An ImageJ macro for the quantification of ciliary accessible cholesterol

Output Details

ALOD4, a toxin-based probe, can be used to visualize the accessible pool of cholesterol (PMID: 30790255). Here we describe an algorithm for segmenting fluorescently-labeled cilia using SSTR3 as a ciliary marker. The segmented masks are used to determine the intensity of ALOD4-bound accessible cholesterol in the ciliary membrane. The algorithm asks for a directory path and searches for .tif-files. Each identified .tif-file is cropped for a user defined region of interest (ROI) which must contain the cilia in the image series. Then the Z-stack is maximum intensity projected, the contrast of channel 1 (SSTR3-mApple, red) is optimized for visualization, and both channels are duplicated. The MorphoLibJ library helps to identify the SSTR3-positive cilia. SSTR3 signals are then thresholded, binarized, and intensity-corrected. A shape filter prevents false positive signals by preventing non-ciliary structures to be assigned as objects. The identified objects are applied onto the channel 2 signal (mNeon-ALOD4, green) and intensities are measured and saved.
Tags
  • Cholesterol
  • Cilia
  • Ciliogenesis

Meet the Authors