Thank you very much for responding, I do appreciate it. I didn’t write back immediately, as I wanted to make sure I had a clear set of questions for you.
I’m not sure I understand everything that you wrote, so I’ll add questions & commentary to quotes from your post:
“Once the segmentation algorithm has identified expressing vs non-expressing pixels…”
Ok, so this algorithm must be using the luminosity and/or some color information in the original ISH image to determine the expressing/non-expressing state for that pixel. I understand from the documentation that this algorithm is masking away non-tissue areas of the image and may be accounting for occulsions, tissue tears, etc. So does the algorithm use color information at this stage and if so, how?
“…the expressing pixels are colorized based on the luminosity of the ISH image (0.21 R + 0.72 G + 0.07 B) over some small local neighborhood”
Ok, so that sounds like for a given pixel that is determined to be expressing, you then go back to the ISH image, and perhaps position a 2-D Gaussian hill over the pixel and sample the ISH color information around this hill to gather a number for the expression image pixel. I don’t understand (0.21 R + 0.72 G + 0.07 B). That color axis in 3D color space is more or less green and none of the ISH images seem to have greenish color in them. When I plot expressing and non expressing ISH pixel colors, they seem to be arranged along a line in color space which is roughly grey. Here are the points, plotted as in my first post, along with the 0.21R + 0.72G + 0.07B line in thick green.
Ok, so the expression map is then plotted with the jet colormap, that is helpful. I notice that in the Brain Explorer application, you can actually find some numbers associated with the jet colormap. Are these accessible via the API? That is, is the range which the jet map represents available via the API? Do these numbers have any physical meaning, or are they arbitrary?
Finally, do you know if the luminosity of the stain in the ISH image is roughly linear with the number density of mRNA strands that code for the given protein, or is it some other relationship, such as exponential?