Influence of the Background in Single Molecule FRET TIRF Microscopy

Abstract

Single-molecule total internal reflection fluorescence (TIRF) microscopy combined with Forster Resonance Energy Transfer (FRET) is a powerful tool that allows direct observation of structural dynamics and heterogeneities without the need for ensemble synchronization. The weak signals in single-molecule TIRF microscopy experiments imply that background determination can have a critical influence on the measured fluorescence intensity.Here we compare and evaluate the performance of different background estimators in single-molecule FRET experiments in samples having different molecular densities. We found that not all background estimators perform well in samples with high molecular densities and propose a new and simple background estimator, called the local statistical percentile (LSP), which is suited for single molecule studies in dense biological samples. This background estimator is implemented as a new toolkit in the single molecule FRET microscopy software package iSMS (1). We illustrate how the LSP background estimator provides consistent single-molecule information over a wide range of molecular densities.(1)S. Preus, S. L. Noer, L. L. Hildebrandt, D. Gudnason, and V. Birkedal. 2015. iSMS: single-molecule FRET microscopy software. Nat Methods 12:593-594.