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- W4246100876 abstract "Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time, etc.) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume, closely related to the size of the confocal Point Spread Function (PSF). However the measurement parameters scale with the observation volume, which makes it difficult to compare measurements obtained with different samples (solutions, cells, tissues, etc.) and in different environments (temperature, substrates). Preliminary calibrations of the observation volume are usually performed with fluorophore solutions, with well known diffusion constant and concentration. However, since optical aberrations depend upon optical alignments, refractive index of the solvent, observation depth, etc., these calibrations are unusable if not performed under the exact same conditions of the experiment of interest. Optical aberrations can induce small deformations of the confocal PSF, to which FCS measurements are much more sensitive than conventional confocal imaging. We quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics (AO) system for aberration corrections. Optical aberrations are gradually introduced by focussing the excitation laser beam at increasing depths in fluorescent solutions with various refractive index mismatches, which lead to a drastic depth-dependent bias in the estimated FCS parameters. Aberration corrections with a Deformable Mirror (DM) stabilizes these parameters within a range of several tens of μm above the cover slide - solution interface. In the near future, such an AO system should improve significantly the robustness of FCS measurements in environments of various optical properties (crowded solutions, cellular media, tissues, etc.)." @default.
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- W4246100876 date "2012-01-01" @default.
- W4246100876 modified "2023-10-18" @default.
- W4246100876 title "Adaptive Optics for Fluorescence Correlation Spectroscopy" @default.
- W4246100876 doi "https://doi.org/10.1016/j.bpj.2011.11.2297" @default.
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