Optimal Practices for Surface-Tethered Single Molecule Total Internal Reflection Fluorescence Resonance Energy Transfer Analysis

Single molecule fluorescence microscopy can be used to follow the mechanics of molecular biology processes in real time. However, many factors, from flow cell preparation to improper data analysis can negatively impact single molecule fluorescence resonance energy transfer (smFRET) experiments. Here, we describe some best practices for ensuring that smFRET data are of the highest quality. In addition to instrumentation, we describe sample preparation and data analysis.