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A Brief Introduction to Single-Molecule Fluorescence Methods

  • Siet M. J. L. van den Wildenberg
  • Bram Prevo
  • Erwin J. G. PetermanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1665)

Abstract

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

Key words

Microscopy Confocal fluorescence TIRF Wide-field epi-fluorescence Fluorophore 

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Siet M. J. L. van den Wildenberg
    • 1
    • 2
  • Bram Prevo
    • 1
  • Erwin J. G. Peterman
    • 1
    Email author
  1. 1.LaserLaB and Department of Physics and AstronomyVrije UniversiteitAmsterdamThe Netherlands
  2. 2.Équipe de VolcanologieObservatoire de Physique de GlobeClermant-FerrandFrance

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