I’m attaching two links to separate images. These images are merged red and green filter images from fluorescent microscopy. The green stain was intended to identify the membrane of the skeletal muscle cells in the cross-section, and the red stain was intended to identify a protein target predominantly localized to the membrane.
The primary outcomes of this analysis are to count the number of skeletal muscle cells in the cross section (circular objects separated by membranes) and then divide the number of the red pixels above an appropriate intensity by the number of fibers in the cross section as a surrogate of an increase or decrease in the protein’s expression at two separate time points.
Hence, I need good segmentation and separation of the cells, based on the membrane stain. Second, I need an accurate count of the red pixels that are not “background flourescence” so that I can confidently infer the expression of the protein target of interest. I’ve employed smoothing, image math, and thresholding strategies in pilot pipelines but the outcome images are not as pristine and accurate as I would like. I am hopeful you can provide assistance with this issue, as most of the other images look similar to these, and this is a relatively straightforward analysis.
Here are the links to the images.
Thanks so much for any assistance!