RNA 72 hrs

For live cell imaging, no
contrast or background subtraction was applied to any frames analysed. For each
condition and timepoint, 3 FoV movies from 2 biological replicates (n=6 movies
from each condition) were acquired with a total of 49 images per movie (500 ms exposure,
images captured every 5 seconds). Image stacks were imported into FIJI analysis
software where drift alignments were performed (do I reference this??). For
each FoV image, ROIs were drawn around single cell outlines for LD tracking
analysis. LD tracking was performed using the ‘TrackMate’ plug-in on FIJI analysis
software with the following parameters: estimated blog diameter= 0.5 μm, threshold=
500. Simple linear Assignment Problem (LAP) tracker was used to link together identified
tracks with the following parameters: Linking max distance= 1 μm, gap-closing max
distance= 1 μm, and gap-closing max frame gap= 0 μm to ensure no false positive
tracks were linked. Data was extracted for mean speed, displacement, and track quality.