Bimodal single molecule microscopy: Multiparameter spectroscopy gives insight into photodegradation processes

The triumphal course of optical single molecule studies mainly focuses on fluorescence based techniques. However, the structural insight, which can be gained by these methods, is often rather limited due to the broad and unstructured fluorescence spectra. This restriction may be overcome by surface enhanced Raman spectroscopy (SERS), which provides a chemical fingerprint of the investigated species. Nevertheless, for complex systems such as e. g. proteins, the interpretation of the obtained spectra is often too intricate. We present a novel bimodal microscopy approach to correlate the information content of the two spectroscopic modes on the single molecule level. By performing both, fluorescence and SERS spectroscopy on the same individual bichromophoric autofluorescent protein, we are able to assign distinct Raman bands to isolated fluorescence forms. On basis of these data we compare Raman spectra of native and photobleached proteins independently for the two distinct spectral forms. This in turn enables us to study the individual photodegradation processes and to open the field of elucidating the chemical structure of these compounds by spectroscopic methods in the single molecule level.