Fluorescent markers in microscopy: photophysical characteristics and applications in cell biology

In the fluorescence microscopy of biological specimens the structures in cells and tissues usually need to be labelled with various fluorescent markers. The three main groups of fluorescent markers are small organic fluorochromes, fluorescent proteins and quantum dots. Fluorescent markers differ according to photophysical properties and binding specificity for the selected target structures in the sample. For the labelling of specific structures with small organic fluorochromes or quantum dots it is usually necessary to conjugate them with target-specific macromolecules. For the labelling with fluorescent proteins it is necessary to introduce a fluorescent protein gene into the observed cells or organism. The most important photophysical properties of fluorescent markers are absorption and emission spectra, Stokes shift, extinction coefficient and quantum yield. Currently, various super-resolution fluorescent microscopy techniques exploit special fluorochromes that enable fluorescence modulation by specific wavelength illumination, to achieve the resolution below the diffraction limit.