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This page describes a detector for TrackMate that creates objects from a grayscale image (it can be one channel in a multi-channel image). You have to specify a threshold value to segment the objects.

Step-by-step tutorial

Download the tutorial dataset from Zenodo:


  • Open Fiji.
  • Open your image in Fiji.
  • Open TrackMate PluginsTrackingTrackMate. The start panel will open and display information about your image dimensions. Click Next.
  • The Select a detector panel opens. From the pull-down menu, select Thresholding detector. Click Next.


  • A panel with the description of the detector opens. You can set the threshold yourself or click on the Auto button for automatic thresholding. With our test image, the automatic thresholding value is 92. You can also select to Simplify the contours to smoothen the edges of the segmented objects. By clicking on the Preview button, you will see a preview of the segmentation. On the left, the number of detected objects is displayed. Once a suitable threshold has been chosen, click on Next.
  • The objects will be detected. Click Next again.


  • A panel to filter the detected spots according to their quality opens (more information about this filtering can be found here). With our test image, this part can be ignored. Click Next.
  • Next, a panel to filter spots according to their properties (i.e. size, shape, location, or signal intensity) opens. In this exercise, do the following:
    • Click the green + sign at the bottom of the panel - a filter appears.
    • Click the pull-down menu and select Area. Here we will filter out the smallest detected objects. Make sure the Above button is selected.
    • Drag the horizontal line (pink dashed line) to 20. The small objects become invisible.
  • Click Next.


  • A tracking panel opens. In this panel, you can select a method for tracking your objects. In this exercise, we use the LAP tracker. Please select it from the pull-down menu, and click Next.
  • A panel for the LAP tracker settings opens. First, with the Frame to Frame linking parameter, you give the maximum distance to link two objects between frames. Here use 20 microns. Then, you can choose how many spots can be missing, and they could still be the same track. Tick the Allow gap closing box and add values: Max distance: 20 microns and Max frame gap: 4. Next, you let TrackMate know if the tracks are allowed to split. Splitting can be caused, for example, due to cell division. Tick the box Allow track segment splitting and insert value Max distance: 20 microns. Below you will also see settings for Track segment merging. Here this box should remain unticked. Click Next.
  • A Track filter panel opens. In this panel, you can filter tracks according to their properties (i.e., length, speed, or location). With our test image, we do not need to filter any tracks. Click Next.


  • A Track filter panel opens. In this panel, you can filter tracks according to their properties (i.e., length, speed, or location). With our test image, we do not need to filter any tracks. Click Next.

  • Next, a panel with multiple display options appears. Here you can define how the objects and tracks are labelled. In this exercise, we will label the objects and tracks according to their mean speed.

  • First, make sure that the Display spots, as ROIs and Display tracks boxes, are ticked.
  • Select Track mean speed from the Color spots by and Color tracks by pull-down menus and click auto.
  • In this panel, you can also export the results as CSV files. Please do so by clicking Tracks at the bottom of the panel. A window with a lot of data shows up. Ensure you export the Spots (information about the spot) and the Tracks (information about the tracks) files. Close the results window and click Next.
  • You will see a plot features panel - again, click Next.


  • In the next panel, you can perform multiple operations. For this exercise, we will export a tracking image of our experiment. Then from the pull-down menu, please select Capture overlay. As you click on Execute below, a pop-up window opens. Here you can define the time interval you want to save. You can hide the image if you wish by ticking the box Hide image and click OK. TrackMate will generate a video of the experiment. Remember to save the image with FileSave as….

Joanna W. Pylvänäinen - July 2021