Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

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• 作者：Michael J. R. Previte ; Serge Pelet ; Ki Hean Kim ; Christoph Buehler ; Peter T. C. So
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：May 1, 2008
• 年：2008
• 卷：80
• 期：9
• 页码：3277 - 3284
• 全文大小：554K
• 年卷期：v.80,no.9(May 1, 2008)
• ISSN：1520-6882

文摘

Multicolor fluorescence correlation spectroscopy has beenrecently developed to study chemical interactions ofmultiple chemical species labeled with spectrally distinctfluorophores. In the presence of spectral overlap, thereexists a lower detectability limit for reaction products withmulticolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamicproperties such as dissociation constants to be measuredfor chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photoncounting sensitivity to acquire spectral and temporalinformation from multiple chemical species. Further, wehave developed a global fitting analysis algorithm thatsimultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysisapproach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite thepresence of overlapping emission spectra.