A brief introduction to single-molecule fluorescence methods

Siet M.J.L. van den Wildenberg, Bram Prevo, Erwin J.G. Peterman*

*Corresponding author for this work

Research output: Chapter in Book / Report / Conference proceedingChapterAcademic


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.

Original languageEnglish
Title of host publicationSingle Molecule Analysis
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc
Number of pages21
ISBN (Electronic)9781493972715
Publication statusPublished - 2018

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Confocal fluorescence
  • Fluorophore
  • Microscopy
  • TIRF
  • Wide-field epi-fluorescence


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