Imaging with total internal reflection fluorescence microscopy for the cell biologist

Alexa L. Mattheyses, Sanford M. Simon, Joshua Z. Rappoport

Research output: Contribution to journalComment/debate

192 Scopus citations

Abstract

Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ∼100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

Original languageEnglish (US)
Pages (from-to)3621-3628
Number of pages8
JournalJournal of cell science
Volume123
Issue number21
DOIs
StatePublished - Nov 1 2010

Keywords

  • Evanescent field microscopy
  • Evanescent wave microscopy
  • Fluorescence
  • Total internal reflection fluorescence microscopy

ASJC Scopus subject areas

  • Cell Biology

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