Hi all,
I have developed a hydrogel based on silk, and I need to do some fluorescent imaging of the encapsulated cells. When I stain the cells, for example, using CalcinAM, the background is huge and does not allow good-quality images, and I think it is because of the inherent autofluorescence of silk fibroin. Do you have a way to circumvent this?
Thanks
Dear Masoud Hasany
usually autofluorecence is worst in the green channel, so I would think about changing the cell dye to a more red one. I have good experience with CellTracker CMPTX.
Another approach quick and dirty approach could be to acquire the autofluorescence in another channel (i.e. DAPI or Cy3) and to acquire your CalceinAM staining. You might than be able to use image calculation im ImageJ to divide the CalceinAM image by the autofluorecent image. The result should look like a low background hydrogel with your CalceinAM signal more or less untouched. This will give you at least an idea if you want to order another dye for really sufficient imaging.
kind regards
Sönke
Hi Masoud,
I would try with some autofluorescence quencher like True Black or Sudan Black. Applied before doing the staining should reduce the background without affecting the intensity of the target areas.
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