Abstract
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/cm2 up to about 200 J/cm2 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.
Highlights
In fluorescence microscopy considerable improvements in resolution have been reported in recent years
Methods of super-resolution microscopy, e.g., stochastic optical resolution microscopy (STORM) or photoactivated localization microscopy (PALM), have been reported [8 10], which commonly are based on single molecule detection, and which permit a resolution below 20 nm
Photosensitization and light-induced generation of cytotoxic reactive oxygen species, e.g., singlet oxygen or superoxide radicals, has been related to various intrinsic molecules, e.g., flavins [16] or porphyrins [17,18], as well as fluorescent dyes, e.g., organelle markers or photosensitizers used in photodynamic therapy (PDT) [19]
Summary
In fluorescence microscopy considerable improvements in resolution have been reported in recent years. This holds in particular for confocal [1] and multiphoton [2,3] laser scanning microscopy (LSM). Including 4-Pi and stimulated emission depletion (STED) microscopy, where a lateral resolution of about 30 nm as well as a high axial resolution have been obtained [4]. Those techniques, require exposure to very high light doses, such that damage to living cells may occur. The high light doses needed for those methods may again limit their application to living cells
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