My work is to fibertype muscle sections using immunofluorescence. This is what I stained for: Blue for the edges of individual fibers, green for a specific type of fibers (type IIa) and red for type IIb fibers. The attached figures were obtained using TileScan functions (several pictures joined together) for Blue. For Green and Red figures I uploaded only the individual images.
My pipeline works well when identify the individual number of fibers (Blue image) but sometimes individual fibers are splited into 2. I should have overcome this using MaskImage but the problem remains. Overall this is not a major issue as I can refine the staining or obtained a better picture quality. This pipeline attached was done for this purpose.
When I try to identify objects manually or corrected those that were split, the coloured edges of the object does not come out. The only available option is to press F. Please, let me know what I am doing wrong.
Individual fibers may either be positive to green, red or both. When positive to both colours they may colocalise or not. The staining for green or red is diffuse and fibers present different intensity to the staining. Some are very bright while other do not. Inside the fiber types, they also differ in their morphology. Furthermore, the green or red staining may or may not cover the complete size of the fiber.
Please, I’d like you to help me with:
The pictures need to be in high quality and be analysed as a total. The issue is that they are very heavy (100Mb) and the processing takes time. I tried reducing the quality or rescale but does not work properly. When I save the images in PNG I do not get the same quantification of fibers. What else could I do?
As I have the total number of fibers and their properties (size, etc), I want to have the same for the green, red and those positive for both. The segmentation of each of them is not proper as you can see if look at the blue channel.
Please note that the pipeline attach is for the Blue channel of the Tilescan no for the individual images, which have the red and green colour. If provide me an email I will send the TIFF image of the Tilescan.Or you may suggest how to do it with the images attach and I modify the diameter.
I sincerely hope you can give me some clues.