The Telepresence Add-In The Trackit! Add-In

The trackIT!
The
Add-In
telePresence
Add-in
Any copyrights relating to this manual shall belong to Olympus Soft Imaging Solutions GmbH. We at Olympus
Soft Imaging Solutions GmbH have tried to make the information contained 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 herein.
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 written permission of
Olympus Soft Imaging Solutions GmbH.
Windows, Word, Excel and Access are trademarks of Microsoft Corporation which can be registered in various
countries. Adobe and Acrobat are trademarks of Adobe Systems Incorporated which can be registered in
various countries.
© Olympus Soft Imaging Solutions GmbH

All rights reserved
Olympus Soft Imaging Solutions GmbH, Johann-Krane-Weg 39, D-48149 Münster, Tel. (+49)251/79800-0, Fax: (+49)251/79800-6060
Contents
trackIT! Performance range ............................................. 3
Installation ......................................................................... 5
The trackIT button bar ..................................................................................... 5
Background correction with trackIT! ............................................................. 6
The "Define Automatic Tracking" dialog box ............................................... 7
Tracking objects ............................................................... 9

Automatic tracking of a simple object ......................................................... 10
Suitable parameters for the task ...................................................................... 12
Automatic tracking of several objects ......................................................... 13
What information is contained in the generated sheets ? ......................... 16
The relationship between the Object Overview sheet and the Object
Information sheet ............................................................................................. 18
Having the sheets displayed anew ................................................................... 18
Saving results ................................................................................................. 19
Exporting results .............................................................................................. 19
Automatic tracking using the object separator ........................................... 21
Automatic tracking of unsteady objects ...................................................... 23
Edit tracking results ....................................................................................... 25
Altering the display of an object trajectory ....................................................... 25
1
Hiding objects ................................................................................................... 26
Clearing uninteresting objects .......................................................................... 28
Selecting interesting objects ............................................................................ 28
Deleting uninteresting objects .......................................................................... 28
Filtering objects ........................................................................................................ 30
Which filter criteria are there? .......................................................................... 32
Inverting filter definitions .................................................................................. 33
Combining filter definitions ............................................................................... 33
Saving and re-using filter definitions ................................................................ 34
Setting filter definitions interactively in the image. ........................................... 35
Merging trajectories .................................................................................................. 36
Contents
Execute manual tracking ...............................................................................37

How to track an object manually .......................................................................38
Tracking a further object manually ...................................................................39
Correcting set object positions subsequently ...................................................39
Deleting an object position ...............................................................................40
Key configuration during manual tracking ........................................................40
Deleting tracking results ...................................................................................41
Administration of tracking configurations ...................41

Saving the configuration ................................................................................41
Loading the configuration .............................................................................42
Overview of the configuration options .........................43

Detector tab .....................................................................................................43
Tracker tab ......................................................................................................45
The Edit Tracking Results dialog box ...........................................................47
Suggestions and questions with regard to trackIT! ....52

Does it make any difference whether objects are detected larger or smaller? .52
What can I do with objects whose direction of movement is also detected? ....52
What can I do about objects which are detected too small? ............................52
What can I do with two objects which are recognized as one object? ..............52
What can I do when one object is recognized as several objects? ..................52
How can I track certain objects only? ...............................................................53
How can I see that an object as been lost? ......................................................53
2
What can I do when my object is not detected? ...............................................53

How can I recognize a correctly set object separator? .....................................54
What do I do when two objects cross? .............................................................54
What do I need the Preview for detection for? .................................................54
How can I look at the tracking overlay in an RGB image and save it? .............55
Index..................................................................................57
trackIT! Performance range

With the trackIT Add-in you can track and analyze the movements of objects
(e.g. cell movements). This can be one or more objects. You can decide
whether or not objects which appear or disappear from the image should
also be tracked.
Depending on the task, the object movements can be tracked automatically
or manually. When you use automatic tracking, the position of the object on
each image will be determined by the software. You can subsequently check
and alter these values. When you use manual tracking, you determine the
position of the object yourself by mouse click. You can do it on every image
or, e.g., only on every fifth image.
Preconditions The precondition for the measurement is an image stack in which the object
movements can be recognized. These image stacks can be created with the
Add-Ins fast image acquisition (FIS) and image sequence processing (isp).
They are saved as files in multipage.tif format, the file name extension is TIF.
An image stack contains all the images of an acquisition series.
Results Following successful tracking, the trajectory of the object is displayed
graphically in the overlay, thus permitting a rapid overview. Every object is
given an unmistakable identity number and another color to represent the
segment covered.
The trajectories are colored
differently for each object.
The results are also displayed in several sheets. They allow the detailed
3
analysis for example in relation to the distance covered, the direction and
the speed. The relevant unit (for example µm for the distance covered) is
adopted from the image calibration.
You can find detailed infor-
mation in the sheets which
are set up automatically.
Additionally, you can see the results in various histograms which graphically
display the tracked objects according to, e.g., speed or trajectory length.
In this histogram the objects

found are grouped
according to their speed.
Save Results The results of a tracking will be saved together with the image. Therefore,
you will not need to save the sheets separately if you save the image stack
immediately after the tracking has been performed.
These sheets can be saved in CSV file format for use in other programs.
trackIT! and the image trackIT! is completely integrated in the image analysis program.
analysis program
Requirements for Not all image stacks are suitable for use with trackIT! Please take note of the
image stacks when following prerequisites:
using trackIT! • The sample must be focused.
• The sample should be illuminated as uniformly as possible. If this is not
possible, then you can carry out a background correction before
tracking, see p. 6.
• The image acquisition frequency should be adjusted to the speed of the
object movements, so that (in the case of continuously moving objects)
4
each image represents a recognizable but not too large a movement

step.
• There must be a manageable number of objects to be dealt with.
• The stage may not move, so as to permit correct documentation of the
object movements.
• The images must be correctly X/Y calibrated. Only then can trackIT!
provide correct information with regard to trajectories and speed.
• For the automatic tracking, the images must be available as 8- or 16-
bit gray value images. 24-bit or RGB images will be automatically trans-
formed into 16-bit images following a relevant query before tracking
takes place.
Installation
Installation
The installation of the trackIT! software is no different from a "normal"
installation of your image analysis program. You must have Administrator
rights. The dongle must be inserted in your PC's USB or parallel port before
installation, otherwise an error message will appear.
To install the software, proceed as follows:
1) Put the program CD into your computer's CD-ROM drive.
> The setup window normally opens automatically. If it does not, or if
you have closed the window, then you can reopen it at any time by
clicking the setup.exe on the CD-ROM.
2) Select the appropriate menu item to install the software.
3) Follow the instructions given by the installation program.
The trackIT button bar

Following the start of trackIT!, you will see the trackIT button bar. The
buttons are inactive as long as no image stack is loaded.
Background Correction (p. 6)
Define Automatic Tracking (p. 7)
Execute Automatic Tracking
Execute Manual Tracking (p. 37)
Merging trajectories
(p. 36)
Edit tracking results (p. 25)
5
Should you have inadvertently closed the trackIT button bar, you can reopen
it at any time. To do so, click any button bar with your right mouse button,
and select the trackIT option in the context menu.
Background correction with trackIT!
Background correction with trackIT!

With background correction you can improve any uneven illumination of the
object. The background correction is carried out on all images in the image
stack without the need to extract the images for this purpose.
In an image stack which exists as a gray value image, background correc-
tion is always recommended when the objects cannot be detected because
they are not really distinguishable from the background. You can carry out
tracking without background correction first and if the results are unsatisfac-
tory, then make a new attempt with a background corrected image stack.
You can select whether the corrected image is to be written into the same
image buffer (the original image will be overwritten!) or whether it is to be
written into the target image buffer. Before carrying out the background
correction, make sure that the target image buffer is either empty or can be
overwritten.
Note Ensure that the automatic settings for contrast for 16-bit images are acti-
vated. Otherwise, the image stack may only contain white images following
background correction. To do so, open the Display Properties dialog box
using the [CTRL + ALT + 2] key combination, select the Visualization tab
and select the Automatic contrast check box.
In the case of image stacks which exist as RGB images, background correc-
tion is always carried out before automatic tracking. The image stack is con-
verted into a gray value image in the process. The resulting image is always
written into the target image buffer, so that the original image is retained.
Before carrying out the background correction, make sure that the target
image buffer is either empty or can be overwritten.
Note Use the functionality of the Overlay button bar to look at the results of the
automatic tracking on the RGB image (instead of on the gray value image).
You can find further information under "How can I look at the tracking over-
lay in an RGB image and save it?" on page 55.
6
The "Define Automatic Tracking" dialog box

Before you can track objects you must configure the automatic tracking. The
values to be defined depend on the characteristics of the objects. These
values are generally different for each image stack. An object will only then
be detected and tracked when the defined values match. Detected objects
are displayed hatched in green in the overlay.
You define the settings in the Define Automatic Tracking dialog box:
The Define Automatic

Tracking dialog box is
divided into three sections:
the button bar, the tab Button bar
section and the navigator.
Tabs
Navigator
7
Button bar The button bar is in the upper section of the dialog box. Among other
buttons, it contains the one which starts the tracking operation.
Preview for detection

Automatic preview
Start tracking
Reset move factor
Save/Load tracking options
Tabs In the middle of the box you will find the Detector and Tracker tabs. Here,
you define all the settings you need to detect and track the required objects.
Note A detailed explanation of all of these tabs' settings can be found in the
chapter "Overview of the configuration options" on page 43.
The Navigator The navigator is located in the lower section of the dialog box. Use the
navigator to look at all the images in a stack as in a film (scrolling forwards
and backwards) or click from image to image manually. This is generally
necessary before a tracking is performed, to provide you with an overview
of the type and number of objects which are to be tracked. Only in this way
can the correct configuration settings be carried out.
If you want to jump to a particular image, enter the number in the display field
and press the [Enter] key.
By using the navigator
buttons you can move
quickly within an image
stack.
The navigator can always be used, irrespective of which tab is currently

active.
8
Tracking objects
Tracking objects
Below, you will find a description of the most important working steps which
are required to carry out an automatic tracking.
Most working steps are necessary in order to define the appropriate settings
for the object detection, so that the objects of interest are being detected.
These settings depend on the characteristics of the sample. There are no
values that are generally true.
The tracking is started by clicking the respective button, in the Define Auto-
matic Tracking dialog box.
PLEASE NOTE Only detected objects are tracked, all other objects are ignored. As long as
no object has been detected, it does not make sense to start the automatic
tracking, as it won't deliver any results.
Overview
The flowchart shows the
important steps for the
preparation of a tracking C lick o n a p o in t w ithin
operation. The steps in the th e o b je ct
rectangular text boxes must
always be carried out.
In contrast, the configura-

tion options provided in the D e fin e th e
oval text boxes need only to m in im u m /m a xim u n
be used when the sample o b je ct size
characteristics require it.
If n e ce ssa ry, click o n

If n e ces sa ry, fu rth e r p o in ts w ith in If n e ces sa ry,
d e fin e the th e o b je ct u n til it is a p ply th e
o b jec t s ep arator d e tecte d o b jec t m e rg er
9
If n e ces sa ry,
D e fin e th e se a rch If n e ces sa ry,
d e fin e the
d e fin e the
varia tion ra d iu s
“h idd en in m a x.“
If n e ces sa ry,
s ele ct th e ch ec k
S ta rt tra ckin g b o x „trac k a pp ea -
rin g ob jec ts“
Note Four demonstration files are supplied together with the software, to facilitate
the learning of the most important working steps and configuration options.
These demonstration files begin with a simple movement of an object
(Demo 01.tif) and then demonstrate increasingly complex processes (Demo
02, Demo 03 and Demo 04.tif).
Automatic tracking of a simple object
You can follow the instructions given below with the help of these demon-
stration files. We recommend you to gain your first experience with object
tracking in this way, before applying your knowledge to your own images.
You can find all demonstration files in the Images subdirectory of your
trackIT! installation directory.

How to... Track a simple cell movement:
1) Load the image stack Demo 01.tif into an image buffer. You can find
this file on the ..\Images\trackIT path, as long as you have not renamed
the installation directory.
2) Open the Define Automatic Tracking dialog box.
3) To gain an overview of the demonstration file, click the Play button in
the Navigator group. Click the First frame button in order to return to the
beginning of the image stack.
4) Activate the Automatic preview function. Only then will the display be
automatically refreshed when you change the settings in this dialog
box.
> trackIT! checks whether any objects can already be detected with
the currently defined settings. If so, the detection will be indicated
by a green hatching.
5) Define the object which is to be tracked (in the demonstration file this
is only one cell). Your data is then correct when trackIT! displays the
required object(s) as being detected (i.e. hatched in green).
In this example, one object
has been detected. You can
recognize it by the hatching.
Only this object can be
tracked.
10
• The object definition takes place in several steps and differs from
object to object. It must only be carried out on one image in the image
stack. Generally you take the first image. For object definition, first click
the Set/Add point from which the move factor will be calculated button
and then click on a point within the object, in the image window. In this
way you define any pixel which is to be tracked.
This should preferably take place using a pixel in the foreground of the
object area, i.e. in the direction in which the object moves. Further-
more, the pixel should preferably be a pixel that changes its gray value
in the next image, so that the movement is clearly recognizable for
trackIT!
> The defined pixel is marked with a red cross.

> The value in the Move factor field is refreshed. The move factor
provides the degree of probability that the pixel is moving (and is
not part of the background).
In the majority of cases, the object will already be detected, i.e., displayed
hatched in green. If this is not the case, check whether you have activated
Automatic preview and define the object sizes as described below.
If the object is detected, you can leave out the object size and define the
search radius immediately (see step 6).
• Click the Set minimum object size button.
> This moves you to the image window, where the cursor is displayed
as a circle.
• Draw this circle with pressed left mouse button to a size that is just
smaller than the object which is to be tracked. Click the right mouse
button to conclude the size definition.
> The circle must not be centered over the object, you can draw the
circle in any section of the image window.
> Only the absolute value of the area is relevant for the size filter, not
the form.
• Now define the maximum object size using the Set maximum object
size button.
> This moves you to the image window. Now, draw the circle to a size
that is somewhat larger than the object which is to be tracked and
click the right mouse button to conclude the size definition.
6) Go to the Tracker tab to define the search radius. The search radius
defines the maximum distance which an object may have covered
between two consecutive images (not the distance between the first
and the last image). trackIT! only searches for the object within this
distance.
• To define the search radius, click the Set search radius button. This
moves you to the image window. With the left mouse button pressed,
draw a circle of the required size and finish the definition by clicking the
right mouse button. This circle must not be centered on the object, you
11
can draw it in any section of the image.
> Now, all of the preconditions for a successful tracking have been
set. The object has been detected, i.e. it is displayed hatched in
green. The focal point is displayed as a red square.
> If the object has not been detected, check whether the Automatic
preview function has been activated. If the object is then still not
marked, select further points within the object that is to be tracked
using the Add point from which the move factor will be calculated
button.
7) To start the tracking, click the Start tracking button.
> All the images in the image stack are now displayed in succession.
Simultaneously, all the movements of the marked pixel are
registered.
> The tracking finishes when the last image has been displayed. The
trajectory is displayed in the overlay of the image. The first image in
the image stack will then be on display again.
The overlay displays the
object trajectory.
> The tracked object is given the marker "0" and the trajectory is
displayed in color. Although the serial numbering of the objects is
irrelevant for this example, it does make the results clearer when
several objects are tracked. The starting point of the object is
marked with a square, the end is marked with a circle.
The color of the trajectory display depends on the background: in
case of light background, dark colors are used and in case of dark
background, light colors are used. The colors themselves are
defined by trackIT! and cannot be changed.
> Several results sheets are generated automatically after each
tracking. These provide detailed information about the tracked
object(s) (see p. 16).
12
Suitable parameters for the task

If you do not obtain the required results when following the instructions given
above, use the values from the table below. These values have been proven
to be suitable in internal tests. Set the values by entering the given numbers
into the appropriate fields and pressing [Enter]. The object should now be
detected. Now repeat the tracking.
Parameter Value to be entered

Size filter min. (Detector tab ) 2000 µm2
Size filter max. (Detector tab) 6000 µm2
Object separator (Detector tab) deactivated
Search radius (Tracker tab) 90 µm
Automatic tracking of several objects
Parameter Value to be entered

Variation (Tracker tab) 0,1
All further trackIT! setting options can be ignored for
this example.

This task is about tracking several objects. To begin with, only those images
which are already present in the first image of the image stack are to be
tracked. The object which appears later is ignored for a moment.
How to Do the following:
the Navigator group. Click the First frame button in order to return to the
beginning of the image stack.
> As you can see, this demonstration file contains 3 moving objects
at the beginning of the image sequence. A fourth object appears in
image number 9 and disappears after image number 15.
4) Activate the Automatic preview function. It should remain active
throughout the entire task, since you then do not need to refresh the
display manually.
5) Define the objects which are to be tracked.
• To begin with, click the Set/Add point from which the move factor will
be calculated button and then on a pixel within the object, the move-
ment of which is to be tracked.
> The value in the Move factor field is refreshed.
PLEASE NOTE It is not necessary to click on every object that is to be tracked. A object will
13
be tracked, as long as it is detected, not depending on whether or not you
clicked on a pixel within this object.
• Define the minimum Object size. trackIT! will only find objects that are
the same size as, or larger than, the value entered here. To do so, click
the Set minimum object size button.
> This moves you to the image window, where the cursor is displayed
as a circle. Pressing the left mouse button, draw this circle to a size
that is somewhat smaller than the smallest object which is to be
tracked. Click the right mouse button to finish the size definition.
• Now define the maximum object size using the Set maximum object
size button.
> This moves you to the image window. Now draw the circle to a size
that is somewhat larger than the largest of the objects which are to
be tracked. Click the right mouse button to finish the size definition.
> All objects which lie within the defined minimum and maximum size
can already be displayed hatched in green.
> If this is not the case, check once again whether you have activated
Automatic preview and read on.
6) Now go to the Tracker tab to define the search radius. The search
radius defines the maximum distance which an object may have
covered between two consecutive images (not the distance between
the first and the last image). trackIT! only searches for the object within
this distance.
• To define the search radius, click the Set search radius button. This
moves you to the image window. With the left mouse button pressed,
draw a circle of the required size and finish the definition by clicking the
right mouse button.
> Now, all of the preconditions for a successful tracking have been
set. The objects will probably be detected now, i.e., displayed
hatched in green. The focal points are displayed as red squares.
> If the objects have not been detected, first check whether the Auto-
matic preview function is set.
• If all of the objects which are to be tracked are not yet detected, click
the Set/Add point from which the move factor will be calculated button
and then on other objects which are to be tracked.
> Using the Remove last added point button, you can delete the last
added point at any time and select another point.
> The Reset move factor button enables you to reset the move factor
to the default value of 0,1 and start again.

All the movements of the detected objects are registered. The focal
points are displayed as red squares.
distances covered are displayed in the image overlay. The first
image in the image stack will then be on display again.
> Three objects are displayed in the ObjOverview sheet.
14
into the appropriate fields and pressing [Enter]. The objects should now be
detected. Now repeat the tracking:
Value to be entered/
Parameter selected
Track appearing objects deactivated
(Tracker tab)
Parameter selected
this example.
Tracking appearing In a second tracking session, the object which appears in image 9 is now
objects also to be tracked. To do so, just one further setting is required. Do the
following:
1) Change over to the Tracker tab and select the Track appearing objects
check box.
2) Click the Start tracking button and watch what happens.
> The appearing object will now also be detected. The ObjOverview
sheet will now list four objects instead of three.
Note If you want to track appearing objects, you can carry out the object definition
in an image where the object or objects are clearly visible. In this way you
can be sure that objects which appear later will also be detected. In the
Demo 02.tif file for example, you could carry out the object definition in
image 11.
15
What information is contained in the generated sheets ?

Sheet name Content
Displays for each object:
ObjOverview_<file • the distance between starting and finishing point (straight line),
name> • the trajectory length,
• the extent (this is equivalent to the diameter of the operating range),
• the direction of the movement in ° (in relation to the Y
axis):
• the average speed,

• the X/Y coordinates for starting and finishing point,
• if applicable, the image number in which the object was 'lost' (e.g. because
it moved out of the image section)
In the lower half of the sheet you will find a statistics (in color). This is very
helpful when numerous objects have been detected.
In the Mean row, the data for all the objects is added up and divided by the
number of objects. In this way you can, for example, see the mean
trajectory length or speed, at a glance.
Object This sheet will only be shown when you have double-
Information_<file- clicked on an object in the ObjOverview sheet_<file-
name> name> (on the line number in the column on the far left).
The Object information_<filename> sheet shows the

information given below for the object selected: This data will be displayed for
every image in the image stack, i.e. in the case of an image stack containing 50
images, the sheet will consist of 50 lines.
• the information whether the object has been tracked in this image or not. With
objects that have been tracked, there will be a plus sign in the Valid column.
• the X/Y position of the object (or of the marked pixel),
16
• the time (since the acquisition began) at which the image was acquired.
• the time difference between this image and the previous image,
• the distance covered between this image and the previous one,
• the speed in this image.
Sheet name Content

Graph The graph is also empty as long as no object in the ObjOverview_<filename>
has been selected with a double click. A route time diagram is displayed for the
selected object in the Graphs window: Here, the distance covered is compared
to the time.
PLEASE NOTE Use the sheet/object link to see which object in the ObjOverview sheet
represents which object in the overlay.
Make sure that the image window is completely visible. Then doubleclick the
line number in the column on the far left, in the ObjOverview sheet. The
relevant object will be highlighted by flashing circles.
17
Closing all sheets If you would like to close all of the created results sheets without enquiry,
quickly use the keyboard shortcut [Shift+Ctrl+D]. The image in the image window
will remain on display.
The relationship between the Object Overview sheet and the

Object Information sheet
The content of the Object Information sheet relates exclusively to the object
that has been selected with a doubleclick in the ObjOverview sheet.
If, for example, you mark object 2 in the ObjOverview sheet (with a double
click on the line number in the far left column), then all data in the Object
Information sheet relates to object number 2.
If you subsequently select, e.g., the object 4 in the ObjOverview with a
doubleclick, the Object Information sheet will then be updated. Then, all of
the data will relate to the object number 4.
Doubleclick on the number
of the object you want.
The Object Information will

then show precise details on
how this object was
detected in each of the
images in the image stack
(e.g., in the sixth image).
18
Having the sheets displayed anew

Both the ObjOverview and the Object Information sheets will be automa-
tically closed when you have opened the Edit Tracking Results dialog box
and then closed it again.
To have the sheets displayed again, click the Edit Tracking Results button
once more.
Saving results
Saving results
Results will be saved The results of a tracking will be saved together with the image. Therefore, it
together with the is extremely important that you save an image stack immediately after a
image! tracking operation has been successfully complete!. Only then can you
display all results again when opening the image at a later time.
To do so, click the Edit Tracking Results button, in the trackIT button bar.
The results sheets generated for an image stack that has not been saved
after the tracking has been completed, will be lost when either the image
stack or the program are closed.
Saving results In addition to saving the results with the image, you can also save the Object
separately overview as a separate file in the SFS format. The graph can be saved as
SGD format file. These SFS and SGD files can be opened again with
trackIT!
On the other hand, it generally makes little sense to save the object informa-
tion as an SFS file. This file, when opened at a later date, will only contain
the detailed information about the object that was last doubleclicked in the
ObjOverview sheet. Just create the sheet anew, if necessary.
Exporting results
To be able to import the results of a tracking operation into a spreadsheet
program, where you can then edit them further, you will need to export the
results in the CSV (comma separated values) format.
In the Edit Tracking Results dialog box, click the Export Results... button to
create the following CSV files:
- ObjOverview_<filename>.csv
- Positions_<filename>.csv.
The Positions_<filename>.csv sheet contains all of the detailed information
on the objects that have been tracked. Here, the results that in the Object
Information sheet only relate to the currently selected object, will be written
into a sheet one after the other.
19
Saving results
General detailed information detailed information etc.

information regarding object 0 regarding object 1
Contents of the Positions_<filename> sheet
How to export the results:

1) In the trackIT button bar, click the Edit Tracking Results button.
> The Edit Tracking Results dialog box will open.
2) Click the Export Results... button.
> The Select directory dialog box will open.
20
3) Enter the directory in which the files are to be saved and click OK.
> The following files will now be written into the specified directory:
- ObjOverview_<filename>.csv
- Positions_<filename>.csv.
The file names are allocated automatically.
> For checking purposes, the Positions_<filename> sheet will be
opened in trackIT! You can then additionally save them in the SFS
format.
4) Close the Edit Tracking Results dialog box.
5) If necessary, switch to Windows Explorer to change the file names
(e.g., with [F2]).
Automatic tracking using the object separator

In this task, the aim is to track objects successfully, even if they are moving
past each other very closely. This can be done by defining an object
separator.
3) Activate the Automatic preview function.

the Navigator group.
> As you can see, this demonstration file contains 2 moving objects
These objects are moving past each other very closely in images
8-14, so that trackIT! cannot follow the trajectories correctly without
a defined object separator.
5) It is best to define an object separator in one of the images where it will
come into effect. Therefore, change to image number 8 in the
Navigator.
6) Click the Set/Add point from which the move factor will be calculated
button and then on a pixel within the object, the movement of which is
to be tracked.
> It is possible that both of the objects will already have been
detected. However, without the steps mentioned below, the
tracking would not render the correct results, i.e., the detection of
only two objects and the continuous tracking of their movements.
7) Define the minimum object size using the Set minimum object size
button.
21
8) Now define the maximum object size. To do so, it is not enough in this
case to define the object size using the biggest object. Define the
maximum object size in a way that both of the objects are included
(see illustration).
9) Select the Critical size check box. Then, define the Critical size of the
object separator, so that it approximately represents the size of the
larger of the two objects.
Make sure you don't make the Critical size larger than necessary. Other-
wise, the object separator will not render the desired results. In this example,
the Critical size should not exceed the value of 4000 µm.
10) Now go to the Tracker tab to define the search radius.

The focal points of the marked objects are displayed as red
squares. Some images display red circles. This indicates that the
object separator is being used there.
distances covered are displayed in the image overlay. The first
image in the image stack will then be on display again.
In this image, the two red > If the object separator has worked properly, two objects will have
circles show that the object been tracked, whose trajectories cross. Please enter the values
separator is active. from the table given below and repeat the tracking, should you have
got different results.

22
Parameter selected
Size filter min. (Detector tab ) 3 000 µm2
Size filter max. (Detector tab) 15 000 µm2
Object separator (Detector tab) activated
Critical size 3 500 µm2
Object merger (Detector tab) deactivated
Automatic tracking of unsteady objects
Parameter selected
Track appearing objects (Tracker tab) deactivated
this example.

In this task, the objective is to track an object successfully, even if it is an
unsteady object, i.e. if it stands still in the process of tracking and then starts
moving again. This can be done by defining a variation factor.
For this example, the variation factor must be set to 0,1. If the variation factor
was 0, then trackIT! would either recognize just one object that is lost in
image 35, or two objects (if the Track appearing objects option is marked).
Variation factor The variation factor checks whether a stationary object is identical to an
object that is moving in a later image. The identity check takes place through
a cross correlation of the gray value histograms of both images. The image
areas, in which the variation factor comes into effect, are marked by a dark
green rectangle.
You can find more information about the variation factor on page 46.
the Navigator group.
> As you can see, this demonstration file contains a moving object
which stops moving in images 36-41 and starts moving again from
image 42. In order for trackIT! to recognize and track this object as
one object throughout the entire image stack, a variation factor for
unsteady objects must be set.
23
4) Activate the Automatic preview function.
5) Define the object which is to be tracked step by step:
• To begin with, click the Set/Add point from which the move factor will
be calculated button and then on a pixel within the object.
• Define the minimum and maximum object size.
• Change over to the Tracker tab and set the value 0.1 in the Variation
field.
6) Define the search radius.
7) If the object has not yet been detected, mark other pixels in the object.
> In this example, the object to be dealt with is a very inhomogeneous
object, i.e. the gray values within the object vary greatly. For this
reason it is likely that you will have to mark further pixels. Should
this not lead to the result you want, enter a smaller move factor,
e.g., 0,0002.

9) Check the number of objects found.
> Based on your settings, only one object should have been found.
Parameter selected
Move factor 0,0002
Variation factor (Tracker tab) 0,1
Tracking appearing objects activated
this example.
24
Edit tracking results

During a tracking operation, all objects that have been detected will be
followed. You cannot exempt any of the detected objects from the operation
from the start. When the tracking has been completed, you can, however,
alter the display of the results.
• You can alter the display of the object trajectories.
• You can fade out uninteresting objects, or delete them.
• You can have the object trajectories displayed in a histogram.
Altering the display of an object trajectory
Smooth Trajectories You can smooth the displayed trajectory a little. To do so, select the Smooth
trajectories check box. You can see the result in the image overlay.
Original trajectory Smoothed trajectory
25
PLEASE NOTE These settings change the results in the ObjOverview_<filename> sheet. In
particular, smoothing the trajectory will shorten it.
Display distances only Here the direction and the distance covered are displayed as a straight line.
All "wobbles" in the object movement are ignored:
Trajectory Length Distance
This setting does not change the results in the ObjOverview_<filename>

sheet, only the way the trajectory is displayed in the image overlay will
change.
Hiding objects
You can hide the results of detected objects. By default, the trajectories of
all detected objects are displayed.
Mode
In the Object display options group, the following options are available in the
Mode field:
26
All objects This is the standard setting. The trajectories of all detected objects are
displayed.
No objects Only displays the objects without trajectory. The results sheets are empty.
In this way, the initial situation before detection can be displayed.
Selected objects This mode is appropriate when you are only interested in some of the
detected objects. The trajectories of all other objects are then no longer
shown in the overlay and their results are no longer shown in the sheets. The
data will not be deleted, however, and you can see it again at any time by
using the All objects option.
As soon as you have selected the Mode entry, in the Selected objects list,
the Edit Tracking Results dialog box will display the Object Selection group.
Here, the All, None and Interactive... buttons are available.
With these buttons you can determine whether you want to select all of the
objects that interest you, or prefer to clear the objects that don't interest you.
In the Selected objects

mode, the Edit Tracking
Results dialog box will be
expanded by the Object
selection group.
A description of the procedure for selecting objects can be found in the chap-
ters "Clearing uninteresting objects" on page 28 and "Selecting interesting
objects" on page 28.
Filtered Objects This mode is appropriate when you are only interested in the part of the
detected objects that fulfill certain criteria. As soon as you have selected the
Filtered objects entry, in the Mode list, the Edit Tracking Results dialog box
will additionally display the Object filter group. Here, you will find numerous
criteria with which you can filter the objects. You will find further information
in the chapter "Filtering objects" on page 30.
In the Filtered objects mode,
the Edit Tracking Results
dialog box will be expanded
by the Object filter group.
27
Clearing uninteresting objects

1) Select the Selected objects mode.
2) If you want to clear all of the objects that don't interest you, first click
the All button, then the Interactive... button.
> The cursor appears in the image window. All of the object trajec-
tories are selected. The red marking shows you this.
3) While keeping the [Shift] key depressed, click one after the other on the
objects you are not interested in.
> These objects' trajectories will then be displayed in their original
color again.
4) Press the right mouse button to finish the selection process.
> Then, only the trajectories of the objects you are interested in will
be displayed, the trajectories of the others will have been faded out.
> The sheets will also only display the interesting objects.
5) Save the object selection by saving the image (again).
> From now on, only the currently selected objects will be shown
when opening the image at a later time. You can, however, use the
All objects mode to have all the objects displayed again.
Selecting interesting objects

1) Select the Selected objects mode.
2) If you want to select all of the objects that interest you, first click the
None button, then the Interactive... button.
> The cursor appears in the image window. No object trajectory is
selected.
3) Click the objects that interest you, one after the other.
> The trajectories of these objects will be displayed in red.
4) Press the right mouse button to finish the selection process.
> Now only the trajectories of the selected objects will be displayed.
> The sheets will also only display the results of the selected objects.
5) Save the object selection by saving the image (again).
> From now on, only the currently selected objects will be shown
28
when opening the image at a later time. You can, however, use the
All objects mode to have all the objects displayed again.
Deleting uninteresting objects

Using the Selected objects or the Filtered objects options will result in the
uninteresting object trajectories being faded out. Their results in the sheets
will also be faded out. As soon as you again select the All objects mode, all
of the detected objects will be displayed once more.
However, you can also definitely delete the results of uninteresting objects.
Do the following:
1) Make sure that, e.g., only the interesting trajectories are on display.
2) Click the Delete... button to definitely delete the results of uninteresting
objects.
> You will receive the following query:
3) Click the No button, to delete all of the trajectories that are not currently
on display.
29
Filtering objects
Filtering objects
1) Should you only want to see the results of certain objects, open the Edit
Tracking Results dialog box when the tracking has been completed. In
the Mode list, select the Filtered objects entry.
> The Edit Tracking Results dialog box will be expanded by the
Object filter group. In it you will find numerous filter criteria. By
default, no filters are set. You will find further information in the
chapter "Which filter criteria are there?" on page 32.
> The values that are displayed in the Object filter group's fields will
have been taken from the ObjOverview_<filename> sheet. In each
case, the highest and the lowest values found will be taken. In this
way you will initially still see the results of all of the objects that have
been tracked.
30
The sheet ObjOverview_Demo 02 shows the results for the tracked objects.
When you open the Edit Tracking Results dialog box, the Object filter group shows the smallest
and greatest value calculated in each case, in the Distance, Length, Extent and Speed fields.
Filtering objects
2) Now you can activate the filter or filters with which you want to filter the
results. To do so, select the respective check box.
3) Enter a minimum and/or maximum value in the edit field. To do this, you
can either use the small arrow buttons next to the edit field, or use your
keyboard to enter a value, then press the [Enter] key.
> Your entry will only be accepted when it further limits the value that
has already been given:
In this example, several
objects have been tracked
and the following values
were calculated: The
shortest distance that one of
the objects traveled was
2 µm. The longest distance
In this field, all values that In this field, all values that are
was 8 µm. Your filter defini-
tion can limit these calcu- are bigger than 2,0 µm (and smaller than 8,0 µm will be
lated values further, but it smaller than 8,0 µm) will be accepted.
can't extend them. accepted.
4) Take a look at the results of your filter definition. Click the Apply button.
> Then, only the objects to which the filter criteria apply will be
displayed in the overlay and in the sheets.
Update display > When the Update display automatically check box has been
automatically selected, the image overlay and the ObjOverview_<filename>
sheet will be continuously updated. In this case the Apply button is
not required, and is therefore inactive.
1) select check box 2) enter a value
31
3) click
"Apply"
Filtering objects
Which filter criteria are there?

Distance
• The distance that an object has traveled from its starting to its end
point.
End
Start
Length
• The trajectory length that an object has traveled from its starting to its
end point.
End
Start
Extent
• The diameter of the operating range
Taken together, all of the positions of an object trajectory make up the
operating range. The largest distance between two positions deter-
mines the diameter of the operating range, which is also called Extent.
Start End
Direction
37°
• Direction in which the object moves. The direction is determined by
centering the starting point and by defining the end point's position on
End a circle in° (in relation to the Y axis).
32
Start
Speed • The mean minimum and maximum speed of the object between two of
the image stack's images.
Filtering objects
Inverting filter definitions

By default, your filter definitions will be applied in such a way that only those
objects that fulfill these filter criteria will be displayed. You can also invert a
filter definition. Then, only the objects that do not fulfill the filter criteria will
be displayed. To do so, select the Invert check box.
You can decide separately
for each filter whether it is to
be inverted or not. In the
illustration, you can see that
the Distance and Speed
filters have been switched
on. And that the Distance
filter has been used
inverted, i.e., in the example
shown, only objects whose
distance lies outside the
minimum and maximum
values will be displayed.
Combining filter definitions

Should you have selected several filters, you can decide whether an object
is to fulfill all of the filter criteria, in order to be displayed, or whether it need
only fulfill one of the filter criteria.
Should it have to fulfill all of the filter criteria, the Combine AND option must
be selected. This is the standard setting.
With this setting, only
objects will be displayed
where the distance traveled
and the speed, both lie
between the minimum and
maximum values.
33
Filtering objects
Should an object only need to fulfill one of the filter criteria, the Combine OR
option must be selected. When the filter definition remains the same, you will
have more objects displayed when you use the Combine OR option, than
when you use the Combine AND option.
With this setting, objects
where either the distance
traveled or the speed, (or
both), lie between the mini-
mum and maximum values,
will be displayed.
Saving and re-using filter definitions

You can save your filter definitions for later use with other samples.
Saving filter definitions 1) To do so, click the File... button.
> The Save/Load tracking options dialog box will open.
34
2) Assign a meaningful filter name and click the Save button.

> Your filter definition will be saved in the TrackIt subfolder, in your
root directory.
> From then on, you will be able to use this filter definition for every
tracking operation. When you do so, while you are loading it,
trackIT! will check whether the minimum and maximum values that
have been saved are applicable for the current tracking operation.
If this is the case, the values will be loaded. If not, the values will be
ignored. You will receive a corresponding message. In this case,
only the options in selected check boxes and the Combine AND or
Combine OR settings will be adopted.
3) Click the Close button.
> The dialog box will be closed. Filter definitions that have not been
saved will be ignored.
Filtering objects
Loading a filter 1) To do so, click the File... button.

definition > The Save/Load tracking options dialog box will open. It contains all
of the filter definitions that have been saved.
2) Select a filter definition, and click the Load button.
> The values will be entered in the corresponding fields in the Object
Filter group.
Setting filter definitions interactively in the image.

You can also set the filter values (except the speed) interactively in the
image overlay. This always makes sense if you intend to use structures in
the image as an orientation guide when you are setting values.
Using the [Shift+F4] keyboard shortcut, you can make the scale bar appear
and use it to help you judge a distance's length. This makes only sense if the
images have been correctly calibrated.
You define the filter values in two steps: in the first you set the minimum
value, in the second, the maximum value. The procedure is the same for the
Distance, Length and Extent, filter values.
Here, you can see the
mouse clicks that are
select filter define minimum value define maximum value
necessary to define the
Distance filter interactively
in the image.
35
select automatic update of display
Defining Distance, Length and Extent interactively

1) Make certain that the image window in which the tracked objects are
displayed is fully visible.
2) Activate the filter by selecting the check box next to it.
3) Click the button located to the right of the minimum filter value.
> The pointer moves to the image window.
4) Draw a line that is equivalent to the minimum distance, length or extent.
> The direction and form in which you draw the line is of no impor-
tance, since only the value of its length will be saved.
5) Finish the definition by depressing the following mouse button:
left mouse button to define the minimum distance,
right mouse button to define the minimum length or extent.
> Your entry will be adopted, provided it further limits the value that
was previously calculated. Should, however, your entry exceed, or
fall short of, the values that were previously calculated, it will be
ignored. Instead, the smallest calculated value will be inserted.
Defining the direction interactively

1) Activate the filter by selecting the check box next to the Direction filter.
2) Click the button located to the right of the maximum filter value.
> The pointer moves to the image window.
3) Click a point on the first leg of the angle.
1 > A line will appear.
3 4) Position the line by clicking on a point on the line.
> The first leg of the angle has been defined. The point that was last
set will at the same time become the angle's vertex.
2 5) Move your mouse to the left or to the right.
> A second line will appear.
6) Set the second line by clicking on a point on the line.
> The second leg of the angle has then been defined. The current
angle will be indicated by an arrow.
PLEASE NOTE The angles will always be measured in a clockwise direction.
1
3
If an object is lost in an image and is tracked as a new object in a later image,
you can correct the result later, using this function to merge the two object
36
trajectories.
PLEASE NOTE Keep to the correct chronological order when selecting the trajectories:
select the trajectory which shows the beginning of the covered segment first
(e.g. in images 1-20) and then the one for the second part of the segment
(e.g. in images 21-50). You will receive an error message if you try to merge
trajectories which are not in the correct chronological order.
1) Click the Merge Trajectories button.
> This moves you to the image window.
2) Move your pointer over the first trajectory that is to be merged. When
you do this, it must be the part of the trajectory that was first set up.
> The trajectory is marked red.
3) Click on the second trajectory to be merged.
> The object trajectories are connected. The object is summarized in
the ObjOverview sheet.
4) Press the right mouse button to finish the process.
PLEASE NOTE You can cancel the merge mode by using the [Esc] key.
Execute manual tracking

Manual tracking of objects is always preferable when the image quality or
the object behavior is unsuitable for automatic tracking. In this case you can
define the current position of an object in the image, by clicking the mouse.
Manual tracking is useful for tracking objects which are only visible in a few
of the images in an image stack. Then, you only mark the object position on
these images and skip the other images without marking a position.
In addition, you can also use the manual tracking functions to alter the
results of an automatic tracking operation.
The results of a manual tracking will be, exactly as those of an automatic
tracking, displayed in the ObjOverview_<filename> sheet.
In this example several
objects have been tracked
manually.
Using the Add new button,
you can track further
objects.
Using the Remove All but-

ton, you can delete all the
trajectories displayed in the
Object IDs field, irrespective
of whether the trajectories
were created using manual
or automatic tracking. The
sheets will also be cleared
and you can start from the
beginning again.
Note For users of the Windows XP operating system:
The pointer (in the form of a selection cross) will only be correctly displayed
in manual tracking if the display of mouse trajectories is deactivated. Check
your mouse configuration in the Windows Control Panel if necessary.
37
How to track an object manually

If you want to track an object manually, you can decide whether you want to
determine the object position in every image in the image stack, or, for
example, only in every fifth image. In the second case, scroll the mouse
wheel after you have marked a position, or use the arrow buttons on your
keyboard. Then, the next images in the image stack will be skipped. Click
the left mouse button when you want to mark the next position.
The illustration shows how
an object is followed
manually. The white spots
represent the clicked object
positions.
In this example, the object

position was only deter-
mined in every fifth image.
The images between these
were skipped.
38
1) Load the required image stack (e.g., the Demo 03.tif file) and click the
Execute Manual Tracking button.
> The Manual Tracking dialog box opens.
> If you have previously carried out automatic tracking on this image
stack, the objects which have already been tracked will be listed in
the Object ID field.
2) Click the Add New button to track an object manually.
> The image window will be activated and the cursor becomes a
cross. Always the first image in the image stack will be displayed. If
you want to track an object which appears later manually, you will
therefore need to first skip a few images before you can define the
object position.
> An information window, that explains the way the mouse buttons
and some keyboard keys work, will appear at the bottom left of your
screen (see also "Key configuration during manual tracking" on
page 40):
As soon as you start manual

tracking, the key configu-
ration will be displayed.
3) Click on the (approximate) center of the object which is to be tracked.

> In this way you will define the object's current position in this image.
The position will be marked with a square.
> Then, the next image in the image stack will be displayed.
4) Click once more on the center of the object, to define the object posi-
tion. Alternatively, you can skip several images without marking a posi-
tion, by scrolling your mouse wheel, or by using the arrow keys on your
keyboard.
> For object positions that you have skipped, trackIT! will calculate an
X/Y position by using the averaged values of the previous and the
following X/Y position.
5) After you have marked the object position in the last image, you will be
asked whether you want to track another object manually.
> If you answer positively, the image window becomes active again
and you can define the first position of the next object.
> If you answer negatively, the manual tracking is canceled. You can,
however, track further objects at a later time.
> The same sheets are created following manual tracking as those
after automatic tracking. Further information can be found in the
chapter "What information is contained in the generated sheets ?"
on page 16.
Tracking a further object manually

1) If, after completing manual tracking, you decide to track a further object
manually, click once again on the Execute Manual Tracking button.
> The Execute Manual Tracking dialog box opens.
2) Click the Add New button to track a new object.
39
Correcting set object positions subsequently
You can, at any time, correct object positions that have been determined by
a tracking operation. This is always possible, regardless of whether the posi-
tion you want to alter has been automatically or manually set.
1) Select the object you want to alter in the Object IDs list in the Manual
Tracking dialog box.
2) Click the Edit button.
> The image window will be activated. The first image in the image
stack will be displayed. The set position will be marked with a red
square. The position number will be displayed.
3) Then move to the position you want to correct. There are two ways of
doing this:
• You can either have all of the positions that have been marked shown,
one after the other, by scrolling your mouse wheel, or by using the
arrow keys on your keyboard. The position number will be displayed.
• You can click the spot on the trajectory that you want to change. While
you are doing this, keep the [Ctrl] key pressed. Then, the image in the
stack in which the position was marked will be displayed. The position
number will also be displayed.
4) Then release the [Ctrl] key, and using your mouse's left button, drag the
square that is shown in red, onto the new place.
> This object's trajectory will then have been changed. The data in the
sheets has also been updated.
> The next image in the image stack will be displayed.
5) Then you can, if necessary, correct other positions, or finish editing the
object trajectory by using your right mouse button.
Deleting an object position

You cannot completely delete an object position. It is, however, possible to
delete the values that have been given for a position. Then trackIT! will
smooth this trajectory and calculate the new values for the X/Y position by
using the averaged values of the previous and the following X and Y posi-
tion. The values for distance and speed will be adopted from the object
position that follows.
EXAMPLE: The position 10 on an object trajectory is to be deleted. The upper sheet
shows the value for the position 10 before the deletion. The lower sheet
shows the value after the deletion. The new X/Y position for the position 10
corresponds to the averaged values from the positions 9 and 11. The
values for distance and speed have been adopted from position 11.
40
Key configuration during manual tracking

• Marking an object position: click the left mouse button.
• Moving to the next image: Scroll the mouse wheel, or use the arrow
keys on your keyboard. The can move backwards and forwards
through the image stack.
• Deleting a position To do so, press the [Shift] key at the same time as
the left mouse button. The cursor will change into a cross. Then click
on the positions you want to delete.
Administration of tracking configurations
• Opening a position Click the [Ctrl] key at the same time as the left
mouse button, to load the image that corresponds to the currently
marked spot on the object trajectory. You can then check this position,
and, if necessary, correct it.
• Finishing Manual Tracking: After the marking of the object position in
the final image, manual tracking finishes automatically. If you want to
stop manual tracking before this, press the right mouse button. All of
the positions that have up till then been marked, will be saved and
shown in both the overlay and in the sheets.
• Abort the Manual Tracking: Press the [ESC] key to abort the tracking
operation. The positions that have up till this point been marked, will not
be saved.
Deleting tracking results

In the Manual Tracking dialog box you can delete all, or individual, trajecto-
ries of a tracking. This applies both to trajectories that have been created by
automatic, as well as by manual, tracking.
When you use the Remove All button, all of the trajectories displayed in the
Object IDs field will be deleted without any further query. The sheets will also
be deleted.
When you use the Remove button, only the trajectories that are currently
marked will be deleted.
Administration of tracking configurations

You can save your configuration files and reload them at any time. This
makes sense, for example, if you are working with similar samples and
therefore want to keep using the same configuration parameters. Further-
more, you can use the configuration parameters to document which settings
you have used to carry out tracking. You can delete any unnecessary
configuration files at any time.
Saving the configuration
41
How to To save a trackIT! configuration, do the following:
2) Click the Save/Load tracking options button.
> You can see the Save/Load tracking options dialog box.
Loading the configuration
3) Enter a name for the configuration. The CTO file name extension will
be added by trackIT! automatically and need not be entered.
> The Save button is activated.
4) Press Save, to save the configuration and to close the window.
> Your configuration will be saved in your installation directory in the
TrackIt subfolder. If the folder does not exist, it will be set up
automatically.
Loading the configuration

1) To load the configuration, click the Save/Load tracking options button
in the Define Automatic Tracking dialog box.
> You can see all the configuration files which are in the TrackIt
subfolder in your installation directory.
2) Select a configuration with a double mouse click.
> The configuration will be loaded and the dialog box closes automat-
ically.
Note The path for the configuration files is predefined and cannot be altered. The
files must be saved in the installation directory in the TrackIt subfolder and
can only be loaded from there.
42
Overview of the configuration options
Overview of the configuration options

In this chapter you will find an explanation of all the trackIT! configuration
options.
Detector tab
43
Move factor This factor provides the degree of probability that a "move" event is taking
place in the selected pixel. This is manifested by a gray value alteration over
a period of time. The move factor is based on a complex formula which
cannot be discussed in detail here.
Size filter Enter the minimum and maximum sizes that the object to be tracked can
have into the Min. and Max. fields. All objects which have an area that is
greater or smaller than the value defined here will be ignored.
Thus the size filter is a central element to exclude irrelevant objects from the
detection process. Ensure that the size filter is correctly set before every
detection.
The entry must be in the units squared that the image is calibrated in, e.g.
µm2. Only the absolute value of the area is relevant for the size filter, not the
form.
Detector tab
Object separator It could be that trackIT! detects two or more individual objects as one large
object. In this case you can separate the objects by defining the maximum
size that an object may have. To do so, select the Critical size check box. All
objects which are larger than this size are automatically separated into
several objects. The entry must be in the units squared that the image is
calibrated in, e.g. µm2.
The value of the object separator must be within the parameters of the set
minimum and maximum size filter. If you do enter a higher value, this will
automatically be replaced by the value in the Max. field as soon as you click
into another field.
Object merger
It could be that trackIT! unintentionally detects one object as two different
objects. You recognize this by the fact that two focal points are displayed,
instead of just one (recognizable by the red squares). In this case you can
use the object merger, so as to retain just one object.
Note You can also use the object merger to detect objects which have been
correctly recognized as an object, but which have been incompletely
detected (e.g. with notches on the edges). In this case, object merging
produces the same results as the manual selection of further pixels in the
object.
If you select the Use check box, the "dilatation" morphological filter is used
during tracking. This completes pixels at the edges of objects. In this way
several separately detected objects are merged to one object.
The filter examines the area around a single pixel and compares the value
with the values of the pixel in the set near vicinity. The central pixel is given
the maximum gray value of all the pixels from the vicinity. The size of the
vicinity can be defined with the Small , Medium and Large option. These
settings are the equivalent of a hexagonal grid of 3x3, 5x5 and 7x7 pixels.
44
Tracker tab
Tracker tab
Search radius Here you define the maximum radius within which an object can move from
one image to another before it is found again. trackIT! only searches for the
object within this radius. You can either draw the radius interactively in the
45
image window using the mouse, or enter a fixed value in the Search radius
window. A search radius that is too small leads to trajectories being
interrupted.
"Hidden in In this field you define the number of images within which trackIT! checks
max. frames" value whether an object is moving or not.
Example:
An object moves in images 1-5, then disappears from the focal area and
reappears in images 13-50. With the default value of "hidden in
max. frames" being set to 5, trackIT! would set the object from image 10
onwards to "dead" status and then no longer track the object.
In order that trackIT! still checks whether the object is still moving in image
13, you must enter a minimum value of "hidden in max. frames" of 8 (= 13-
5) images.
Raise the "hidden in max. frames" value only when you can assume or know
that the later moving object is really the object that either stopped moving or
disappeared out of the focal area in an earlier image. It does not make sense
to set the "hidden in max. frames" value at a high level as a matter of course.
Unlike tracking an object using the variation factor (see below), with the
Tracker tab
"hidden in max. frames" value there are no "identity checks". An object that
reappears in the vicinity of the stationary object can, in the case of a high
"hidden in max. frames" value, be recognized as that object, even if it looks
quite different (assuming that it is within the defined object sizes).
Tracking appearing With this check box you define whether only objects that are visible on the
objects first image of the image stack should be tracked, or also those which appear
in a later image. Objects which are stationary in the first images of an image
stack and then "run off" at some time will only be tracked using this check
box.
Unsteady objects Unsteady objects are objects which begin to move and then stop moving at
some time.
Variation The variation factor checks whether an object that starts to move is identical
to an object that has stopped moving in the same image area. The image
areas, in which the variation factor comes into effect, are marked by a dark
green rectangle (during the tracking only).
The level of the variation factor defines the extent to which, in percent, the
gray values which are within the dark green rectangle may vary, so that
trackIT! may interpret the object as an identical object (and therefore
continue to track it). A variation factor of 0.1 means that the gray values may
vary by a maximum of 10%, so that the object may be further tracked.
The higher the variation factor is, the more probable it is that tracktrackIT!IT
continues to track objects which have stopped moving. A low variation factor
leads, inversely, to greater loss of objects.
The variation factor can have a maximum value of 1. With this value trackIT!
will definitely track an object that appears later, even if it looks completely
different and perhaps does not match the object that stopped moving at all.
Thus a variation factor that is too high increases the risk that two different
objects may be interpreted and tracked as one single object.
46
The Edit Tracking Results dialog box
Object display options Using the various object display options, you can control whether all or only
certain defined results of a tracking should be displayed.
All objects The default setting is All objects. The display of the results relates to the
display of the trajectories in the overlay and the entries in the sheets. By
selecting a display option you simply mask results. No data is deleted and
you can display all results again at any time.
The following possibilities are still available in the Mode field.
No objects Only displays the objects without trajectory. The results sheets are empty.
In this way, the initial situation before detection can be displayed.
Selected objects This option is appropriate when you are only interested in some of the
detected objects. The trajectories of all other objects are then no longer
shown in the overlay and their measurement data is masked in the sheets.
The data will not be deleted, however, and you can see it again at any time
by using the All objects option.
As soon as you have selected the Mode entry, in the Selected objects list,
the Edit Tracking Results dialog box will display the Object Selection group.
Here, the All, None and Interactive... buttons are available.
With these buttons you can determine whether you want to select all of the
objects that interest you, or prefer to clear the objects that don't interest you.
47
Filtered Objects Displays only the objects that correspond to your filter definition. You will find
further information on filtering in the chapter "Filtering objects" on page 30.
Smooth Trajectories Here you can smoothen the displayed trajectory somewhat, so that the
display is then clearer. The data in the sheets are updated, which is easily
recognizable by the shorter trajectory length.
Display distances only Here the direction and the distance covered are displayed as a straight line.
All deviations from the shortest distance between the starting and finishing
points will be ignored (see the illustration on page 26).
Display trajectories The results of tracking are displayed graphically in the overlay. Additionally,
the values are given in the sheets. And the Display trajectories group offers
more display options, too. Click the corresponding button to activate one of
these display options.
Original trajectories: This way, a new image will be written into the next free image buffer. It will
only show the object trajectories, without the image:
Centered trajectories: This way, a new image will be written into the next free image buffer.
Displays the trajectories of all objects so that they all start from the center of
the image window:
48
Histograms Graphical display of the object classes in a bar graph.

Histograms can only be created if at least two objects are shown. If only one
object is shown, this button will remain inactive.
In this histogram the objects

found are grouped
according to their extent.
The classification is made according to a variety of parameters. By setting a

class size you define how coarsely or finely the objects should be classified.
For example, a fine classification is recommended for the Mean Speed
parameter, when all the objects were of approximately the same speed, and
a coarser classification if there were greater differences in the speed of the
objects.
You can create histograms for the following parameters:
- (covered) distance (e.g. in µm)
- trajectory length (e.g. in µm)
- extent (e.g. in µm)
- direction (as a direction diagram that will be written into the next
free image buffer, see p. 50).
- mean speed (e.g. µm per second)
The unit of length is taken from the image calibration. The unit of time
derives from the time stamp of the image stack.
Classes When creating histograms, you can define the number of classes. The
default setting is for five classes.
49
With the number of classes, you define the number of groups that the rele-
vant result parameter is subdivided into (e.g. the speed). In the case of track-
ing many objects, the results of which are very close to each other, it makes
sense to increase the number of classes to, e.g., 20. Inversely, it is better to
reduce the number of classes to, for example, 2 when the tracking leads to
very different results. You can change the number of classes for each histo-
gram separately. Furthermore, you can create the diagrams as often as
required, without renewed tracking, and also change the number of classes.
Save Results Since the results will always be saved together with the image, you can at
any time recreate the histograms anew, and do not need to save them
separately. However, it is possible to save the histograms you have created
in the *.SFD format. The direction diagram will be written into the next free
image buffer as an image. It can be saved in different graphic formats. The
default suggested by the program is the file name extension *.TIF.
Note If you would like to close all created results sheets and histograms without
query and without saving, you can do so by using the key stroke
[Shift+Ctrl+D]. The image in the image window will remain on display.
Direction diagram The direction diagram displays the direction in which the objects have
moved. The radius of 360° is subdivided into the selected number of
classes. The number of objects which have moved within this degree range
is given for each area.
50
If you selected a division

into 4 classes when creating
the direction diagram, this
means that each class has a class 4: class 1:
degree range of 90°. 270°-360° 0-90°
2
Number of objects found
in this class
1
class 3: class 2:
180°-270° 90°-180°
If you have selected a

division into more classes,
each section will again be class 8: class 1:
315°-360° 0-45°
divided finely. For example,
each class has a degree
range of 45° when you
divide into eight classes. class 2:
Thus the display of the class 7: 45°-90°
270°-315° 1 1
results in the direction
diagram always depends on
1
51
the division of the classes.
class 3:
class 6:
90°-135°
225°-270°
Invisible further
division of the radius
into 8 classes
class 5: class 4:
180°-225° 135°-180°
Suggestions and questions with regard to trackIT!

Does it make any difference whether objects are detected larger
or smaller?
An object that is detected larger does not have the same focal point as a
smaller detected object. In this way the coordinates for the distance, trajec-
tory length and the speed are distorted.
Ensure therefore that the objects are not detected larger than necessary by
only making use of the object merger when really required.
What can I do with objects whose direction of movement is also

detected?
First check whether the object merger is activated, if this is the case, switch
it off.
Otherwise this result from the detection cannot be influenced later since it is
linked to the properties of the image stack: trackIT! always uses three
images for the detection. If the object in these three images moves in steps
that are too large, this leads to a detection as shown in the illustration. The
only thing that helps is to ensure during the next new image acquisition that
the chronological distance between the acquisitions is shorter (and thus the
object movements turn out smaller).
What can I do about objects which are detected too small?
Click on further points within the object.
Alternatively, you can switch on the object merger on the Detector tab.
What can I do with two objects which are recognized as one

object?
52
If you want to detect both objects, use the object separator.

If you only want to detect one object, reduce the maximum object size. Clear
the Object merger option or reduce it by one level (e.g. from Large to
Medium or Small.
What can I do when one object is recognized as several

objects?
Check whether the maximum object size is set too small.
Check whether the Object separator is activated and either switch it off or
adjust the Critical size.
How can I track certain objects only?

When tracking automatically, all objects that have been detected will be
tracked. An object will be detected when it matches the defined configura-
tion parameters. You can, however, fade out those objects after tracking
which are of no interest to you (see p. 26).
In contrast, when tracking manually, you can freely choose which objects
you want to track. Simply click on the object which is of interest. In the case
of several objects, select the next object once you have completed the first
tracking, and also track it manually.
How can I see that an object as been lost?

The beginning of a trajectory is marked by a square, the end by a circle. If
an object has been lost, the trajectory ends without a circle (see the illustra-
tion).
In the ObjOverview sheet, you will find an entry in the "Lost in frame No."
column.
A frequent reason for the loss of an object is that the search radius was not
defined large enough. In such a case, increase the search radius and start
tracking anew.
If, however, an object has been lost because the object runs out of the
image, this is correct behavior and cannot be influenced.
What can I do when my object is not detected?

First check whether you have switched on the Automatic preview or whether
you updated the display manually following your last alterations (using the
Preview for detection button).
Note Objects are best detected when you select a pixel which lies in the front area
of the object, i.e. in the direction in which the object is moving. Furthermore,
the pixel should preferably be a pixel that changes its gray value in the next
image, so that the movement is clearly recognizable for trackIT!
53
However, it may well be that an object will not be detected correctly if just
one pixel is defined. In this case, click using the Set/Add point from which
the move factor will be calculated button into the object several times until it
is selected. In the process, select preferably those pixels which have a gray
value that changes in the next image, making the movement clearly
recognizable for trackIT!.
If the object has still not been detected, then check also:
• whether you have defined the object size correctly,
• whether you have switched on the Automatic preview (or whether you
have manually updated the display using the Preview for detection
button).
How can I recognize a correctly set object separator?

An object separator is always correctly set when a focal point (in the form of
a red square) is displayed for each separate object. It does not matter
whether the green hatching for the detection is also separated or not, as is
shown in the example on the left.
Below, you will find two examples of object separators that have been set
incorrectly.
separator too large (1 object) separator too small (7 objects)
When you use an object separator, remember that trackIT! only takes
objects into account which are within the defined maximum size. For the
detected objects, trackIT! checks in a second step whether an object sepa-
rator is to be taken into account. If this is the case, then trackIT! separates
the large detected object into two or more objects. Therefore, when working
with the object separator, the added up size of the individual objects must
always be defined as the maximum size. The individual object size must be
entered into the Critical size field.
What do I do when two objects cross?

The result depends on how closely the objects run together. In the best
54
situation, trackIT! recognizes both objects and tracks the trajectories

correctly (see the illustration on the left). Possibly an object separator needs
to be used.
It is also possible that trackIT! only continues to follow one trajectory, then
you must complete the other trajectory using manual tracking. When neither
of the two trajectories is tracked further, you can also complete the trajecto-
ries using manual tracking.
What do I need the Preview for detection for?

The Automatic preview uses the parameters defined in the Define Automatic
Tracking dialog box on the selected image stack. If the parameters are
correct, the objects will be detected. Otherwise you must alter the parame-
ters. For every altered parameter, the Automatic preview checks again
whether the objects will now be detected.
The Preview for detection only checks the set parameters if called to do so
by a click on the button. Here you can change several parameters at once
and then check whether the objects have been detected.
We recommend that you always work with Automatic preview because it

ensures that objects are displayed as detected as soon as the parameters
have been correctly set.
You should only switch off this option in a situation when very many objects
mean that the new calculation of the detection following each alteration of a
configuration parameter takes too long.
If you leave the Automatic preview switched on at all times, then you only
need the Preview for detection to look at any image in an image stack,
before or after tracking to see how the objects were detected. To do so, go
to the required image using the Navigator. The display of the detection is not
altered here when Automatic preview is switched on, so as to save calcula-
tion time. To see how the object or objects in this particular image were
detected, you must click the Preview for detection button:
detection in image 1 after change to image after clicking on Pre-

12 view for detection
How can I look at the tracking overlay in an RGB image and

save it?
How to To do so, copy the overlay of the gray value image and paste it into the RGB
55
image. To do so, use the buttons in the Overlay button bar only.
1) Activate the Overlay button bar and load the gray value image with the
overlay that you want to copy.
2) In the Overlay button bar, click the Layer button and then on the Lock
Data Layer option.
> The check disappears from this option and the data layer is no
longer locked.
3) In the Overlay button bar, first click the Select All and then the Copy
s
Objects button.
> Now all the overlay elements are in a special internal clipboard.
4) Load the RGB image.
5) In the Overlay button bar, first click the Paste Objects button and then
the Select None button.
> The overlay is now displayed with the RGB image
6) Save the RGB image.
> In this way the overlay is saved together with the RGB image.
56
Index
Numerics Filter definitions

Combine 33
24-bit RGB images 4
Interactive 35
Invert 33
A Load 35
Appearing objects 15 Save 34
Automatic preview 7, 54 FIS 3
Automatic Tracking Focal point 14
Configure 7
Start 7 G
Graph 17
B
Background Correction 6 H
Hatching, green 7
C Histograms 3, 48
Cell Movements 3 Classes 49
Centered trajectories Save 50
48
Circles I
Flashing 17
Illumination, uneven 6
Red 22
Image Calibration 3
Configuration files
image sequence processing (isp) 3
Load 42
Image sequences, requirements 4
Path 42
Image Stack 3
Save 41
Installation 5
Configuration options, overview 43
Correcting object positions 39
Critical size 21 K
CSV file format 19 Key configuration 40
57
D L
Define search radius 11 Lost objects 16
Demonstration files 9
Parameter 12
Detected Objects 7, 9 M
Direction diagram 50 Manual Tracking 37
Dongle 5 Execution 38
Key configuration 40
E Sheets 39
Maximum object size 13
Edit results 47 Minimum object size 13
Extent 32 Move factor 11
multipage.tif 3
F
Fast Image Acquisition 3
Filter criteria 32
Index
N Requirements for trackIT! 4

Reset move factor 7, 14
Navigator 8
Reset point 14
Number of classes 49
S
O
Saving results 4, 19
Object definition, steps for 10
SFS file format 19
Object ID 37
SGD file format 19
Object information 16
Sheet/object link 17
Object overview 16
Sheets
Object separator 21, 54
Close all 17
Objects
Content 16
Appearing 15
Export 19
Correcting positions 39
Object information 16
Critical size 21
Object overview 16
Crossing 54
Positions 19
Define size 13
Sheets, export 4
Detect only certain objects 53
Smooth Trajectories 28
Detected smaller or larger 52
Spreadsheet program 19
Filtering 30
Focal point 14
Lost 16, 53 T
Not detected 53 Tab
Separator 21 Detector 43
Tracking several objects automatically 13 Tracker 45
Unsteady 23 trackIT button bar 5
Operating range 32 Trajectories
Original trajectories 48 Centered 48
Overview of working steps before tracking 9 Colors 12
Display options 25
P Edit 39
Finishing point 12
58
Positions 19
Interrupted 53
Preview for detection 7, 54
Merge 36
Original 48
Q Smooth 28
Questions Starting point 12
Direction of movement also detected 52 Update display automatically 31
Object not detected 53
Objects detected too small 52 U
One object recognized a several objects 52
Unsteady object 23
Two objects recognized as one object 52
Working with trackIT! 52
V
R Variation factor 23
Rectangle, dark green 23
Red square 14
Remove last added point 14

The Telepresence Add-In The Trackit! Add-In

Diunggah oleh

Informasi Dokumen

Judul Asli

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

The Telepresence Add-In The Trackit! Add-In

Diunggah oleh

Hak Cipta:

Format Tersedia

The trackIT!

© Olympus Soft Imaging Solutions GmbH

trackIT! Performance range ............................................. 3

Tracking objects ............................................................... 9

Execute manual tracking ...............................................................................37

Administration of tracking configurations ...................41

Overview of the configuration options .........................43

Suggestions and questions with regard to trackIT! ....52

What can I do when my object is not detected? ...............................................53

trackIT! Performance range

In this histogram the objects

each image represents a recognizable but not too large a movement

The trackIT button bar

Define Automatic Tracking (p. 7)

Execute Automatic Tracking

Execute Manual Tracking (p. 37)

Edit tracking results (p. 25)

Background correction with trackIT!

The "Define Automatic Tracking" dialog box

The Define Automatic

Preview for detection

The navigator can always be used, irrespective of which tab is currently

In contrast, the configura-

If n e ce ssa ry, click o n

Automatic tracking of a simple object

> The defined pixel is marked with a red cross.

Suitable parameters for the task

Parameter Value to be entered

Parameter Value to be entered

Automatic tracking of several objects

7) To start the tracking, click the Start tracking button.

What information is contained in the generated sheets ?

• the average speed,

The Object information_<filename> sheet shows the

Sheet name Content

The relationship between the Object Overview sheet and the

The Object Information will

Having the sheets displayed anew

General detailed information detailed information etc.

Contents of the Positions_<filename> sheet

How to export the results:

Automatic tracking using the object separator

3) Activate the Automatic preview function.

10) Now go to the Tracker tab to define the search radius.

Suitable parameters for the task

Automatic tracking of unsteady objects

8) To start the tracking, click the Start tracking button.

Edit tracking results

Original trajectory Smoothed trajectory

This setting does not change the results in the ObjOverview_<filename>

In the Selected objects

Clearing uninteresting objects

Selecting interesting objects

Deleting uninteresting objects

1) select check box 2) enter a value

Which filter criteria are there?

Inverting filter definitions

Combining filter definitions

Saving and re-using filter definitions

2) Assign a meaningful filter name and click the Save button.

Loading a filter 1) To do so, click the File... button.

Setting filter definitions interactively in the image.

Defining Distance, Length and Extent interactively