Macros>Install. The plugin can be obtained from:https://
imagej.nih.gov/ij/macros/.- In our experience, setting noise tolerance to 10 enables better
identification of spots. The operator should optimize this
parameter for his or her images. - Be sure to invert the “black spot” images into “white spot”
images and save as image sequence format before MATLAB
analysis. - Based on our smFISH results, we determined that discrete
RNA transcripts are distributed at least 1 pixel from one
another. Therefore, a script should be written to compare the
intensity of each local maximum in each slice with the intensity
of the neighboring pixels in that slice and the slices immediately
above and below (nine pixels in the slice above, eight surround-
ing pixels in the same slice, and nine pixels in the slice below).
Each 3D maximum represents a single RNA transcript. The
operator should determine how many slices and pixels are
needed for RNA analysis within his or her images. - After RNA transcripts in smFISH and MB images are deter-
mined as described inNote 21, a script should be written to
assess the extent of colocalization between the two signals in
3D. In our program, an MB to smFISH colocalization event
occurs if within a 3 3 3 voxel of a smFISH local maximum
there is an MB 3D local maximum. Vice versa, we consider an
smFISH to MB localization event if within a 3 3 3 pixel
cube of an MB local maximum there is a smFISH local maxi-
mum. A 5 5 5 pixel cube can also be used for comparison
between MB and smFISH signals to reduce the impact of local
maxima uncertainty.
Acknowledgments
This project was supported by grants from the National Basic
Research Program of China (2016YFA0100702), the National
Natural Science Foundation of China (81371613) and the Beijing
Natural Science Foundation (7162114).References
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