RNA Detection

by breaking excessive lines by pressing the left mouse button and

the Alt key simultaneously. Go to “Label inspection” mode to

check whether you have made all necessary corrections (Fig.5).

12. Use “get Masks” or simply close the Annotation control win-
    dow to finalize your masks image and save it (seeNote 41)
    (Fig.5).

3.5.2 Transcript
Detection

1. Open the original z-stack image that you acquired on the
   microscope. In addition, open the Cell mask image that you
   generated in the previous steps in Fiji if you would like to assign
   transcripts to cells (seeNote 42).


2. Start the “Cell transcript analysis” plugin which is part of the
   MS-ECS-2D update site (plugins >Cell transcript > Cell
   transcript analysis). Select the smFISH image and cell mask
   and adjust the settings to your sample (Fig.6a).


3. First, run the Transcript analysis plugin once to generate a plot
   of maximum distributions (‘MaxDistrib*.png’) (seeFig. 6b).
   This plot will help you to determine the appropriate transcript
   detection threshold. The threshold should be set between
   background and transcript signal peaks (seeFig. 6b). You can
   zoom into the plot by dragging a rectangle around the area of
   interest (seeFig. 6b 2 ). Identify the transcript detection thresh-
   old that is appropriate for your smFISH sample.

Fig. 5The Cell annotation plugin. The Cell annotation pipeline in Fiji can be used

to correct over- and under-segmentations (Correction mode) by drawing missing

lines and breaking excessive lines. In the Annotation mode, cells can be

assigned to a specific group, for example based on cell type. In the Label

inspection mode (depicted here), the final cell segmentation results can be

checked

154 L. Carine Stapel et al.