Scanning Fluorescence Correlation Spectroscopy: A Tool for Probing Microsecond Dynamics of Surface-Bound Fluorescent Species
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- 作者：Ying Xiao ; Volker Buschmann ; and Kenneth D. Weston
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：January 1, 2005
- 年：2005
- 卷：77
- 期：1
- 页码：36 - 46
- 全文大小：535K
- 年卷期：v.77,no.1(January 1, 2005)
- ISSN：1520-6882
文摘
In this report, a combined imaging and fluorescencecorrelation spectroscopy (FCS) method is described andits ability to characterize microsecond fluctuations in thefluorescence emission of a sample is demonstrated. Asample scanning laser confocal microscope is operatedin the customary way while recording the time that eachphoton is detected with a time resolution of 50 ns usinga low-cost counting board. The serial data stream ofphoton detection times allows access to fluorescencesignal fluctuations that can be used to characterizedynamics using correlation methods. The same datastream is used to generate images of the sample. Usingthe technique, we demonstrate that it is possible tocharacterize the kinetics of transitions to and from nonemitting or "dark" states of the fluorescent dyes DiIC16and ATTO 520. Results are similar to, but deviate slightlyfrom, a model that has been frequently used for extractingsinglet-triplet: conversion rates using conventional solution-based FCS. Like conventional FCS, the concentration,or in our case the areal density of coverage, of fluorescentspecies can also be obtained. Many single-moleculefluorescence experiments aim to extract kinetics fromintensity trajectories; this method may be used as a rapidand convenient technique for characterization of surface-linked or thin-film samples prior to performing the moretime and effort intensive single-molecule measurements.Besides the capacity to measure photophysical phenomena, the surface-sensitive FCS method could also beapplied for measuring conformational changes or interaction kinetics for species immobilized on a surface. Onepossible scenario is measurements of the frequency andduration of association of ligand-receptor pairs where afluorescently labeled component is freely diffusing and theother is surface immobilized. Given that microarrays ofcustom-designed, surface-immobilized peptides and nucleic acids are now readily available, the ability to sensitively measure association and dissociation rates of thesurface-linked species with a freely diffusing species couldbe a useful extension to what has already become anextremely important tool for characterizing the interactions of biomolecules.