tracyspot

Tracy (Beta)

Note: Tracy is currently in beta — features and workflows may change regularly.

Table of Contents

• Installation

• Getting Started

  • Launching Tracy
  • Loading Your Movie
  • Browsing Spots
  • Generating Kymographs
  • Generating Trajectories
  • Browsing Trajectories

• Additional Features

  • Drift Correction
  • Colocalization
  • Step Finding
  • Reference Image
  • Custom Columns
  • Color by Value

• Saving & Loading

  • Save Trajectories
  • Load Trajectories
  • Import TrackMate Data

• License

Installation

Install Tracy with your terminal:

# Create and activate a dedicated environment
conda create -n tracy python=3.10 -y
conda activate tracy

# Install Tracy
pip install tracyspot

Tip: On Windows, you can download Anaconda and use the Anaconda Prompt as your terminal.

Getting Started

1. Launching Tracy

conda activate tracy       # if not already active
pip install tracyspot --upgrade   # update during beta
tracy &                    # run in the background

Note: The first launch may take a few seconds.

2. Loading Your Movie

• Click Load Movie (or use Load » Movie).
• Select a single- or multi-channel TIFF movie.
• If necessary, enter pixel size and frame interval when prompted.
• Pan with the middle‑button drag (or Ctrl/Cmd + drag), zoom with the mouse wheel.
• Switch channels by clicking the channel label (shortcut: 1, 2, …).
• Toggle the maximum projection with the button below the movie (shortcut: m).
• Adjust the contrast using the slider.

3. Browsing Spots

• Click on the movie to detect a spot at the click location.

  • Blue square: search region.
  • Magenta circle: fitted spot (radius = 2σ).

• Hover over the inset to view a 3D fit (scroll to zoom, drag to rotate).

• Hold r + scroll (or use Spot » Search Radius) to adjust the search radius.

• Navigate frames with the slider under the movie.

The spot histogram shows intensities in the search area and highlights values in the spot.

4. Generating Kymographs

• Switch to Line mode (slider under movie or n).
• (Optional) Toggle max‑projection (m) to guide line placement or load a reference image.
• Draw a segmented line by clicking anchors (press Esc to cancel).
• Double‑click to finish and generate the kymograph.
• For multi‑channel movies, a kymograph is generated for each channel (toggle with 1, 2, …).
• Cycle through kymographs with , (previous) and . (next).

Tip: Apply a LoG filter via Kymograph » Apply LoG filter for clearer tracks (applies to subsequent kymographs).

5. Generating Trajectories

A. From a Kymograph

• On the kymograph, draw a segmented line (blue anchors) to follow the track.
• Double‑click to finish and compute a trajectory using your search radius and tracking mode (see tracking options).
• Click any kymograph or plot point to jump to that spot; use →/← to step forward and backward.
• Press spacebar to animate the trajectory.
• If you want to recalculate the trajectory with new tracking options, press Enter (or Trajectory » Recalculate).
• (Optional) Fill gaps via Kymograph » Connect Spot Gaps.

Trajectories are displayed on kymographs dynamically, so overlapping kymographs may share trajectories.

B. Direct Movie Tracking

• Click an initial spot on the movie.
• Skip a few frames and click the next spot. Repeat until the track is covered.
• End a click sequence with Enter or cancel with Esc.
• Browse/select tracks as above.
• If you want to recalculate the trajectory with new tracking options, press Enter (or Trajectory » Recalculate).

You can use “video‑game” controls: w/a/s/d to move the cursor, l/j to change frames, k to select the spot.

Tracking Options

• Search Radius: adjust with r + scroll

• Tracking Mode (t):

  • Independent (default): fits each frame independently.
  • Tracked: uses previous frame’s spot as center.
  • Smooth: independent + post‑filter outliers.

• Tracking options are set for any subsequent analysis. An existing trajectory can be recalculated using the currently set options by pressing Enter (or Trajectory » Recalculate).

• If a spot looks wrong, you can invalidate it with x when it's highlighted.

• Avoid using spots in existing tracks via Spot » Avoid previous spots.

Plots

• Intensity Plot: integrated spot intensity at each frame.
• Speed Histogram: frame‑to‑frame speeds with the net speed overlayed (i.e. only considering start and end position).

Browsing Trajectories

• New trajectories append to the Trajectory Table.
• Click trajectories in the table or use the arrow keys (↑, ↓) or click; right‑click for options (e.g., Go to kymograph).
• Delete a trajectory with Backspace.

Additional Features

Drift Correction

• Identify a stationary reference spot.
• Ensure it’s detected by clicking it (magenta circle).
• Use Movie » Correct Drift to track it from start‑to‑end and apply the frame shifts.
• Review and save if satisfactory.

Colocalization

• Determines colocalization if a spot exists within 4 pixels in the other channel.
• Toggle under Spot » Colocalization for multi‑channel movies.
• Existing trajectories prompt analysis
• Results appear as new table columns.

Step Finding

• Calculates steps in the intensity profile.
• Enable Trajectory » Calculate Steps.
• Adjust rolling‑average window and minimum step size.
• Existing trajectories prompt analysis.
• Results appear as steps in the Intensity Plot.
• Detected steps and sizes are saved in Per‑Trajectory sheet; each point’s step ID in Data Points.

Reference Image

• Useful for overlaying filaments or guides during kymograph creation.
• Load via Load » Reference Image
• Toggle with the icon under the movie.

Custom Columns

• Right‑click any column header or use Trajectories » Add Column.

  • Binary: Yes/No flags.
  • Value: any numeric/text value.

• Assign via right‑click on table row or kymograph label.

Color by Value

• If custom column or colocalization data exists, under Trajectories » Color By choose binary, value, or colocalization.

Saving & Loading

Save Trajectories

• Save » Trajectories exports an Excel file with three sheets:

  • Data Points: all spot measurements.
  • Per‑Trajectory: summary statistics per trajectory.
  • Per‑Kymograph: stats grouped by kymograph.

⚠️ If a trajectory can be found within two kymographs, the per‑kymograph stats will be wrong.

Load Trajectories

• Load .xlsx files with the above sheets or similar formats (requires columns: Trajectory, Channel, Frame, Search Center X, Search Center Y).
• Use Kymograph » Draw from Trajectories to redraw embedded lines.

Import TrackMate Data

• Load .csv from TrackMate via Load Trajectories; uses TRACK_ID, FRAME, POSITION_X, POSITION_Y to perform a search.

License

This project is released under the MIT License — see LICENSE.txt.