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A test system for cell viability based on colony formation has been established and applied to high resolution fluorescence microscopy and single molecule detection. Living cells were irradiated either by epi-illumination or by total internal reflection (TIR) of a laser beam, and light doses where at least 90% of irradiated cells survived were determined. These light doses were in the range of a few J/cm(2) up to about 200 J/cm(2) depending on the wavelength of illumination as well as on the presence or absence of a fluorescent dye (e.g., the membrane marker laurdan). In general, cells were less sensitive to TIR than to epi-illumination. However, comparably high light doses needed for repetitive excitation of single molecules limit the application of super-resolution microscopy to living cells.

Light Dose is a Limiting Factor to Maintain Cell Viability in Fluorescence Microscopy and Single Molecule Detection