(f) Detect spots using the Laplacian of Gaussian (LoG) detector in Trackmate. Spot size (0.36–0.4 μm) and thresholds
should be optimized empirically.
(g) Detected spots are joined into trajectories using simple
linear assignment problem (LAP) tracker in Trackmate.
The parameters for tracking such as linking max distance,
gap-closing max distance, and gap-closing max frame gap
should be optimized empirically. Typical values for these
parameters are 0.4 μm, 0.7 μm, and 1 frame, respectively.
Any tracks that are less than three frames should be
discarded.
(h) Visually inspect the tracks to ensure appropriate tracking.
(i) Export tracking data as a spreadsheet.
2. For RNA particles undergoing directed movement:
(a) Particle tracking is generally performed for typically
40 frames in the region of interest (cytoplasm) (similar
to steps 1a and 1b of previous section; the only exception
is to change the option in the Slice Keeper to 40 frames
instead of 5 frames).
(b) FFT band-pass filter in Fiji can be used to filter out noise
that is below 3 pixels and above 40 pixels (similar to step
1c of previous section).
(c) Go to Image > Properties and set the values for number of
channels, number of frames, and pixel size (in μm). Click
the box for the global option at the bottom of the pop-up
window for applying these calibrations to all images.
(d) Use Plugins > Tracking > TrackMate to launch Trackmate and confirm that the number of frames, pixel size,
dimensions of the region of interest, and time interval are
correctly read by Trackmate.
(e) Detect spots using the Laplacian of Gaussian (LoG) detector in Trackmate. Spot size (0.36–0.4 μm) and thresholds
should be optimized empirically.
(f) Detected spots are joined into trajectories using the linear
motion LAP tracker in Trackmate. The parameters for
tracking such as initial search radius, search radius, and
max frame gap should be optimized empirically. Typical
values for these parameters are 0.4 μm, 0.7 μm, and
0 frame, respectively. Any tracks that are less than
20 frames should be discarded.
(g) Visually inspect the tracks to ensure appropriate tracking.
(h) Export tracking data as a spreadsheet.
Tethering Assay for RNA Mobility
275
should be optimized empirically.
(g) Detected spots are joined into trajectories using simple
linear assignment problem (LAP) tracker in Trackmate.
The parameters for tracking such as linking max distance,
gap-closing max distance, and gap-closing max frame gap
should be optimized empirically. Typical values for these
parameters are 0.4 μm, 0.7 μm, and 1 frame, respectively.
Any tracks that are less than three frames should be
discarded.
(h) Visually inspect the tracks to ensure appropriate tracking.
(i) Export tracking data as a spreadsheet.
2. For RNA particles undergoing directed movement:
(a) Particle tracking is generally performed for typically
40 frames in the region of interest (cytoplasm) (similar
to steps 1a and 1b of previous section; the only exception
is to change the option in the Slice Keeper to 40 frames
instead of 5 frames).
(b) FFT band-pass filter in Fiji can be used to filter out noise
that is below 3 pixels and above 40 pixels (similar to step
1c of previous section).
(c) Go to Image > Properties and set the values for number of
channels, number of frames, and pixel size (in μm). Click
the box for the global option at the bottom of the pop-up
window for applying these calibrations to all images.
(d) Use Plugins > Tracking > TrackMate to launch Trackmate and confirm that the number of frames, pixel size,
dimensions of the region of interest, and time interval are
correctly read by Trackmate.
(e) Detect spots using the Laplacian of Gaussian (LoG) detector in Trackmate. Spot size (0.36–0.4 μm) and thresholds
should be optimized empirically.
(f) Detected spots are joined into trajectories using the linear
motion LAP tracker in Trackmate. The parameters for
tracking such as initial search radius, search radius, and
max frame gap should be optimized empirically. Typical
values for these parameters are 0.4 μm, 0.7 μm, and
0 frame, respectively. Any tracks that are less than
20 frames should be discarded.
(g) Visually inspect the tracks to ensure appropriate tracking.
(h) Export tracking data as a spreadsheet.
Tethering Assay for RNA Mobility
275