Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. |
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Authors: | J?rg Enderlein Ingo Gregor Digambara Patra Thomas Dertinger U Benjamin Kaupp |
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Institution: | Institute for Biological Information Processing 1, Forschungszentrum Jülich, 52425 Jülich, Germany. j.enderlein@fz-juelich.de |
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Abstract: | Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function. |
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Keywords: | confocal microscopy fluorescence spectroscopy laser spectroscopy photophysics single‐molecule spectroscopy |
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