Hi All,
This is my first post and a bit of a last resort since I've tried to search high and low for an answer; I hope someone experienced can spare a few minutes to help! The goal of my project is to determine any sorts of color-shifting that occurs from stimulating cells adhered on top of biofilms. I've taken two images of the cells before and after stimulation and would like to quantify this small blue shift.
Currently, I've been playing around with the Image Calculator > Subtraction function but am still not sure how to interpret the resulting data. The areas where I expected the most color change seem to remain black. The outline of the cells themselves appear to have the most color change, most likely (and unfortunately) due to their slight movement as I change buffers. Perhaps this intensity washes everything else out?
And so the main goal I have now is to just quantify any color shifting that has occurred, even if it's just around the cell walls. I was hoping that the result from the subtraction would give a quantifiable shift, but I don't think it can provide anything besides the image. Is there a way to quantify color shifts with ImageJ? I.e. I would like to say, OK, after stimulating my cells, there is an overall blue shift in peak reflectance of
X nanometers.
Of course, the ideal experiment would be to have a spectrometer built into a microscope so I could get reflectance spectra before and after the stimulation, but that set-up would cost a pretty penny!
Here is a link to my pictures in case you wanted to fiddle with the images:
https://www.dropbox.com/sh/mscr657d9o185gk/AAAN4bJ6Otra88vkhe-CcXkkaThe first image is before stimulation, the second is after stimulation, and the third is the subtraction (#1-#2) that I get. Again, most of the subtraction is black. I expected to see more of a noticeable color difference in the areas that are widely orange and blue shift to yellow.
Thank you for any help that you can provide! I appreciate your time and efforts!
Best,
Long Phan
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