Quickstart

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This Quick start manual assumes that you have successfully installed TLM-Tracker on your computer. This page describes the analysis of a time-lapse movie, i.e., a sequence of sample images, in a straightforward manner. To get more detailed information please use the main manual page. A good practical introduction into time-lapse microscopy and sample preparation is available here.

Contents

Download sample files and start the program

Download a sequence of example images of a growing fluorescent Bacillus megaterium microcolony. Unzip the archive into one folder and start the program as described on the Download page.

Load images

  • Select File -> Load Images/Movie, navigate to the folder containing the image files and select all images.
  • Choose automatic when asked how to assign the images to the bright field and fluorescence channels.

Preprocessing of images

  • Cut images to the relevant sections:
  1. Use the control elements in the General panel to navigate to the last image of the movie (i.e., the image that is most covered with cells).
  2. Click the scissors Scissors.png and mark an area on Screen 1 containing all cells of this image.
  3. Click the Cut Section button.
  • Adjust the brightness of all images to one common level:
Click the Adj. Brightness button.
  • Remove images that are not suitable for analysis:
  1. Enter the numbers of those images that should be removed into the Tools panel (from ... to ...)
  2. Click Remove Image(s).
In the example, this refers to the images 22 and 30 which are blurred.
  • Edit parameters:
  1. Click Editparams.png in the toolbar.
  2. Adapt parameters as applicable.
For the example movie, the following values are suitable:
time interval = 300 s
pixel scaling = 0.1026 μm/pixel
exposition time = 50 ms
basic fluorescence = 1222
rate constant of photobleaching = 0.0756 s − 1
3. The option correction of fluorescence results in automatic correction of the photobleaching effect after tracking.
4. Close the dialogue with the OK button.
  • Click Continue to enter the segmentation section.

Segmentation of cells

  • Load a predefined segmentation process:
  1. Navigate to the first image of the sequence.
  2. Load a predefined segmentation process by selecting the corresponding radio button (Watershed, Threshold, Active Contours - Chan-Vese, Active Contours - GAC or Active Contours - Hybrid). Execute the selected process for all images with the Execute all button.
  • Check the results of the segmentation:
  1. Go through the image sequence one by one and examine the position of the calculated frames covering the cells in 'Screen 2 - Objects'.
  2. Check the Overlap checkbox in 'Screen 1' to perform a comparison between the previous image and the current image.
  • Manually edit the segmentation:

In case of improper recognition of single cells by the segmentation algorithm you can manually correct the segmentation process. Navigate to image 9 of the example image sequence. Compare image 9 with image 8 and image 10. In one case, you will see that 2 cells in image 8 are recognized as one cell in image 9. In image 10, these cells are recognized as 2 cells again. Correct this error by manually dividing these cells in image 9:

  1. Zoom into the image by clicking the Zoom in button Tool zoom in.png in the toolbar and drag a frame on the region to zoom on 'Screen 1 - Edited Image'.
  2. Use the paint tool from the Manual correction panel by clicking the Paint Pixels button or from the toolbar by clicking the Paint button Tool shape stroke.png. Now you can divide the cells manually by right-clicking on 'Screen 1' and painting a border between the cells (left mouse-button: white pixel, right mouse-button: black pixel).
  3. Re-execute the segmentation process for this image by clicking the Execute button in the Manual correction panel.
  4. Check your results: 'Screen 2' should display the results of the segmentation, whereas the cells of interest are recognized as 2 single cells.
  5. Check (and, if necessary, correct) all other images in the same manner.
  • Background correction of fluorescence:
  1. Navigate to the last image (i.e., the image that is most covered with cells).
  2. Select the Fl Bg Correction button from the Manual correction panel or the corresponding button from the toolbar (Tool pushpin.png) and mark several regions that show background in all frames.
  • Assign user defined data:

Optionally, you can assign a string or number to every single recognized cell. These annotations will be saved in the Export step together with other cell specific properties.

  1. Right-click on a cell in 'Screen 2' to open a panel.
  2. Enter some text or a number.

Click the Continue button to reach the tracking section.

Tracking

  • Execute the tracking: Press the Execute button to use the standard method for tracking.
  • Customize the tracking method: Specify alternative tracking methods and different parameters by pressing the Edit button.

As a result of the tracking a lineage tree is shown on 'Screen 2'.

  • Inspect the lineage tree for logical correctness: Examine the lineage tree. Use the Zoom tool from the toolbar to enlarge the tree. Move through the image sequence and regard the association of cells from image to image on 'Screen 1'.
  • blue rectangle - cell in the current image
  • green rectangle - cell in the previous image
  • yellow line - assignment of cells between the current and the previous image.
  • Correct erroneous assignments: If there are erroneous assignments or background objects which were recognized as cells, the tree can be edited using the tools in the Edit lineage tree panel as described here. Connections and cells can be removed, missing connections can be added.
  1. Modify connections and/or remove cells by selecting them on 'Screen 1' and 'Screen 2'. To draw a new connection, two cells/nodes can be selected by holding down the Strg/Ctrl-key or by using the left (first cell) and right (second cell) mouse button.
  2. Re-calculate the layout using the Layout button.
  • Correct logical errors: If there are interruptions or incorrect assignments of cells within one lineage, e.g., 2 cells from the current image are recognized as 1 cell in the subsequent image, manual editing is required. This edit necessitates to go one step back and correct the segmentation process.
  1. Click the Back button to return to the segmentation process. 'Screen 1' now displays the segmented cells in black an white.
  2. If necessary, use the Zoom button Tool zoom in.png to enlarge the relevant section of the image.
  3. Use the paint tool from the Manual correction panel by clicking the Paint Pixels button or from the toolbar by clicking the Paint button Tool shape stroke.png. Now you can divide the cells manually by by right-clicking on 'Screen 1' and painting a border between the cells (left mouse-button: white pixel, right mouse-button: black pixel).
  4. Re-execute the segmentation process for this image by clicking the Execute button in the Manual correction panel.
  5. Click Continue to proceed with the tracking process.
  6. Reexecute the tracking process.

When the lineage tree is completely correct continue to the export section using the Continue button.

Export

Export raw data: Click the Rawdata - per Image button to export all achieved data for every object in every image as a file in CSV- or XLS format. In this file every cell gets an index. Each cell of one lineage has the same index in subsequent images. The following annotations are saved for each cell:

  • index of the cell [-]
  • index of the parent cell [-]
  • time of the image [h]
  • x- and y-position of the cell [μm]
  • length and width of the cell [μm]
  • area of the cell [μm²]
  • mean fluorescence value [AU]
  • maximal fluorescence value [AU]
  • standard deviation of the fluorescence values [AU]
  • age of the cell [cell divisions]
  • userdata [-]

Export growth and product formation rates: TLM-Tracker also calculates growth and product formation rates for every single cell, which can be exported using the

  • Calculated rates - per Image - save data for every cell in every image
  • Calculated rates - per Cell - save data for every cell of one image.
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