RNA Detection

16. Image your samples on an epifluorescence microscope with a
    high NA objective and a sensitive camera (seeNotes 6and 33 )
    (Fig.1b 5 ). Use z-spacing of 0.3μm or less to capture each
    transcript in multiple z-slices (seeNote 34).

3.5 Image Analysis Here, we describe all steps required for image analysis. For more
background information on the image analysis tools, you may
consult the documentation associated with Stapel et al. 2016 [15].

3.5.1 Membrane
Segmentation

These steps can be skipped if one is not interested in assigning

transcripts to individual cells.

1. Open your image in Fiji and duplicate the central membrane
   slice of the z-stack (e.g., slice 10 for a z-stack of 19 slices)
   (Fig.1b 1 ) using the function “Duplicate” (seeNote 35).


2. If you are analyzing multiple images at the same time, resize all
   images to the exact same size. This is a requirement for the
   membrane prediction pipeline in KNIME that we will use in
   the next steps. You can use Fiji function “Canvas size” for this.
   Save the image in ‘.tiff’ format and collect all membrane images
   for which you want to generate segmentations in a single
   folder.


3. Start KNIME and open the workflow “MS-ECS-2D_2.0”
   (Fig.4). Double click on the node “Image reader” at step 2.1
   (Fig.4) and select the membrane images that you generated in
   the previous steps (seeNote 36).


4. Reset the node “Prediction” at step 2.2 (Fig. 4) by right-
   clicking on the node and selecting “Reset”.


5. Double click on the node “Prediction” and then on “Run cas-
   caded RF” to open these nodes. Now double click on the node
   “Image Resizer” to configure this node. Set X, Y, and Z to
   downsample your images and speed up the analysis (seeNote 37).


6. Wait until the prediction is finished. The node will show a green
   check mark. Now use the node “Image Writer” at step 2.3 to
   write the result images to a folder on your computer (Fig.4).


7. Sort the membrane probability and the vertex probability
   images (Fig.1b 2 ) into separate folders.


8. Open Fiji and start the plugin PathFinder which is part of the
   MS-ECS-2D update site (seeNote 38). Select the folder with
   your original membrane images (seestep 2), the folder with the
   membrane probability images (seestep 7), and the folder with
   vertex probability images (seestep 7) as prompted (seeNote
   39 ). While the PathFinder plugin is running, several image
   windows will pop up and disappear again.


9. Once the run is finished, all pop-up windows will have disap-
   peared and you will find a folder “results” inside the folder with

152 L. Carine Stapel et al.