How are proteins on the surface of cells able to be studied by TIRF microscopy, without becoming "stuck" to the microscope slide surface?
I do no know the answer to your question, but it would be helpful to the person who answers it to know whether it is plant, animal or protozoa.
I was thinking of mammalian cells typically used in cell culture, like HEK293 or CHO
I'm not sure I fully understand the question, but TIRF limits excitation to a thin plane directly above the coverslip. The exact depth depends on the wavelength and angle of incidence, but is typically in the range of 100-200 um. So cell surface proteins that are visible by TIRF are within this range of the surface. I would think mechanical changes of adhesion complexes and internalization allow some proteins to dissociate or turnover without sticking to the surface, but to some extent surface proteins do get stuck to the glass. Close examination of the path of migrating cells will reveal a trail of membrane and protein left behind.
The reason I am puzzled is because I have found many examples in the literature where people are able to observe the mobility of proteins on the outer surface of the cell plasma membrane by TIRF microscopy, even ones containing large ectodomains. Apparently sticking to the cover slide is not an issue, so I was wondering what the reason for that is... do the structures that adhere the cell to the slide leave enough room between the slide and the cell membrane for the height of the typical extracellular protein, so it is able to move unencumbered? And what is that height?
That is interesting about migrating cells.
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