Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution

Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. For filter-based experimental systems, where signal at different wavelengths is acquired sequentially, photostability has been required in addition. Herein, we systematically analyzed our spectral fitting models and bleaching correction algorithms which mitigate both limitations. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores. To demonstrate how small spectral shifts and changes in bleaching rates can be exploited, we analyzed vesicles in different lipid phases. Our findings suggest that a wide range of dyes, commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy due to the above-mentioned restrictions, can be efficiently applied also in FMS.