Object tracking

In the commercial image analysis software "Volocity", automated measurement protocols can be constructed by dragging, dropping and configuring a sequence of individual "tasks".

By combining the "Find Objects" task with a subsequent "Track" task, 3D objects can be identified and followed over time. The initial "Find Objects" segmentation can be refined, e.g. using "Separate Touching Objects"; and tracking results in the form of "Measurement Items" can be viewed in tabular form, as a graph, etc.

Generation of Kymographs using 2D+t images. In the generated kymographs, objects can be tracked and the results are visualized.

Microtubule end tracking in live cell fluorescent images of Drosophila oocyte involves overcoming the following challenges, which can be tackled by a series of preprocessing steps and tracking described in Parton et al (2011)

• illumination flicker & photobleaching: suppress by normalising intensities, e.g. using Image->Adjust->Bleach Correction in Fiji/ImageJ
• uneven illumination: Fourier bandpass filtering (e.g. Process->FFT->Bandpass Filter) preserves features within a selected size range
• high background / poor contrast: foreground filter, e.g. Temporal Median filter
• tracking: e.g. TrackMate in Fiji/ImageJ (segmentation using DoG detector)

This macro and plugins suite for ImageJ (and Fiji) serves to measure the velocity of moving structures and visualize them, from image time series (2D over time).

The module can be installed in ImageJ as a Macro Menu and each function/component can be called separately. The full workflow consists in calling some, or all, the functions sequentially in order to get from the image preparation (e.g. filtering and visualization of tracks) to the production of the kymographs (time vs. distance plot) and their analysis (retrieving the velocities).

Here is the full workflow sequence:

• Load image sequence
• Crop and time-filter the image sequence ("Walking average" plugin)
• Generate tracks by z-projection ("Stack difference" plugin)
• Select tracks and restore them in the original stack.
• execute plugin "multiple kymograph"
• Analyse: select edges of moving tracks graphically and quantify movement in a table.

input: 8-bit, 16-bit stacks, 2D in time. Calibrated is better for meaningful velocity measurements.

ouput: the kymograph image, the velocity measurements tables.

Requires ImageJ version: 1.33.n minimum.

Example of applications:

• velocity of moving objects/ structures with sharp edges, incl. the velocity of microtubules (and their plus ends),
• the velocity of vesicles or particles along a 2D path
• the velocity of migration of the edge of a cell or a multicellular group
• retraction velocity of contractile bundles (e.g. actin fibers) or multicellular tissues after mechanical disruption (e.g. laser surgery)

The workflow contains a Matlab package (plusTipTracker) for segmentation and tracking of microtubule tips, based on fluorescence time-lapse movies from microtubule tip markers such as EB-GFP. The tracking model accounts for the specific movement characteristics of microtubules Moreover, scripts for secondary analysis of detected microtubule paths are provided.

plusTipTracker is part of u-track 2.0 package. The workflow is described in the reference.