RNA Detection

10. Gently add 350μL wash buffer, incubate for 5 min, and
    carefully pipette off. Repeat this step once more.


11. After gently removing the wash buffer, add 250μL of the
    TAMRA-labeled d2EGFP mRNA smFISH probe sets
    (250 nM in hybridization buffer).


12. Store the chambered cover glass in a humidified chamber at 37C
    for at least 16 h covered with Parafilm to minimize evaporation.


13. Gently pipette out the d2EGFP smFISH probes.


14. Quickly and gently wash each well by adding 350μL of wash
    buffer and incubating for 5 min. Repeat this step twice. Do not
    allow wells to dry between washes.


15. After the second wash, gently add 400μL wash buffer to each
    well. Incubate the samples in a humidified chamber at 37C
    for 30 min. The samples should be covered with tinfoil.


16. Aspirate wash buffer and wash the samples twice with 2SSC.
    Do not let the samples dry between washes.


17. Aspirate the 2SSC and gently add another 300μLof1PBS
    to each sample. It is optional to include DAPI in the 1PBS.


18. Image in three dimensions with 0.25μm increments in thez-
    direction using Cy5 and TRITC filter sets.


19. Save the images for each channel as a TIFF image stack.

3.4 RNA
Identification and 3D-
Colocalization
Analysis

1. Open the image stack for each channel using Fiji 8.


2. Draw a Region of Interest (ROI) around the cell of interest
   using the ‘Freehand selections’ tool.


3. Open the ROI Manager by selecting Analyze>Tools>ROI
   Manager.


4. Add the ROI to the list in the ROI Manager. This ROI will be
   used after completingsteps 5– 9. Be sure not to close the ROI
   Manager toolbar before obtaining the spot information for
   both Cy5 and TRITC channels. (This ROI is used for both
   channels and should not be deleted.)


5. Enhance particulate objects by selecting Process>Subtract
   Background and set the rolling ball radius to 2.0 pixels. Make
   sure to process all images (seeNotes 13and 14 ).


6. Identify particles in an image sequence by selecting Plugins>
   LoG3D. Set sigma X and sigma Y to 1, and sigma Z to 0. Select
   ‘Process slice per slice’ (seeNotes 15– 17 ).


7. Filter out particles contributed by background noise by select-
   ing Image>Adjust>Threshold. Adjust the threshold value so
   the puncta resemble the bright spots visible in each slice of the
   original image sequence. Apply the threshold, with back-
   ground pixels set to NaN, to all images in the stack.

Ratiometric Bimolecular Beacons 237