Optical density of tissue section

The pipeline can be used for calculation of end point optical density (OD) of formazan product formation, such as succinate dehydrogenase (SDH) activity in histological sections.
Input: e.g. tiled brightfield image of the entire cross section of stained heart, taken at 540 nm.
Reference images: This analysis requires blank and dark current reference images of identical frame size to the input. 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 with median calculation to remove debris from images. 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.
2. Using a pipeline on a blank only recording: load a single blank image and execute the “Create reference image for OD BLANK using median” pipeline. Alternatively, to do it manually: Load the blank images by checking the “Positions as Time Lapse” checkbox in the Multi-Dimensional Open dialog. If the recording contains input and blank frames, set the range of blank frames in the “Settings” tab Positions filed. Note: If doing the latter, don’t forget to undo these settings when processing input images below.
3. Median project the blank images using the “Editing/T or Z-project” with “Type=Median”.
4. Using the context menu of the projection image select “Set as Reference Image” and “Blank for OD/Normalization”. Minimize this image and close the original image.
5. Open the input recording as a Multi-Dimensional Open dialog, and re-activate this pipeline by selecting it in the Pipelines menu.
6. Open Excel Data Window from the Tools main menu
7. 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

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.