Tracking of microglial processes.

Open your microglial process tracking video by dragging it into the Fiji toolbar. This loads the video file for subsequent analysis.

Check the video for drift, contrast, and movement of processes to ensure adequate quality for tracking. This preliminary assessment confirms the video is suitable for analysis.

Insert your calibration relative to your experimental setup and specify the time between frames. Click Global and press OK to apply these settings.

Launch the M-Track J plugin and zoom in on a selected microglial process to prepare for detailed tracking analysis.

Press Add and manually track the process by clicking on it in each frame. The software automatically advances to the next frame for continuous tracking.

Once tracking is complete, press Measure to generate two output files containing M-Track Points and M-Track J tracks of your measured relative tracks.

Import the generated M-Track files into Excel for further statistical analysis and quantification of microglial process movement.