Spectroscopic and microscopic techniques allow analysis of various aspects of biological systems on molecular level. One of the common problems of such studies is overlapping of absorption or fluorescence spectra of biological molecules of interest. Several examples of how to separate them are shown for in vitro and in vivo systems. These examples include analysis of competitive binding of two molecules (anticancer drugs, mutagens and biologically active substances) to nucleic acids via absorption spectroscopy, separation of fluorescence spectra of Photosystems via time-resolved fluorescence spectroscopy and microscopy and analysis of Forster Resonance Energy Transfer (FRET) between voltage-sensitive fluorescence proteins. As a result constants of binding of anticancer drugs and mutagens to nucleic acids are determined for molecules that absorb light in the same region with nucleic acids. Also composition and ratio of Photosystems is determined in leaves, which opens room for non-invasive “express” analysis of these parameters in plants, allowing determination of optimal growing conditions and presence of diseases. FRET studies of voltage sensitive fluorescent protein help to figure out the mechanism of its action and improve its functionality. In general, it is shown that number of different approaches can be used to analyze overlapping absorption and fluorescence spectra of biological molecules on different levels of organization in vitro and in vivo.
