Fluorescence Comes Of Age: Measuring Angstrom-Level Distance Changes Within Single Filaments Of Regulated Actin

Epifluorescence imaging reveals that samples of reconstituted regulated actin (rAc) filaments contain a heterogeneous mixture of dissociated troponin and tropomyosin, unregulated actin, single rAc filaments, an bundles of rAc filaments. Sample heterogeneity confounds the interpretation of spectroscopic measurements of rAc. We have addressed the issue of sample heterogeneity by combining the particle sorting capability of confocal imaging with time-resoled FRET spectroscopy. To perform the measurements, rAc is sparsely immobilized to a glass coverslip. Non-immobilized regions of rAc do not contact the glass and undergo Brownian motion that is apparent in fluorescence video-microscopy. We perform one (or more) single point TCSPC measurements on an isolated rAc filament that is identified in a confocal image pre-scan. Each point measurement involves ∼50 rAc-bound Tn molecules within the confocal volume. We observe ∼20 photons/Tn/second. Thus, a single 20 second point measurement produces ∼20,000 photons, enough for reliable determination of fluorescence lifetimes. We have used this technique to determine Ca2+-induced distance changes in troponin with rAc filaments. This method may be a robust and scalable platform for the screening of drugs that bind to rAc and modulate activation.