Nanotopology of cell adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM).

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Because of the low penetration depth of an evanescent electromagnetic field, total internal reflection fluorescence microscopy (TIRFM) proved to be a powerful technique to examine fluorescent dyes or photosensitizers in close vicinity to the plasma membrane of living cells. In addition, on variation of the angle of incidence of exciting laser light, the penetration depth is varied, so that cell-substrate topology can be examined with nanometer resolution. Using a specific illumination device for TIRFM and a highly sensitive electron multiplying (EM) CCD camera, fluorescence of the photosensitizer protoporphyrin IX (PPIX) was studied in human cancer cells after application of 5-aminolevulinic acid (5-ALA) prior to and after irradiation with sublethal light doses (635 nm, 4 J/cm2). For cells growing on microscope cover slides, cell-substrate distances varied between approximately 20 and 250 nm with a mean distance of approximately 120 nm. On light exposure, these distances generally decreased, and a mean value below 100 nm was attained. Moreover, focal contacts visualized with a fusion protein of yellow fluorescent protein and focal adhesion kinase were maintained on light exposure, i.e., light-induced detachment of cells from their substrate was not likely to occur.