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DNA Nanotechnology

Volume 749 of the series Methods in Molecular Biology pp 273-289

Date:

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

  • Matt V. Fagerburg
  • , Sanford H. LeubaAffiliated withDepartments of Cell Biology and Physiology and Bioengineering, University of Pittsburgh School of Medicine and Swanson School of Engineering, Petersen Institute of NanoScience and Engineering, University of Pittsburgh Cancer Institute Email author 

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Abstract

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