Oil-red-O Fat droplet analysis

Analysis of size and density of fat droplets stained with Oil-Red-O and counterstained with hematoxylin.

Input: Fluorescence image of Oil-Red-O (Texas Red channel) and brightfield image of hematoxylin in a single grayscale channel (Texas Red emission filter in brightfield illumination). The recording is medium resolution (40x lens or 0.4 um/pixel).
Output: parenchyma area in pixels, number and average size of oil droplets.


Reference images: This analysis requires a blank and dark current reference images for hematoxylin, and optionally a positive control for Oil-Red-O. All of identical frame size to the input. Use the “Create reference image for OD BLANK and FLUORESCENCE BG using median” pipeline to create a blank. If no dark current image available use a value in “Dark current intensity” instead. To use a positive control scaling the Oil-red-O channel set the “Positive control reference image operation” parameter to “Make reference image” when processing a positive control image, and “Use reference image” to process all samples. Use a recording with a lot of fat droplets for positive control. Blank and Dark Current Images: The blank images must be recorded in the same session with the input to ensure that the alignment of the optics matches between the input image and the reference image. Use multiple blank images recorded into a separate multi-position data set to remove debris by median calculation provided in the pipeline. Dark current images can be reused from older recordings, if camera parameters or frame size are not changed. Channel numbers of reference images must match the channel number of the input image. Channel numbers can be changed using the Edit/Rename command.

Analysis protocol:
1. Load the dark current reference image, and using the context menu select “Set as Reference Image” and “Background/Dark Current”. Minimize this image. Alternatively set the “Dark current intensity” parameter to the value you normally measure in an image with no illumination.
2. Using a pipeline on the blank recording: load a single blank image and execute the “Create reference image for OD BLANK and FLUORESCENCE BG using median” pipeline. This calculates the blank required to calculate hematoxylin OD to remove vignetting during thresholding the parenchyma, and calculates the fluorescence background for Oil-red-O.
3. Open the input recording as a Multi-Dimensional Open dialog, and re-activate this pipeline by selecting it in the Pipelines menu.
4. Choose a position in the recording as positive control where are a lot of fat droplets. Set the “Positive control reference image operation” parameter to “Self-referenced”.
5. Adjust pipeline parameters for fluorescence and size of oil droplets and parenchyma threshold until re-running the pipeline provides the desired results.
6. Re-run the pipeline by setting “Positive control reference image operation” parameter to “Make reference image” , and then set it to “Use reference image”/
7. Open Excel Data Window from the Tools main menu
8. Use the pipeline automation button (double blue arrowhead) with selecting “Run Pipeline … on All Stage Positions” to process images
8. Save contents of Excel Data Window from the File main menu


Adjustment of sensitivity:
• If debris is detected a fat droplets, increase the “Minimum seed fluorescence (% of scaled image)” or the “Min Spherical Mean”.
• If dimmer cells are missed, decrease the above values, or decrease the “Debris cutoff” value.
• If bright debris outshines cells, decrease debris cutoff percentile.

This pipeline was used in:
Wong HS, Mezera V, Dighe P, Melov S, Gerencser AA, Sweis RF, Pliushchev M, Wang Z, Esbenshade T, McKibben B, Riedmaier S, Brand MD. Superoxide produced by mitochondrial site IQ inactivates cardiac succinate dehydrogenase and induces hepatic steatosis in Sod2 knockout mice. Free Radic Biol Med. 2021 Feb 20;164:223-232.