2) Objects between frames are linked such as that the linking costs of all links between the two frames are minimized as a total. Using the LAP tracker the cost is calculated proportional to the squared distance between the linked spots. The basic principles for linking are the following: 1) Linking a detected object at t = i to a detected object at t = i+1 has a cost. TrackMate has three main trackers called “simple LAP”, “LAP” and “Linear Motion LAP” (LAP: linear assignment problem). Once detected the objects have to be linked from frame to frame to an entire track. The final detection result (bottom) is achieved after adding two filters, the spot intensity filter (C top) and quality filter (D top). d) More filters can be added to increase the precision of detection, such as the filtering by quality of the signal (top). The resulting image (bottom) contains fewer spurious detections. c) Detected spots in B are filtered by increasing the intensity threshold value to 10 within the LoG detector step (top). b) Setting an estimated object diameter, but no intensity threshold using the LoG detector (top) results in many detected spots, marked by magenta circles (bottom).
TrackMate GUI and spot detection.Ī) Screenshot of frame 15 of the example image stack FakeTrack.tif. In summary, by tuning a few, well explained parameters the user can detect and filter the objects, and visually inspect the result of the detection. The final detection result is visible in figure 1d, bottom. When adding further filtering (“Set filters on spots” fig 1d, top) we add an additional quality filtering step, this time with a higher quality threshold – the automatically calculated one is fine. We then go with the automatic “initial thresholding” for the quality. By setting a threshold 10 on the peak-detection we reduce the number of detected spots dramatically (compare fig. In our example we now tune our first parameters for the LoG detector (fig.1c): Estimated blob diameter - 5 pixels threshold: 10. mean intensity) and the effect of the filtering is directly visible in the image for easy visual control. The criteria for filtering out detected objects are intuitive to understand (e.g.
To avoid including background noise as spots, TrackMate filters detected spots in several steps: The LoG detector applies a Laplacian of Gaussian (LoG) filter before detecting local maxima for a sharper intensity profile of the spot signal. The default detector used in TrackMate is the LoG detector. DetectionĪs discussed above, the first step is to detect the spots. The final output will be in pixels and timeframes. In our example, however, there is no physical scaling. When running TrackMate on an active time-lapse sequence TrackMate automatically reads in the image scaling in order to give the final tracking results in calibrated physical units.
I suggest the reader to open up the image and try out the tracking while reading this article. In addition, there is a moving - but not dividing - object, three objects with a weaker intensity that are not moving, and some background noise.
MANUAL TRACKING IMAGEJ MOVIE
In this synthetic time-lapse movie one object undergoes one division and one merging event. FakeTrack.tif is available within Fiji (File > Open samples > Tracks for TrackMate). I will use the sample image file Fake-Tracks.tif as an example to introduce you to the tracking using the TrackMate GUI. TrackMate as Fiji-plugin has a graphical user interface (GUI) that guides the user through a few decisions and parameters handling these difficulties in particle detection and linking. For example: objects moving out of and into the field of view, particle fusing (e.g. Even in the hypothetical case of perfect detection, difficulties in the process of linking itself are present. Mistakes during the detection will have a considerable impact on the linking process. In the second step, detected objects are linked from frame to frame to estimate their trajectories. In the first step, objects are detected and their positions are identified. Single particle tracking in general consists of two steps. This article gives an overview of the functionalities of TrackMate. A very powerful but still easy to use tool for single particle tracking is TrackMate – which comes as a plugin within Fiji.
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