(c) Incubate for 10 min at room temperature and then for
20 min at 37
C and 5% CO 2 .
(d) Subsequently, wash two times with 2 mL DMEM + 10%
FBS + 1% Pen/Strep to remove unattached cells.
(e) Incubate for 1 h at 37
C and 5% CO 2 .
(f) Aspirate the media, add 2 mL of fresh DMEM + 10%
FBS + 1% Pen/Strep containing 1 μg/mL doxycycline,
and incubate for 1 h at 37
C and 5% CO 2 .
3.3 Image
Acquisition
1. Equilibrate microscope imaging chamber to 37
C and 5% CO 2
prior to imaging.
2. Select cells for imaging using GFP channel by identifying cells
that contain well-resolved diffraction-limited RNA particles at
densities that facilitate single-particle tracking. Use low laser
power to reduce photobleaching when selecting cells for imaging (see Note 5).
3. Exposure times of 40–50 ms are generally applicable for singlemRNA tracking. Adjust laser power and camera gain for optimal signal-to-noise of RNA particles while minimizing
photobleaching.
4. Collect short streaming image series (50 frames) in a single
plane.
3.4 Image Analysis
Due to the difference in mobility between RNA particles that are
tethered to distinct motors, it might be necessary to perform SPT
with different parameters.
1. For static and diffusing RNA particles:
(a) Particle tracking is generally performed for five frames in
the region of interest (cytoplasm). To do this, open the file
in Fiji and use the Image > Stacks > Tools > Slice Keeper
option to generate five frame movies.
(b) Draw the region of interest (cytoplasm) using the Freehand selections tool from the toolbar.
(c) In case of low signal-to-noise ratio, use Process >
FFT > Bandpass Filter in Fiji to filter out noise that is
below 3 pixels and above 40 pixels.
(d) 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.
(e) 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.
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Varun Bhaskar et al.
20 min at 37
C and 5% CO 2 .
(d) Subsequently, wash two times with 2 mL DMEM + 10%
FBS + 1% Pen/Strep to remove unattached cells.
(e) Incubate for 1 h at 37
C and 5% CO 2 .
(f) Aspirate the media, add 2 mL of fresh DMEM + 10%
FBS + 1% Pen/Strep containing 1 μg/mL doxycycline,
and incubate for 1 h at 37
C and 5% CO 2 .
3.3 Image
Acquisition
1. Equilibrate microscope imaging chamber to 37
C and 5% CO 2
prior to imaging.
2. Select cells for imaging using GFP channel by identifying cells
that contain well-resolved diffraction-limited RNA particles at
densities that facilitate single-particle tracking. Use low laser
power to reduce photobleaching when selecting cells for imaging (see Note 5).
3. Exposure times of 40–50 ms are generally applicable for singlemRNA tracking. Adjust laser power and camera gain for optimal signal-to-noise of RNA particles while minimizing
photobleaching.
4. Collect short streaming image series (50 frames) in a single
plane.
3.4 Image Analysis
Due to the difference in mobility between RNA particles that are
tethered to distinct motors, it might be necessary to perform SPT
with different parameters.
1. For static and diffusing RNA particles:
(a) Particle tracking is generally performed for five frames in
the region of interest (cytoplasm). To do this, open the file
in Fiji and use the Image > Stacks > Tools > Slice Keeper
option to generate five frame movies.
(b) Draw the region of interest (cytoplasm) using the Freehand selections tool from the toolbar.
(c) In case of low signal-to-noise ratio, use Process >
FFT > Bandpass Filter in Fiji to filter out noise that is
below 3 pixels and above 40 pixels.
(d) 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.
(e) 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.
274
Varun Bhaskar et al.