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I label my favrorite protein in cells by IF and image by confocal microsopy. My protein exists in mutliple organelle compartments, hence, appears diffused, but certain conditions can cause a sizeable amount to oligomerize.
I am trying to analysis only large puncta subpopulations of my favorite protein.
I find that thresholding each image before puncta count is impossible to acheive uniformly for all my images because of the excess “background” from non-oligomeric forms.
I noticed that by increasing the curve/“gamma” uniformly eliminates the background signal leaving a sharp (but derived) image. I have had mainly negative comments about “changing gamma” in the past.
Ergo, I plan on “gating” for only large and bright punctae by feeding in a fixed gamma (~3.5 is where i see only large bright punctae). My question is, are there any major concerns or forseeable pitfalls to analyzing punctae formation using this workflow I am proposing?
Thanks in anticipation for your feedback.