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Proceedings Paper

Dual-color two-photon fluorescence correlation spectroscopy
Author(s): Keith M. Berland
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Paper Abstract

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.

Paper Details

Date Published: 24 April 2001
PDF: 11 pages
Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); doi: 10.1117/12.424564
Show Author Affiliations
Keith M. Berland, Emory Univ. (United States)

Published in SPIE Proceedings Vol. 4262:
Multiphoton Microscopy in the Biomedical Sciences
Ammasi Periasamy; Peter T. C. So, Editor(s)

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