tracyspot 1.0.27

Spot navigator

Tracy

Note: Tracy is still in beta

1. Install
2. Getting started
   • Start Tracy
   • Load your movie
   • Browse spots
   • Drift correction
   • Kymographs
   • Track using kymograph reference
   • Modify individual points
   • Browse trajectories
   • Save trajectories
   • Load trajectories
   • Load TrackMate data
3. License

Install

1. Open your terminal
   • Mac: just open Terminal
   • Windows: install Anaconda and use the Anaconda prompt as the terminal
2. conda create -n tracy python=3.10 -y
3. conda activate tracy
4. pip install tracyspot

Getting started

Start Tracy

1. Open your terminal
2. conda activate tracy if not already active
3. pip install tracyspot --upgrade always update while tracy is in beta
4. tracy & it might be slow to open the first time

Load your movie

1. Click Load Movie (also in the Load menu)
2. Choose any single- or multi-channel movie (.tif format)
3. If the pixel size and frame interval could not be loaded from the metadata, you will be prompted to enter them
4. Click and drag using the middle button to pan (or Ctrl + left click / Cmd + left click) and middle wheel scroll to zoom
5. Once loaded, toggle between channels with the number keys
6. Use the M shortcut to show/hide the maximum projection
7. Adjust the contrast using the contrast slider

Browse spots

1. Click on the movie to find a spot using the click position as the search center
   • The blue square marks the search area around the center
   • The magenta circle marks the spot that was found (the circle radius is 2 standard deviations)
2. Hover over the top-right inset to see a 3D representation of the fit (scroll to zoom, click and drag to rotate)
3. You can modify the search radius by holding down the R key and scrolling (or in the Spot menu)
4. Use the slider under the movie to browse frames

Drift correction

1. If your movie drifts, find a spot that is present throughout the movie and is stationary
2. Click the spot (any frame) and make sure it has been found (magenta circle)
3. In the Movie menu, click Correct Drift
4. Check the result in the pop-up and save/load the movie if acceptable

Kymographs

1. To generate a kymograph, enter Line mode using the switch under the movie (shortcut: N)
2. Optionally, look at the maximum-projection (shortcut M) to better see where you should draw lines
3. Draw the segmented line by placing green anchors on the movie (Escape cancels a sequence)
4. Double-click to complete the sequence and store a kymograph
5. If your movie has multiple channels, a kymograph will be generated for each, which will show when you toggle between channels (number keys)

Track using kymograph reference

1. To track a spot in the movie using the kymograph as a reference, draw a segmented line by placing blue anchors on the kymograph (Escape cancels a sequence)

2. Double-click to complete the sequence to generate a trajectory, where spot centers are searched in the movie from the linear interpolation between clicks

3. Press the space bar to play a movie of the trajectory

4. Assess the plots

   • The spot histogram shows the pixel intensities in the search range around the spot center and highlights the intensities within the spot
   • The intensity plot shows each spot's integrated intensity
   • The speed histogram shows the frame-to-frame speeds of the spots and overlay sthe net speed (only considering the start and end point)

5. If necessary, modify the search radius (hold down R and scroll) and re-attempt (Enter key)

6. If necessary, toggle tracking modes with the T key and re-attempt (Enter key)

   • Independent: each frame is treated independently using the interpolated search centers
   • Tracked: each frame's search center is based on the previous frame's spot center
   • Smooth: equivalent to Independent mode but goes through a filtering at the end to remove spots that are far off the main track

7. Click any point in the kymograph or in the intensity plot to jump to that point

   !! Trajectories do not belong to kymographs, and their presence within one is determined on-the-fly for visualisation

Modify individual points

• When a point is highlighted, use the X key to either invalidate the spot or re-attempt a fit if it is already invalid

Browse trajectories

• Adding trajectories will append the data to the trajectory table
• Right click a trajectory in the table to show some helpful options, like Go to kymograph ch1-001
• The backspace key removes the selected trajectory(ies)

Save trajectories

• Save trajectories in the Save menu, which saves an excel file with three sheets

  • Data points: All spots and their corresponding data
  • Per-trajectory: Data corresponding to individual trajectories
  • Per-kymograph: Analysis of points belonging to the same kymograph

  !! Make sure two kymographs do not contain the same trajectory or the Per-kymograph statistics will be wrong

Load trajectories

• Trajectories can be loaded back as they were saved by Tracy (.xlsx file) or any similar file with a Data Points sheet with at least Trajectory, Channel, Frame, Search Center X, and Search Center Y (it will recalculate trajecotires when spot centers are missing in this case)
• You do not need to save kymographs since the clicks you used to build them are embedded in the trajectories. Use Draw from trajectories in the Kymograph menu to redraw them after loading trajectories.

Load TrackMate data

• TrackMate data (.csv) can be loaded, which will trigger a calculation using the trackate centers as search centers to generate a trajectory for each TrackMate track

License

This project is licensed under the MIT License - see the LICENSE.txt file for details.