Challenges in modern microscopy are (1) increasing resolution down to the nanometre range and (2) 3D imaging of cells and tissues with multiple light exposures. However, in both cases light doses may exceed the limits of non-phototoxic doses by several orders of magnitude. Therefore, we determined limits for maintaining cell viability upon application of different wavelengths of light as well as various fluorescent dyes or fluorescent proteins. In a further step we adapted wide field and laser scanning microscopy for light doses compatible with live cell imaging.
If in 3D microscopy various layers of a sample are recorded, light sheet fluorescence microscopy (LSFM) is preferred to laser scanning or structured illumination microscopy, since only those planes are exposed to light which are recorded simultaneously, thus limiting the light dose even upon multiple exposure or long-term experiments. Appropriate LSFM modules have been developed which may be combined with any wide field microscope. If in 2D microscopy studies are focused on cell surfaces, e.g. plasma membranes, total internal reflection fluorescence microscopy (TIRFM) appears to be an appropriate method, since the limited penetration depth of the evanescent wave may also reduce phototoxic cell damage. Here, a variable-angle TIRFM method is suggested for measuring cell-substrate topology.