rmation changes during the interval between photons with specified colors. The procedure works even when the photon colors appear to be scrambled (i.e., one cannot identify states by visual inspection of a photon trajectory) because the photophysical properties of the conformers are similar and/or conformational dynamics is on a similar time scale as the photon counts.[unreadable] [unreadable] The above work has played an important role in analyzing recent experiments performed by Dr. W. A. Eaton's group (3). In this project, quantitative analysis of the FRET efficiency distribution of polyproline was performed. The analysis resolved the puzzle about the excess width of the distribution and allowed one to draw important conclusions concerning the flexibility of the all-trans form of polyproline and the distribution of cis isomers. More details are provided in Dr. Eaton's report.[unreadable] [unreadable] Finally, the optimal concentrations for single-molecule measurements of freely diffusing molecules (4) was established. It was found that a concentration as low as 0.1 molecule per observation volume may not be small enough for single-molecule FRET efficiency measurements. This result follows from a rigorous theory that takes many molecules into account. We considered the distributions of the number of photons (photon counting histograms) and showed that multiple-molecule effects are pronounced at large photon counts even at low concentrations. FRET efficiency distributions reveal multiple-molecule effects at large threshold values. This might be misinterpreted as multiple conformational states. Multiple-molecule effects strongly depend on the brightness of fluorophores. A simple test was suggested to determine parameters for which the single-molecule description is applicable.

Objectives: Sensorineural hearing loss (SNHL) is strongly associated with many aspects of military service including blast injury. The objectives of this proposal are to improve the prevention and treatment of SNHL in veterans. Research De