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7 April 2020 Fluorescence speckle image correlation spectroscopy (Conference Presentation)
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Abstract
Fluorescence correlation spectroscopy is used extensively for quantitative characterization of biomolecules at very low concentration. However, light aberration and scattering from the tissues are two major factors that affect the results strongly. Although adaptive optics arrangement can correct the aberrations of light to some extent, it fails completely to eliminate the light scattering effect. Recently, exploiting the fact that autocorrelation of a speckle pattern is a sharply peaked point spread function and the optical memory effect, non-invasive imaging of fluorescent sample through a scattering medium has been possible. However, it is also very challenging to measure the dynamic properties of the fluorescent molecules or particles through a scattering layer due to poor signal to noise ratio. In this study, we employ a modality based on speckle cross-correlation enabled via optical memory effect to study two dimensional (2D) diffusion of fluorescent particles hidden behind a scattering film. We realized a 2D diffusing model system by confining fluorescent polystyrene beads of 1µm diameter at the water/air interface behind a TiO2 diffuser. The experimental set up was built up in an epifluorescence configuration. The fluorescent beads were excited by an illumination speckle generated by the incident light in a plane-wave geometry while passing through the disordered TiO2 film. Similarly, the emitted fluorescent signal also traversed through the same TiO2 film to generate the detection speckle, which was eventually recorded by a high frame rate CMOS camera. The experimental set up has also been modelled numerically, where speckle pattern has been generated by a spherical wave, transmitted through a scattering object in an optical microscope. Moreover, the dependence of the speckle size on the numerical aperture, magnification, and the distance of the focal plane from the bead plane has also been studied. The numerical results have been compared with the experimental values to estimate the speckle size. Furthermore, we have evaluated the 2D diffusion constant by monitoring the widening of the 2D speckle cross-correlation function versus lag time. This result has been compared with that obtained with the single particle tracking method without the scattering layer. Quantitative agreement between the results obtained by the speckle cross-correlations and the single particle tracking technique without the diffuser establishes the potential application of this technique in correlation spectroscopy. Superimposed multiple beads speckle patterns were also studied and the results will be presented in the conference.
Conference Presentation
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Anirban Sarkar, Irène Wang, Aditya Katti, Jörg Enderlein, Jacques Derouard, and Antoine Delon "Fluorescence speckle image correlation spectroscopy (Conference Presentation)", Proc. SPIE 11351, Unconventional Optical Imaging II, 1135108 (7 April 2020); https://doi.org/10.1117/12.2558129
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