Multi-dimensional fluorescence microscopy of living cells.

An overview on fluorescence microscopy with high spatial, spectral and temporal resolution is given. In addition to 3D microscopy based on confocal, structured or single plane illumination, spectral imaging and fluorescence lifetime imaging microscopy (FLIM) are used to probe the interaction of a fluorescent molecule with its micro-environment. Variable-angle total internal reflection fluorescence microscopy (TIRFM) permits selective measurements of cell membranes or cell-substrate topology in the nanometre scale and is also combined with spectral or time-resolved detection. In addition to single cells or cell monolayers, 3-dimensional cell cultures are of increasing importance, since they are more similar to tissue morphology and function. All methods reported are adapted to low dose of illumination, which is regarded as a key parameter to maintain cell viability. Applications include cancer diagnosis and cell tomography under different physiological conditions.