Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins

The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single-molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet-state quenchers greatly promotes the protein photostability, reduces the photobleaching probability, and improves the net autofluorescence detection rate. Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general. Using this approach, we achieved UV fluorescence correlation spectroscopy on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5× fewer than the current state-of-the-art. This strategy greatly extends the possibility of detecting single label-free proteins with the versatility of single-molecule fluorescence without requiring the presence of a potentially disturbing external fluorescent marker. It also opens new perspectives to improve the UV durability of organic devices.