- Rerun the Cell transcript analysis pipeline with the identified
transcript detection threshold (seeNote 43). - Check the file that ends in “_ResultImg.tif” to determine if
transcripts, transcription foci and nuclei were detected cor-
rectly. Adjust the parameters for transcription foci and nuclei
detection if necessary (seeNote 44). - The file that ends on “_cell.txt” (seeNote 45) contains the
quantitative data of the transcript detection pipeline including
cell size, nuclear size, number of transcripts per cell, number of
transcription foci per cell, number of transcripts in each tran-
scription focus, cell type (if you generated a cell type mask) and
cell position. You can import this data into your favorite pro-
gram (e.g., Excel, Prism, R) to analyze it.
äFig. 6(continued) embryos imaged on systems similar to ours. Below, we will explain the function of each
parameter.Nuclear segmentation parameters: (1). Nuclei channel: channel that contains the image of the
nuclei. (2). Nuclei size: radius of the largest expected nucleus in the sample in pixels. This can be measured in
the image with the line tool in Fiji. (3). Nuclei threshold adjustment: this value only needs to be modified if
nuclear detection is poor. This value should be decreased if weak nuclei are not detected properly.Transcript
segmentation parameters: (1) Transcript channel(s): channel(s) containing smFISH results. Separate channels
should be separated by a comma. (2) Transcript typical radius: can be measured in the image and is typically
two or three pixels. (3) Spot (¼transcript) minimum intensity: determine the value of this parameter based on
the spot intensity distribution (seepanel B), after running the pipeline a first time. Before the transcript
detection threshold has been determined, fill in arbitrary values for each smFISH channel, separated by
commas.Foci segmentation parameters: (1). Foci intensity and (2). Volume threshold adjustments can be
made if foci detection is poor. Values should be increased in case of over-detection and decreased in case of
under-detection. A value should be entered for each smFISH channel, separated by commas. (3) Nuclei
enlargement: this value is used to capture transcription foci that are located at the edge of the nucleus and
usually does not need to be changed. (4) Foci maximum radius: this parameter sets a maximum to the foci size
to prevent that large, nonspecific accumulations of probe are detected as foci. The maximum radius can be
measured in the image. The standard setting of 10 pixels works well for our samples.Miscellaneous
parameters: provide options for data display and storage and are self-explanatory. (b) Setting a transcript
detection threshold. After running the Transcript analysis pipeline a first time with an arbitrary transcript
detection threshold, a “Maxima distribution” image will be generated for each analyzed smFISH channel. This
plot can be used to determine the optimal intensity threshold for transcript detection. (b 1 ) The axes of the
original plot are not well suited to determine the transcript detection threshold due to the large number of
background spots that is detected. The user will need to zoom into the area of interest of the plot, which is
located right next to the background signal by dragging a box around this area (red dashed line). (b 2 ) After
zooming in to the area of interest, a unimodal peak for the transcript intensities can be observed. The
transcript detection threshold (black line) should be set between the background signal (left) and the unimodal
peak for the transcript signal (right). The same threshold should be used for all images from the same sample
(coverslip) that were acquired with the same microscope settings
156 L. Carine Stapel et al.