Segmentation

Author

Kaitlin Sullivan

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Video Tutorial

Here, we binarize and multiply our DAPI images to get only cells present in all three images for segmentation. Then, we select the region of tissue we would like to segment and the dilation value for the nuclei after they are segmented.

If using external segmentation methods, save the output as Fiji-readable ROIs named RoiSet.zip, place the file in a folder called analyzedTables, and skip straight to Quantification.

Segmentation

This option takes the registered DAPI images from the nonLinear folder and segments them.
The DAPI from each round will be binarized then multiplied by eachother to remove cells from out-of-focus planes.

TO RUN:
- Drag-and-drop R1_405_DAPI.tif_registered.tif_NL.tif from the nonLinear folder into FIJI and select Segmentation.

Run from the nonLinear folder…
β”œβ”€β”€ max └── crop └── regImages β”œβ”€β”€ composite └── nonLinear__<- drag DAPI file from this folder***__

WHAT HAPPENS:
- Segmentation Type: select DAPI
- Segment based on DAPI signal from every round (to ensure registration in the z-axis)
- If there is a round where DAPI is not segmentable, uncheck it - Threshold Type: binarize the images for segmentation
- Automatic Thresholding
- Manual Thresholding: (select threshold manually with a slider if some DAPI rounds look strange via Automatic Thresholding) - Dialation Value: dilate ROIs to include the surrounding cytosol
- Suggested Value = 3 microns
- For densly packed regions or nuclear expression only, change value to 0 microns

  • Segmented image is saved in an analyzedImages folder
  • Segmented ROIs are saved in analyzedTables folder
  • IF USING EXTERNAL SEGMENTATION ALGORITHMS: for Baysor, Weka, or CellPose, place your Fiji-readable ROIs as RoiSet.zip in analyzedTables and skip straight to Quantification.

⟡ Previous: Registration Next: Quantification ⟢