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

Part of the Methods in Molecular Biology book series (MIMB, volume 749)


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.

Key words

Single pair fluorescence resonance energy transfer Total internal reflection fluorescence microscope Single molecule fluorescence Alternating laser excitation 



This work was supported by RO1 GM077872 (S.H.L.) from the National Institutes of Health.


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Departments of Cell Biology and Physiology and Bioengineering, University of Pittsburgh School of Medicine and Swanson School of Engineering, Petersen Institute of NanoScience and EngineeringUniversity of Pittsburgh Cancer InstitutePittsburghUSA

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