Adaptive Optics for Fluorescence Correlation Spectroscopy

TL;DR

This paper demonstrates how adaptive optics can correct optical aberrations in fluorescence correlation spectroscopy, significantly improving measurement accuracy of molecular parameters in deep samples.

Contribution

It introduces a simple adaptive optics system using a deformable mirror to correct aberrations in FCS, reducing depth-dependent biases in parameter estimation.

Findings

Aberration correction stabilizes FCS parameters up to several tens of micrometers deep.

Molecular brightness scales with the Strehl ratio squared.

Optical aberrations cause significant depth-dependent bias in FCS measurements.

Abstract

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. In this paper, we quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics system for aberration correction. Optical aberrations are gradually introduced by focussing the excitation laser beam at increasing depths in fluorescent solutions with various refractive indices, which leads to drastic depth-dependent bias in the estimated FCS parameters. Aberration correction with a Deformable Mirror stabilizes these parameters within a range of several tens of \mum into the solution. We also demonstrate, both theoretically and experimentally, that the molecular brightness scales as the Strehl ratio squared.