels that were recorded with different optical
acquisition settings (e.g. different excitation filters). The red, green and blue pull-down menus serve to
select the input for the three color channels that are displayed in the respective colors. In order to dis­play a channel (e.g., a transmission channel) in grayscale, select it from the gray pull-down menu.
When having both grayscale and any color selection active, a transmission overlay/display will be used
for the grayscale selection.

The clipping of the RGB display, i.e., the scaling of the image display brightness, can be changed via
the menu point ViewLayout that opens the Image Clipping window.

Image: Processed. Click this button to toggle between the original image and the processed image as
defined in AnalysisEdit AssayAssay Settings/Image Processing. Image processing is used to im­prove the quality of the displayed image but slows down the systems image display. Therefore it is
especially recommended to switch it off for performance when creating galleries . The image process­ing is described in Chapter 4.9,

Image Processing.

12 Chapter 2 – Main User Interface

Row/Column/Position/Time. Use these entries to select a specific image to be displayed.
The up/down arrows allow fast navigation though your image data set by incrementing or decrementing
the Time, Position and/or Well number.

Interactive objects. Select the object type to be outlined in the display when clicking on an object or
data point.

The image viewer is equipped with a tool bar to select different mouse tools:

Zoom

The Zoom mode is used to zoom into the displayed image. A click into the image causes a zoomed-in
view with the cursor position as the center. To zoom out, the Shift key must be pressed simultaneously.

Selection

The Selection mode allows selecting individual objects within the image via mouse click. The object
type to be displayed can be selected in the pull down menu in the bottom right corner of the image
viewer. Main objects are highlighted by a green outline. The data point corresponding to the selected
object is highlighted with a red circle 2-D histograms and a vertical red line in 1-D histograms.

Move

Depending on the zoom factor, only a part of the image will fit into the display. The Move mode allows
moving the visible area via mouse-drag.

The status bar in the lower left corner shows information about the magnification of the displayed im­age, the current x/y position of the cursor and the pixel value(s) at this position.

2.6 Adjusting the Image Displays

ViewLayout opens the Image Clipping window that allows adjusting the display brightness. Note that
these image settings affect the front panel display as well as well overviews and galleries A raw image
will always have a certain background intensity. Also, one will avoid to over saturate images and – es­pecially in fluorescence applications – rather use only a fraction of the camera chip capacity. The con­sequence is that a raw image is usually low in contrast and may even appear entirely black. Clipping is
applied to change the image brightness by defining a range of low pixel counts to be displayed black
as well as a range of high pixel counts to be displayed with maximum brightness.

Clipping Type. This button toggles between Dynamic [%] and Absolute clipping.

Analysis Software Manual Chapter 2 – Main User Interface 13

Dynamic [%]. Define here how many pixels (as a percentage of the total number of pixels) will be dis­played with maximum and minimum brightness. The intensity of the remaining range of pixels will then
be scaled linearly in between. The higher the numbers the stronger the resulting contrasts.

Absolute. Define here the range of pixel counts to be displayed with maximum and minimum brightness
by dragging the red horizontal lines – that represent the maximum and minimum thresholds – with the
mouse. The intensity of the remaining range of pixels will then be scaled linearly in between.

Gray scale palette. The channel selected in the gray pull-down menu in the main GUI can be displayed
in different false-color palettes that can be selected here.

Use scan settings.

2.7 Selecting Wells for analysis

The command ScanSelect Wells opens the Select Wells display. It features a graphical representa­tion of all wells of the well plate or slide.

When the window is opened for the first time, green circles represent wells that were selected for scan­ning. These wells will be considered in the analysis . To change this selection double-click on the wells.
These wells are also removed from the list on the right. A second double-click reactivates the well.
Dragging a rectangle around a group of wells likewise carries out deactivation and activation.

In the list on the right you can also change the name of the wells and group wells by assigning the
same name.

Restore. This function restores the initial well pattern

Name/Description. In certain cases it may be advantageous to group wells (see also Chapter 4.3,

Results

list. The default entry is the alphanumerical code of the well position. The name can be changed at will.

). Wells of one group need to have an identical entry in the Name/Description column of the well

Well

14 Chapter 2 – Main User Interface

Right-click on one of the scanned wells gives you the option Well Overview. For a time series you will
have to set the time point first. The Well Overview will display all the positions you recorded for one
well. Selecting one of the images will load the image in the main display on the front panel.

Well overview.

Analysis Software Manual Chapter 3 – Assays 15

3 Assays

Assays are the recipes to extract the data of interest from the images of a scan. They define which
objects are to be recognized and how and which measurements are to be performed on the found
objects. This chapter explains in detail how assays are to be set up or modified.

3.1

General.......................................................................................................16

3.2 Object Finder: Detecting Main Objects.......................................................16

3.3 Sub-object Finder: Detecting Sub-objects ..................................................18

3.4 Object Finder Modules................................................................................20

3.4.1 Entire Image..............................................................................................20

3.4.2 Intensity Threshold....................................................................................20

3.4.3 Edge Detection.......................................................................................... 21

3.5 Measurement Parameters .......................................................................... 25

3.6 Derived Parameters.................................................................................... 26

3.7 Image Processing.......................................................................................27

3.7.1 Background Correction ............................................................................. 28

3.7.2 XY Shift.....................................................................................................29

3.7.3 Inversion....................................................................................................29

3.7.4 Cut Image..................................................................................................30

3.8 Virtual Channels.......................................................................................... 31

3.8.1 Simple Math..............................................................................................31

3.8.2 Spectral Unmixing..................................................................................... 33

3.9 Tracking – Analyzing Time-Lapse Data...................................................... 35

3.9.1 Tracking Configuration.............................................................................. 37

3.9.2 Track Analysis Parameters.......................................................................38

3.9.3 Trace Viewer............................................................................................. 43

16 Chapter 3 – Assays

3.1 General

An assay defines all steps necessary to extract quantitative data from the acquired images. It usually
starts with some sort of image processing like background correction. Secondly, objects have to be
detected in the images. The analysis, for example different kinds of measurements, e.g. area, intensity,
shape…, is finally performed on these objects and results for the samples (e.g. wells) are generated.

The AssayEdit Assay command opens the Assay Settings window and allows applying or adapting
the assay to the loaded scan. The Assay Settings window contains six tabs: Main Object, Sub-objects,
Parameters, Derived Parameters, Image Processing and Virtual Channels. The tabs are arranged from
left to right, but you can always jump back and adjust the settings of former steps. However, back­ground correction (in the Image Processing tab) should be performed prior to object detection as it
changes the intensity values.

scan^R distinguishes between two kinds of object types: an assay always defines one Main Object
type and up to four Sub-Object types connected to it. To give an example, main objects may be indi­vidual cells while their sub-objects are individual structures within them.

To represent this hierarchical structure, the Assay Settings window’s tabs Main Object and Sub­Objects are used to adapt different set the search algorithms for main and sub-objects in order to ex­tract the structures of interest from the images. This is done by different Object Finder Modules that
implement different rules for object detection.

The Parameters and Derived Parameters tabs contain the information about the kind of information to
be extracted from the objects (e.g. area, shape,…) .

The Image Processing tab allows defining image processing steps that are to be executed before the
object detection and parameter extraction.

In the Virtual Channels tab new channels can be created as a result of post-acquisition image process­ing (e.g. spectral unmixing). To access the Virtual Channels tab you have to navigate through the tabs
to the right using the arrow buttons on the top right.

3.2 Object Finder: Detecting Main Objects

The Main Object tab of the Assay Settings window provides the commands to define the Main Object
detection.

Color Channel. Select the color channel on which the main object detection is to be performed.

Module. Select the method to detect individual Main Objects from the shortlist.

Analysis Software Manual Chapter 3 – Assays 17

Settings list. Each Object Finder Module has a list of preset parameters. The lists can be modified and
stored at will. Individual modifications of these settings are marked as Modified. Select the settings of
choice from the shortlist.

Adjust. This command opens the configuration dialog of the selected Object Finder Module. (For
changing the list of Object Finder Modules see Chapter 3.4,

Object Finder Modules)

Module settings. This field lists the current settings of the selected Object Finder Module.

Add to list. This adds the modified settings to the Settings list. Click the button to open the Add Set­tings to OFL window (Object Finders Library, see Chapter 4.5,

Object Finders Library

) where you have

to give a New Settings Name for the modified settings list.

Image segmentation. This function divides – if activated via the check box – the entire image into seg­ments: as many segments as there are Main Objects where each segment is assigned to the Main Ob­ject in its center. In other words, each image pixel is assigned to the Main Object it is closest to. All
pixels that are assigned to the same Main Object form one segment of irregular shape and size. The

18 Chapter 3 – Assays

View button opens the View Segmentation window that contains on the left a display of the object­circumscribing rectangles and on the right a display of the segments.

3.3 Sub-object Finder: Detecting Sub-objects

Sub-objects are structures that are directly linked to individual Main Objects. The search for Sub­objects takes place on an image mask derived from the corresponding Main Object. This Main Object
mask can be adapted for each Sub-object type separately.

Sub-object finder: Color Channel, Module, Setting list, Adjust, Add to list. These functions are analo­gous to the ones described in the previous Chapter 3.2,

Name. Give a name to each new Sub object. The default name is Obj. 1.

Sub-object list. It gives an overview of the defined Sub-object types. The New and Remove buttons
allow the insertion and deletion of Sub-object types.

Main Object Mask. Each individual Main Object found in an image creates a mask. The individual Sub­objects are associated with this mask rather then with the Main Object itself. Imagine a Main Object is
the cell nucleus and the Sub-objects are structures outside of it. In order to be detected, the original
Main Object Mask – which only covers the area on the nucleus – needs to be modified in order to en­able the detection of the Sub-objects.

Click the checkbox to enable the image mask modification of the Main Object.

Modify button. Click here to open the Modify Object Boundaries dialog to adapt the main object mask
to the needs of the Sub-objects detection.

Distance. The distance is measured from the outer rim of the main object mask (positive and negative
values are valid)

Object Finder: Detecting Main Objects

.

Analysis Software Manual Chapter 3 – Assays 19

Width. Extension of the sub-object mask

The examples below illustrate the effects of these parameters.

Overlap treatment. Options are Segment, Segment (slow) and remove.

Main object mask Distance 0, Width 1 Distance -5, Width 5 Distance 8, Width 8

When sub-objects are used for analysis, a further parameter becomes available in the Pa­rameter tab (see Chapter 3.5,
for sub-objects is used, otherwise it would be

Measurement Parameters

subobjectsname

): Obj. 1 counts (if the default name

counts). This parameter
gives the number of sub-objects detected for each main object and is a parameter of the
main object.

20 Chapter 3 – Assays

3.4 Object Finder Modules

3.4.1 Entire Image

This is a very simple object definition: the entire image is used as object. You may use this for meas­urements of integral intensities of your sample, i.e., for each image and each parameter (see. Chapter

Measurement Parameters

3.5,
age.

) a single value is calculated, independent of the objects within the im-

Ignore frame. This is the only parameter to adjust: the size of a bordering frame to be ignored. The
default value is 0 (no bordering frame).

3.4.2 Intensity Threshold

As the name says, the Intensity Threshold method is based on intensity values: pixels with intensities
above a predefined threshold will be united to one individual object.

The Object Finder: Intensity Threshold dialog has two image viewer displays. The left one shows the
gray value image including all detected objects marked with a red bounding box. The right one shows
the binary mask of each object.

Settings

The settings used when opening the window depend on the Settings list selected in the Main Object or
Sub-object tab (see Chapters 3.2,

Detecting Sub-objects

Finders Library (OFL)).

Threshold. This is the intensity cut-off for objects. Type in a value or use the arrows to adjust it.

) and are loaded from the Object Finders Library (see Chapter 6.3.2, Object

Object Finder: Detecting Main Objects

and 3.3,

Sub-object Finder:

Threshold: Auto. Click this button to automatically evaluate the image background and set a meaning­ful cut-off value.

Watershed. If neighboring objects are so close to together that thresholding does not lead to a clear
separation, they will be detected as one object. (See left image pair below.) The Watershed algorithm
separates these objects along the contractions of the detected masks. (See right image pair below.) Set
the toggle button to On to use this option.

Ignore border object. Check this box to ignore all objects that are cut-off by the image border.

Analysis Software Manual Chapter 3 – Assays 21

Fill holes within objects. Check this box to fill the object mask in case it contains holes.

Minimum/Maximum object size. Check these boxes and adjust the values to apply minimum and
maximum size filters to the objects (in order to ignore objects that are outside these size limits).

3.4.3 Edge Detection

The EdgeSegmentation module is a general purpose edge based particle detector. The idea of the
algorithm is to find a closed contour around each particle. First the edges of the image are extracted.
For those edges which already form a closed contour the algorithm stops. Since the remaining open
edges may be part of a closed contour around a particle, the algorithm then tries to combine these
open edges so that they form a closed contour as well.

The edge detection algorithm yields better results when objects of strongly varying intensity
have to be detected. In these cases the threshold detection will either lead to clusters when
the threshold is set to a low value in order to detect also dim objects. If a higher value for
the threshold is set, then the dim particles will be missed. Furthermore, as edge detection
is intensity independent it is especially suitable for cell-cycle analysis.