Open AccessConfocal fluorescence correlation spectroscopy through a sparse layer of scattering objects
Author(s) -
Anirban Sarkar,
Joe Gallagher,
Irène Wang,
Giovanni Cappello,
Jörg Enderlein,
Antoine Delon,
Jacques Derouard
Publication year - 2019
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.27.019382
Subject(s) - scattering , optics , fluorescence correlation spectroscopy , refractive index , light scattering , materials science , spectroscopy , fluorescence spectroscopy , fluorescence , microscopy , diffraction , confocal microscopy , forward scatter , physics , quantum mechanics
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.