Single-Molecule Microscopy in the Life Sciences

M. Axmann, J. Madl, G.J. Schütz

Research output: Chapter in Book/Report/Conference proceedingsChapterpeer-review

2 Citations (Scopus)


This chapter provides some of the essential considerations when building a single-molecule microscope. Next, the key parameters accessible from a single-molecule measurement are discussed: position, orientation, color, and brightness. The chapter begins with an overview of the specific benefits a single-molecule approach provides. There are multiple parameters that may be used to characterize a dye molecule i: for example, the time-dependent position, the orientation of its excitation or emission dipole, its absorption and emission spectra, or its brightness. The recorded single-molecule parameters can be further processed to obtain insights into the biological sample. Based on real-life scenarios, the strengths and potential pitfalls of single-molecule approaches are explained. The transitions between two conformational states of a protein detected via the concomitant change in the Forster resonance energy transfer (FRET) efficiency EFRET, from which the lifetimes of the states can be directly monitored. Finally, the chapter puts these parameters in the context of biological experiments.

Original languageEnglish
Title of host publicationFluorescence Microscopy
Subtitle of host publicationFrom Principles to Biological Applications: Second Edition
Number of pages40
ISBN (Electronic)9783527687732
Publication statusPublished - 5 Apr 2017


  • Diffraction limit
  • Dipole orientation
  • Fluorescence brightness
  • Forster resonance energy transfer efficiency
  • Molecule's subpixel position
  • Multicolor imaging
  • Optical resolution limit
  • Single-molecule measurement
  • Single-molecule microscopy


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