Fluorescence correlation spectroscopy of GFP fusion proteins in living plant cells

M.A. Hink, J.W. Borst, A.J.W.G. Visser

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)

Abstract

This chapter presents an overview of the fluorescence correlation spectroscopy (FCS) of green fluorescent protein (GFP) fusion proteins in living plant cells. FCS is mainly applied to well-defined in vitro systems. However, the possibility of monitoring molecular dynamics under equilibrium conditions at a single-molecule level makes FCS an attractive technique for intracellular studies. The fluorescence photons pass through a pinhole and are detected by a highly sensitive detector. The signal-to-noise ratio achieved by this method is very high, as signal interference from scattered laser light, background fluorescence, and Raman emission can be largely eliminated. The chapter also mentions the applications of fluorescence cross-correlation spectroscopy (FCCS), including enzyme kinetics, nucleotide hybridization, conformational dynamics in DNA, and protein-DNA interactions. From these studies it is clear that FCCS is an attractive technique to observe very specific molecular interactions. At present, it is a challenge to apply this technique to cellular systems to monitor molecular interactions.
Original languageEnglish
Pages (from-to)93-112
JournalMethods in Enzymology
Volume361
DOIs
Publication statusPublished - 2003

Keywords

  • cross-correlation spectroscopy
  • correlation microscopy
  • subcellular-localization
  • single molecules
  • nod factors
  • membranes
  • binding
  • fluctuations
  • diffusion
  • kinetics

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