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smFISH-analysis-workflow.md

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General workflow of smFISH analysis

Epithelial polarity analysis in SMG buds

  1. Create ROIs from manual drawing and computation

    • For epithelial polarity analysis, manual drawing is often needed to generate ROIs. I have an algorithm to minimize the efforts of manual drawing.
      • First, preprocess the data by cropping out an ROI including one continuous region of the epithelial surface.
      • Second, manually draw the epithelial surface ROI. In most cases, the epithelial surface will touch one or more of the image boundaries. The imageJ macro will automatically expand the image canvas so that the polygon ROI can be drawn beyond the orignal image boundaries to include the entire epithelial ROI. The epithelial ROI will be generated by intersecting the manually created ROI with the orinal image canvas.
      • Third, the imageJ macro will create a new larger image with a 3x3 grid, where the original epithelial ROI image is placed at the center, while other grids were filed with the mirror images. This is a tactic to handle the boundary conditions during the shrinkage of ROIs.
      • Fourth, the binary image is shrunken by half the surface cell height (7.5 µm), where the difference between the original epithelial ROI and the shrunken ROI will be ROI_basal.
      • Lastly, the binary image is further shrunken by another half cell height, where the difference will be taken as the ROI_apical.