TACTICS v3.x by R.S. 2010-2014
Appendix I
TACTICS Toolbox v3.x
User Guide
Interactive MATLAB Platform For Bioimaging informatics
TRACKING
MODULE
TACTICS v3.x by R.S. 2010-2014
Appendix I -17-
Cell Tracking Module – 1 (user interface)
Once the images were successfully segmented, the next step in the processing scheme, is the
cell tracking and manual correction interface:
From the main menu, go to >Cell Tracking Module
The Cell Tracking Module is used to correct segmentation, label cells and tracking. The GUI has
several selection methods for manual image segmentation that allow the user to inspect and
correct cell segmentation manually, and allows for multiple objects tracking correction.
Navigation panel
Secondary Screen
Primary Screen
Segmentation panel
Mark cell division
Mark cell death
Trajectory panel
Editing tracks panel
Screen capture of the Cell Tracking Module interface
Video tutorial
3
Open new MATLAB figure
1. Run the Linker
2. Split cells after divisions
3. Save manual correction of cell associations
4. Show lineage tree
5. Mark cell division
6. Mark cell death
Load experimental data file
Save experimental data file
Drag zoom rectangle
Capture ROI to secondary screen
Zoom in
Zoom 100%
Zoom out
Pan
Error Zoom (temporary solution for Zoom issues)
Functions supported in the icon bar-
TACTICS v3.x by R.S. 2010-2014
Appendix I -18-
Cell Tracking Module – 1 (user interface)
Change the channel and the display mode (raw,
filtered etc.) of the
Primary screen using this pull down menu
Channel and display mode of the
secondary screen
Read and upload images within
selected range to
RAM (dependent on the computer
memory)
Screen capture of the Cell Tracking Module interface
Keyboard shortcuts: Uparrow - Next frame Downarrow - Previous frame Space - label frame again Enter - associate frame overpassing selective operator F - Format painting S - Segmentation mode M - Mark division mode E - Next frame T - Trajectory mode A - Association mode
D - Dead cell mode E - Next frame R - Remove segment mode U - uncheck/check P - Paint Tool mode
TACTICS v3.x by R.S. 2010-2014
Appendix I -19-
Cell Tracking Module – 2 (cell labeling and tracking)
Instructions for cell labeling and tracking:
1.Load an experiment data file to TACTICS .
2.To label the cells- Go to >’Label cells’>> ‘Label cells’ . A labelling function will label each
segment from n to m (n and m are user input). Reference channel can be used to help to interpret
normalized pixel intensities in the quantified channel and allow to track two types of cells at the
same time. After running the Label Cells function, the cells will be detected, identified and labeled
including feature extraction. A bounding box will mark each segment, and all labeled frames will
be marked in red tab under the sliderbar:
3. To connect between the cells over time- Go to >’Label cells’>> ‘Label association‘. An input
box will ask for the range of frames to be associated and maximum distance that the cells can
travel (a new cell will be defined if the distance to a closer point in time (t-1) is above this criteria.
The input units are currently in pixels. Once finished, all associated frames will be marked in
green tab under the sliderbar (note that the first frame cannot be associated):
4.To track the cells, click on the ‘tracking’ icon or Go to >Track cells>> Hungarian
algorithm>>>Tracking linker. The associated points will be connected to create the cell`s
tracks. The user must input a cut off value that sets the possible minimum track length (a value of
1 is frequently acceptable).
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As an alternative to label association followed by Hungarian algorithm, the user can utilize the
Crocker algorithm for tracking multiple objects Go to >Track cells>> Crocker algorithm. The
advantage is the ability to recover for missing points. However, the algorithm is slower and fails
when the number of cells or time points is large (dozens of cells for 1000 frames will fail the
algorithm).
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•Once cells are tracked, trajectories of cell over time can be visualized. The location of the cells in
different time points is marked with a colored point, and the size of the point is proportional to the
timeframe of the track.
•To mark dividing cells, go to cell division mode and click on the cell at the point of division
(one time point before the appearance of two separated cells) with the middle mouse button. X
sign will be added/removed correspondingly.
•To mark dying cell, go to cell death mode and click on the cell at the last point of
appearance with the middle mouse button. + sign will be added/removed correspondingly.
IMPORTANT. Save the experiment file (overwrite or create new file) before exiting TACTICS (or
before loading new experiment file). Otherwise, any operation will be lost.
TACTICS v3.x by R.S. 2010-2014
Appendix I -20-
Cell Tracking Module – 3 (cell labeling corrections)
A. Improving segmentation of a single frame using the secondary screen view
1. Click on the ‘Capture ROI to secondary screen’ icon . Once you have clicked on the icon
a window will come up with an image of the screen you have been working on. Hold down
mouse (left click) anywhere on the new screen. This will create a second figure from the
right side of the screen containing a magnified image of the ROI within the images that is
chosen for zoom.
2. While still holding down left click use the arrow keys to form a box around the cells that you
would like to work with in the secondary screen (Make sure the aspect ratio is 1:1 or you will
not be able to see the cells clearly or correct them with good accuracy). Once you are happy
with the size and placement of your box, double left click to confirm. The selected region will
be magnified to the secondary screen view and the borders of this region will be appeared in
the primary screen view.
3. When using the mouse-wheel up and down, the ROI will be shown in the primary screen
view. User can use the Drag zoom rectangle icon to move the ROI keeping the current
dimensions.
4. Go to Segmentation mode, and select ‘Segment current frame and save to HD’ option in the
popup menu.
5. To load the segmentation settings go to the File menu > import > (import segmentation settings
OR segmentation settings for current frame). The user can then adjust those settings to
improve segmentation of the current frame by altering values on the bottom right frame..
6. The user can remove individual pixels by clicking with middle mouse button (or mouse-wheel)
while in the ‘segmentation mode’.
Secondary screen view Primary screen view
Box containing the ROI
Secondary Screen showing the
zoomed-in ROI
Screen capture of the Cell Tracking Module interface
TACTICS v3.x by R.S. 2010-2014
Appendix I -21-
Cell Tracking Module – 3 (cell labeling corrections)
Separating merged cells.
TACTICS provides multiple options for manually separating merged cells, including:
1. Splitting a single object using watershed or intensity shell algorithms.
2. Splitting a single object by drawing a line through it or deleting individual pixels.
3. Improving segmentation of a single frame (as per previous page).
1. Splitting objects using watershed or intensity shell algorithms.
If a labeled object is clearly two or more adjoined cells, move the cursor over the box surrounding
the object, and left mouse click (watershed) or right mouse click (intensity shell) in the
primary screen view. If the algorithm succeeds, the bounding box should split into two boxes.
If the algorithm fails, the alternative algorithm can be attempted. After separating all the
adjoined cells in a frame scroll forward or back through the movie to save the changes.
2. Splitting objects by drawing a line or deleting pixels.
a. To separate between cells by drawing a line of deleting pixels use the Secondary Screen
(See the previous page for instructions on how to access the secondary screen using the
capture ROI icon ).
b. Draw a line where cells need to be divided. To do this, left click at the first point of where your
line should be and a yellow dot will appear. Left click again at the same point and drag the
blue line to divide your cells. An intensity line will go between the cells to give two labelled
cells.
c. Double left click on the blue line to accept changes.
d. Click middle button (wheel) or CTRL+A to save changes.
e. The properties of each segment that are saved in the .dat experiment file (described in
Appendix 1), and bounding box will appear for each segment (in the Primary Screen). In the
secondary screen, outlining of the cells by different colors indicates successful segmentation.
f. Run tracking algorithm again . This is required to incorporate the newly segmented
cells, and can be performed after resegmenting one or several frames.
I M P O R T A N T: Once this data file is created and loaded into Cell Tracking Module, any
modification in the data file will be logged in the loaded data file. Therefore it is necessary to
save the data file once processing is applied.
Initial problem cell
First point identified
Blue line drawn
Final product (two defined cells)
TACTICS v3.x by R.S. 2010-2014
Appendix I -22-
Cell Tracking Module – 3 (cell labeling corrections)
Changed threshold value
3. Splitting objects by altering segmentation parameters.
Resegmenting the entire frame can also help with splitting two cells, although care must be taken
that further segmentation problems do not occur in other areas of the frame. Below is an example
where two joined cells were separated by increasing the threshold value as per instructions in A
(p 18).
Optional. Changing the LUT is a standard procedure that helps to distinguish between pixel
intensities. An example shown below shows that pixel intensities in the cell perimeter have low
value, causing over-segmentation by Otsu’s method.
TACTICS v3.x by R.S. 2010-2014
Appendix I -23-
Cell Tracking Module – 3 (cell labeling corrections)
Removal of segmented pixels that are falsely identified as cells:
Sometimes a new object appears during segmentation correction that is incorrectly labeled as a
cell. An example of this is shown below:
Instructions for removal:
1. Zoom-in on the problem area in the secondary screen until each pixel is clearly visible.
2. Right click on one of the pixels to remove the colored dots indicating the segment edge.
3. You can now remove each unwanted pixel by right clicking on it. To replace a pixel that you
have deleted right click in the same place. Similarly, right clicking on a black pixel converts it
from 0 to1, and so can be used to link separated cells.
Cell Tracking Module incorrectly recognizes this
as a cell
Shot of secondary screen with pixels
clearly visible After one right click
After pixels had been removed.
TACTICS v3.x by R.S. 2010-2014
Appendix I
The tracking accuracy depends on the quality of the raw data, the imaging system, the biological
system, and the computational approach to analyze the data. Common problems with tracking
are when a cell suddenly moves a large distance. Tracks can be changed manually using several
approaches, that either involve moving track vectors on the primary screen, or use a pull down
menu in which cells can be connected by numbers.
To correct cell association tracks by shifting connecting vectors:
1.Choose (n-1) in the secondary axes.
2.Click association button.
3.Drag the lines between the middle f the cell bounding-box (n-1) to its origin in n.
4.Click on the green icon in the icon panel to accept changes. Cell without match between n and
(n-1) marked by blue centroid. Cells with match are marked in red and id number. Watch the
video tutorial for more information.
Instructions for manual corrections for tracking:
1.Go to edit tracks mode.
2.Point with the mouse curser on required cell and click on the middle button. A selection window
will give several different options.
•IMPORTANT: the manual corrections are applied on the matrix of trajectories. Therefore, when
applying the linker again, the manual corrections are deleted. For this reason, it is always better
to apply manual corrections for cell association.
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Cell Tracking Module – 4 (cell tracking corrections)
Video tutorial
4
TACTICS v3.x by R.S. 2010-2014
Appendix I -25-
Cell Tracking Module –5 (marking cell division and death)
•Marking cell division
•Go to the (Mark cell division) mode
•Go to the last frame before division, where only one cells in visible
•In the primary screen, wheel click on the dividing cell
•Click on the (tracking splitter) icon
•Parental cells will have name ##-**, whereas ## is the name chosen by the user and ** is a
number by order of appearance.
•Daughter cells of parental cells will have name ##-**.1 and ##-**.2, whereas ##-** is the name of
the parental, and 1 and 2 symbolise daughter 1 and 2.
•For each additional cell division a dot and number 1 or 2 will be added. For instance:
##-**.1.2.1.2.2
1- Cell division mode
4-click tracking splitter
Screen capture of the Cell Tracking Module interface
3- wheel click on cell,
Adding red x annotate the
cell as dividing cell
2- cell about to divide
5- (next frame) two cells
identified as two related
daughter cells and the
information is saved in
the experimental file
Frame n
Frame n
Frame n+1
Frame n+1
Frame n : Final frame before division
TACTICS v3.x by R.S. 2010-2014
Appendix I
The painting tool allows manually to draw, fill, and remove pixels on any selected channels. This
can be very useful to correct segmentation when automated segmentation fails.
To apply the drawing tool:
1.Go to drawing tool mode.
2. Select show segmentation to “on”.
3.Select channel to apply segmentation, channel for visualization, and channel for tracking.
Zooming-in is recommended, but not a recruitment. The blue net will appear for each existing
pixel in the binary matrix. 4. Select the size of painting point. 5. Press mouse left click. Hold it and move the mouse curser to fill new pixels. 6. To remove pixels- Press mouse right click. Hold it and move the mouse curser. 7. To accept: click mouse wheel button. This will save the modified image as segmented one (overwrite) and will label again (relabeling should be saved to experimental file otherwise this will be lost). 8. To cancel (before acceptance): just use mouse wheel to refresh frame (go to the next or previous frames)
9. To cancel (after acceptance): currently TACTICS doesn't have this feature (is that useful?). Therefore need to segment the frame again.
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Cell Tracking Module – 6 (drawing tool)
1- drawing tool mode
2- click show segment on
3- select channels
4- select point size
TACTICS v3.x by R.S. 2010-2014
Appendix I
The parameter selection window ddisplay parameters such as intensity, and velocity for a particular selected cell, which allow to improve the cell tracking. To activate the parameter selection window :
1. Select cell. 2. Choose- ‘plot data for selected cell’ option. 3. New Manu will popup. Choose parameter to be plotted. (more parameters can be easily
added). 4. When using the mouse scroll wheel, the plot will be shown in the secondary figure view for the relevant time points.
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Cell Tracking Module – 7 (Parameter selection)
1- select cell
2- select mode
3- select parameters in popup menu
4- parameter is plotted
Display number of objects in the current frame
TACTICS v3.x by R.S. 2010-2014
Appendix I
Complicated switch in tracking can accrues when cells drift and tracking is incorrect for the following tracks. This can strongly influence the lineage tree and its analysis.
To correct tracks for lineage trees:
1. From main Go to >’Tools’>> ‘Generate Lineage tree’. A popup window shows the lineage for any selected cell. In this particular example it can be seen that a problem accrues in the tracking. Click on the lineage window interactively goes to the frame of selection in the Cell
Tracking Module. 2. In some cases other supporting tools can help to allocate events suspected as wrong. For
instance the cell tracks window. From main Go to >’Tools’>> ‘open cell tracks window’. Other tools can be useful Go to >’ Data inspection’ , such as show number of objects over
time, show trajectories matrix and search for undefined association. 3. Adjust the current settings showing for frame n and (n-1). 4. To visualize the association between cells at particular frame click on Centy button. In this
particular example, because (n-x) is set to -1, the cells in DIC in frame 382 but the tracks and bounding boxes are actually shown for frame 381. This provides the locations of the
cells in the previous frame. The Centy button shows that a new cell was defined (blue), and that the second daughter cell was wrongly labelled as cell 1.1.2.2.
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Cell Tracking Module – 8 (corrections of lineage trees)
1- Generate Lineage tree 2- (optional) open cell tracks window
or use other inspection tools
4- Click on centy
3.a.- input frame to show to be -1
3.b.- input channels to
visualize frame n-1
3.c.- input channels to
track and visualize frame n
TACTICS v3.x by R.S. 2010-2014
Appendix I
cell association tracks by shifting connecting vectors
5. The user can move the pink lines, so the pink star * touch the destination location in
frame 383. In this case a line was stretched from cell number 7 to 1.1.2.2, while the line that start. In this example, from the cell between 1.1.2.2 and 1.1.2 was pointed back on itself. This mean that this cell doesn't Have a much and will therefore recognized as a new cell. To accept click the cell association correction button.
6. After the correction, run the linker again. The lineage tree is now corrected. If the user regrets or want to repeat the process, labelling the frame again goes to initial association.
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Cell Tracking Module – 8 (corrections of lineage trees)
5- drag lines to link between cells
6- lineage tree is corrected
Two-dimensional graphic visualization of lineage tree. Bifurcations in the tree represent cell divisions. X-axis
represents the cell index. Y-axis represents the frame (or time) that each cell appear.
TACTICS v3.x by R.S. 2010-2014
Appendix I
Selective operator is method that allows the user to select what frames will be associate or
skipped. The user can select by frequency to be skipped (for instance every 3 rd frame) or/and
certain frames in the movie. The associated frames appear in green bars under the movie
sliderbar. Only the frames between following green bar coded frames are associated.
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Cell Tracking Module – 9 (selective operator)
Selective operator
Number of cells in frame in
selective mode
Green bars shows the associated
frames
Trajectories matrix shows association
between every 10 frames
Selective operator
Modes
Show frames Show tracks
Show tracks
annotations
Non-Selective All frames Only in selected frames Only in selected frames
Selective All frames Only in selected frames Only in selected frames
Only Selective Skip to selected frames Skip to selected frames Skip to selected frames
Partly Selective All frames All frames Only in selected frames
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