Hello,
I am planning an experiment and I would need to encode 4 different fluorescent proteins in mammalian cells, that should be seen and distinguished with a fluorescence microscope in a live imaging experiment and with FACS as well. I don't know if it is possible to do something like this, first of all because the expression of 4 fluorescent protein will for sure create stress in the cells and also because I don't know if they're going to overlap too much or not. My cells already have 2 proteins tagged with GFP and mTurquoise, I will add mCherry and I'm looking for the fourth one. I was checking Sirius, a protein that absorbs and emitts in the blue/UV spectrum, I don't know if it could be distinguished from mTurquoise. Same for EYFP, since I already have GFP. Has anyone ever done something like this? Thank you!
I think you are right that this will probably cause some cell stress, but assuming it works and expression levels are not so much and the cells are OK, you will likely need to do spectral imaging and linear unmixing: https://www.microscopyu.com/techniques/confocal/spectral-imaging-and-linear-unmixing
If you find spectral overlap of four FPs, you can use unmixing to disentangle them. This commonly done by using a confocal with a special spectral detector that samples the emission from the sample in fine steps, and then uses a software algorithm to separate them. There have been some far red FPs published recently. I would look at one of those for the fourth color. Stay away from blue. Imaging live cells with the 405 nm laser causes a lot of UV damage.
Christopher B O'Connell thank you for your answer. I will give a look at the far red proteins, I was also reading something about near infra-red proteins as well. In both cases I don't know if we have the proper emission filter and I'm not sure if it's not going to overlap with mCherry. About the blue, you're right but I would need to take a picture every 15-30 minutes for 24-72 hours. Would it cause too much damages?
You could use a Cy5 filter set, or similar for near IR. Here is a useful website of a fluorescent protein database https://www.fpbase.org/. Based on what I saw there, with a confocal you will be reasonably safe because mCherry is not excited by a 640 nm laser. If you use widefield epifluorescence then you may have a crosstalk issue because excitation filters are spectrally wider than lasers.
That amount of imaging will likely be OK for a blue FP. Some cells are more or less sensitive depending on intensity and exposure time. Far red is generally better because longer wavelengths are lower energy and cells/tissue are less autofluorescent out in the red.
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