Recent Developments in Fluorescence Spectroscopy

Abstract

In recent years there have been significant advances in the instrumentation for time-resolved fluorescence spectroscopy. Development of the frequency-domain method and the availability of suitable laser sources and detectors now provides remarkable resolution of complex intensity and anisotropy decays. At present the applications of fluorescence are more limited by the lack of suitable fluorophores than by instrumentation. In particular, the information content of fluorescence is primarily on the 10 ns timescale, which is comparable to the decay times of most fluorophores.

To circumvent the short lifetime of most available fluorophores, we will describe a new class of fluorophores, ruthenium and osmium metal-ligand complexes, which display decay times ranging from 100 ns to several microseconds. Surprisingly, these complexes display useful anisotropy, and thus can be used to measure protein hydrodynamics on the microsecond timescale. These compounds are highly photostable and should thus be useful in fluorescence microscopy. Importantly, the long decay times of these probes allows off-gating of the prompt autofluorescence and the design of simple instrumentation for lifetime-based sensing and imaging.

The increasing availability of ps and fs lasers has resulted in an interest in two-photon excitation for time-resolved spectroscopy and for intrinsic confocal microscopy. We show that two-photon excitation near 300 nm can be used to excite the intrinsic fluorescence of alkanes, thereby avoiding the usual requirement of vacuum UV excitation. Alkane fluorescence is quenched by water and alcohols, and it is possible that fluorescence from aliphatic groups will be obtained from biological molecules. Additionally, we show that the calcium probe Indo-1 can be excited by simultaneous absorption of three photons at 885 nm, a wavelength conveniently available from Ti:Sapphire lasers.

And finally, we describe a new type of fluorescence experiment which uses multiple light pulses. The initial pulse is used to create the excited state population. We refer to this phenomena as light quenching, which allows changes in the excited state orientation and wavelength-selective removal of fluophores. Light quenching promises to provide an increased information content on the complex intensity and anisotropy decay of biomolecules.