Ratiometric single-molecule studies of freely diffusing biomolecules

We outline recent developments in biological single-molecule fluorescence d etection with particular emphasis on observations by ratiometric fluorescen ce resonance energy transfer (FRET) of biomolecules freely diffusing in sol ution. Single-molecule-diffusion methodologies were developed to minimize p erturbations introduced by interactions between molecules and surfaces. Con focal microscopy is used in combination with sensitive detectors to observe bursts of photons from fluorescently labeled biomolecules as they diffuse through the focal volume. These bursts are analyzed to extract ratiometric observables such as FRET efficiency and polarization anisotropy. We describ e the development of single-molecule FRET methodology and its application t o the observation of the Forster distance dependence and the study of prote in folding and polymer physics problems. Finally, we discuss future advance s in data acquisition and analysis techniques that can provide a more compl ete picture of the accessible molecular information.