Crossing the Border towards Deep UV Time-Resolved Microscopy of Native Fluophores

Abstract

More than 20 years ago, single photon counting based techniques evolved as one recognized standard in fluorescence detection. In combination with confocal microscopy FLIM (Fluorescence Lifetime Imaging Microscopy) and FCS (Fluorescence Correlation Spectroscopy) became established techniques for investigations down to the single molecule level. Up to date, these experiments typically are carried out in the visible up to the near infrared spectral range.Based on recent advances in fiber amplified laser technology [1] and ultrasensitive detection, we present a novel approach to extend time-correlated single photon counting (TCSPC) into the deep UV using 266 nm excitation. Hereby, direct access is granted to the native fluorescence of biomolecules originating from appropriate chromophoric groups such as the amino acids tryptophan and tyrosine within proteins. As first results, we will present label-free FLIM of cells where the aromatic amino acids within the proteins become visible. As a benchmark, also FCS with organic fluorophores in the deep UV will be shown.Another application of time-resolved fluorescence microscopy in the deep UV includes microfluidics and thus enables label-free detection and identification of various aromatic analytes in chip electrophoresis [2, 3]. Fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for different substances in the electropherogram with the aim to identify aromatic compounds in mixtures. Based on the time-correlated single photon counting the background fluorescence can be discriminated resulting in improved signal-to-noise-ratios. In addition, microchip electrophoretic separations with fluorescence lifetime detection can be performed with protein mixtures emphasizing the potential for biopolymer analysis.