Abstract
In the presence of strong light scattering, as often encountered in biological tissue, optical microscopy becomes challenging and technical demanding. Beside image quality, the quantitative determination of molecular properties is also strongly affected by scattering. We have carried out fluorescence correlation spectroscopy (FCS) experiments, in a solution of fluorophores, through a sparse scattering layer made of dielectric beads. We observe that the fluorescence signal steadily decreases as the focus is moved away from the scattering layer. By contrast, the estimated number of molecules recovers its normal value beyond a characteristic distance of about twice the bead diameters, below which it is strongly biased. Accompanying theoretical modeling demonstrates how diffraction and refraction by the scattering layer and their impact on FCS measurements depend on size and refractive index of the beads.
Highlights
Fluorescence Correlation Spectroscopy (FCS) is a widely used technique to measure the absolute concentration, dynamics and mobility of molecules in various environments, including complex biological media [1,2,3]. It uses the temporal autocorrelation function (ACF) of the fluorescence signal collected within a small detection volume of a sample with a confocal microscope
A least square fit of the ACF provides parameters such as the number of molecules within the detection volume and the diffusion time in and out of this volume. These quantities are sensitive to the size and shape of this detection volume, quantitatively defined by the Molecular Detection Function (MDF) that can be approximated by the product of the excitation light intensity distribution and the fluorescence collection efficiency [3]
The fluorescence count rate decreases with focus distance D from the scattering substrate and tends towards a non-zero constant at large D
Summary
Fluorescence Correlation Spectroscopy (FCS) is a widely used technique to measure the absolute concentration, dynamics and mobility of molecules (in the pM to μM range) in various environments, including complex biological media [1,2,3]. A least square fit of the ACF provides parameters such as the number of molecules within the detection volume and the diffusion time in and out of this volume These quantities are sensitive to the size and shape of this detection volume, quantitatively defined by the Molecular Detection Function (MDF) that can be approximated by the product of the excitation light intensity distribution and the fluorescence collection efficiency [3]. FCS measurements in a model turbid medium (suspension of polystyrene beads) have been performed by Zustiak et al [6] who have observed a significant decrease in the molecular brightness together with an increase in the molecule number These observations were attributed to a loss of ballistic photons and to an enlargement of the detection volume, caused by the scattering of the excitation light by the turbid sample. The fluorescence emitted by the molecules is affected by scattering, which induces an additional decrease in the molecular brightness and an increase in the molecular number
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