Pushing Camera-Based Single-Molecule Kinetic Measurements to the Frame Acquisition Limit with Stroboscopic smFRET

Single-molecule fluorescence resonance energy transfer (smFRET) experiments permit detailed examination of microscopic dynamics. However, kinetic rate constants determined by smFRET are susceptible to systematic underestimation when the rate constants are comparable to the data acquisition rate. We demonstrate how such systematic errors in camera-based total internal reflection fluorescence microscopy experiments can be greatly reduced by using stroboscopic illumination/detection, allowing accurate rate constant determination up to the data sampling rate and yielding an order of magnitude increase in the dynamic range. Implementation of these stroboscopic smFRET ideas is straightforward, and the stroboscopically obtained data are compatible with multiple trajectory analysis methods, including dwell-time analysis and hidden Markov modeling. Such stroboscopic methods therefore offer a remarkably simple yet valuable addition to the smFRET toolkit, requiring only relatively modest modification to the normal data collection and analysis procedures.