  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, 6.5x less than
the current state-of-the-art. This strategy greatly extends the possibility to
detect 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.
