Detecting the same individual protein and its photoproducts via fluorescence and surface-enhanced Raman spectroscopic...

We present a novel multiparameter microscopy approach allowing for both fluorescence and Raman imaging and spectroscopy of the same individual autofluorescent protein and its photoproduct by colocalization of the same species in the respective spectroscopic images. For the investigated bichromophoric autofluorescent protein DsRed_N42H we are able to assign different Raman spectra to the photoproducts of the distinct chromophores. Furthermore, a careful analysis of Raman spectra taken from native proteins in comparison to Raman spectra from photobleached species allows for a feasible estimation of the underlying photodegeneration processes of the individual spectral forms.