Assesment of Cellular Damage by Inverted Fluorescent Microscopy

Three fluorescent probes, Calcein AM for the detection of living cells, Ethidium Homodimer for the detection of dead cells and Hoechst 33258 for the detection of cellular nucleus damage, were selected for verification of cellular damage by using inverted fluorescent microscopy after the application of photodynamic active substances with phototoxic effect. Calcein AM does not fluorescent until it is transformed into an intensively green fluorescing calcein by non -specific cytoplasmatic esterase. Ethidium Homodimer penetrates the cells through a damaged membrane, collocates to nucleic acids producing, in dead cells, fluorescence in the red spectral field. Hoechst is used for DNA detection, collocates in a small groove with a preference for A-T vapours, thereby affording detection of cell damage. With the help of this probe we can distinguish between live, necrotic and apoptotic cells.