Authors: Lamb, D. C.; Muller, B. K.; Brauchle, C.

Source: Current Pharmaceutical Biotechnology, Volume 6, Number 5, October 2005 , pp. 405-414(10)

Publisher: Bentham Science Publishers

Abstract:

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Keywords: fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; time correlated single photon counting; pulsed interleaved excitation; time-resolved fluorescence correlation spectroscop; alternating laser excitation

Document Type: Review article

DOI: http://dx.doi.org/10.2174/138920105774370625

Affiliations: 1: Physical Chemistry, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 11 Haus E, 81377 Munich, Germany.

Publication date: 2005-10-01

More about this publication?

• Current Pharmaceutical Biotechnology aims to cover all the latest and outstanding developments in Pharmaceutical Biotechnology. Each issue of the journal contains a series of timely in-depth reviews written by leaders in the field covering a range of current topics in both pre-clinical and clinical areas of Pharmaceutical Biotechnology. Current Pharmaceutical Biotechnology is an essential journal for academic, clinical, government and pharmaceutical scientists who wish to be kept informed and up-to-date with the latest and most important developments.

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