No abstract

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair.