Defining metrics of visual acuity from theoretical models of observers.

Many experimental studies show that metrics of visual image quality can predict changes in visual acuity due to optical aberrations. Here we use statistical decision theory and Fourier optics formalism to demonstrate that two metrics known in the field of vision sciences are approximations of two different theoretical models of linear observers. The theory defines metrics of visual acuity to potentially predict changes in visual acuity due to optical aberrations, without needing a posteriori scale or offset. We illustrate our approach with experiments, using combinations of defocus and spherical aberration, and pure coma.

The random walk of accommodation fluctuations.

The focusing distance of the eye fluctuates during accommodation. However, the visual role of these accommodation fluctuations is not yet fully understood. The fluctuation complexity is one of the obstacles to this long standing challenge in visual science. In this work we seek to develop a statistical approach that i) accurately describes experimental measurements and ii) directly generates randomized and realistic simulations of accommodation fluctuations for use in future experiments. To do so we use the random walk approach, which is usually appropriate to describe the dynamics of systems that combine both randomness and memory.

Intracellular dynamics measurements with full field optical coherence tomography suggest hindering effect of actomyosin contractility on organelle transport.

Intracellular motion can be quantitatively monitored in tissues using coherence-gated microscopic techniques. With full-field optical coherence tomography (FFOCT), the use of high numerical aperture microscope objectives provides a high resolution mapping of intracellular dynamics that are probed with subwavelength sensitivity. In the upper temporal bandwidth that we have used (1-6 Hz) the main contribution to the dynamic signal arises from the overall dynamical, optically heterogeneous cytoplasm. We propose a method to specifically study the impact of actomyosin contractility on the intracellular dynamic signal by performing high throughput, comparative measurements of multicellular aggregates with and without blebbistatin action, a selective inhibitor of class-II myosins that disrupts actomyosin contractile activity. Our results indicate a significant increase in the fraction of the signal that decorrelates within 1 second after inhibition of contractility. This observation mitigates the anticipated importance of actomyosin contractile forces to directly move organelles, but highlights their role in hindering organelle transport via their stiffening effect of the viscoelastic cytoplasm.

Fluorescent correlation spectroscopy measurements with adaptive optics in the intercellular space of spheroids.

In this study we demonstrate the use of adaptive optics to correct the biasing effects of optical aberrations when measuring the dynamics of molecules diffusing between cells in multicellular spheroids. Our results indicate that, on average, adaptive optics leads to a reduction of the 3D size of the point spread function that is statistically significant in terms of measured number of molecules and diffusion time. The sensorless approach, which uses the molecular brightness as optimization metric, is validated in a complex, highly heterogeneous, biological environment. This work paves the way towards the design of accurate diffusion measurements of molecules in thick biological specimens.

Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope.

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues but also when performing FFM measurements through a single cellular layer. This work paves the way for the application of FFM to complex three-dimensional multicellular samples.

Adaptive optics for fluorescence correlation spectroscopy.

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 µm into the solution. We also demonstrate, both theoretically and experimentally, that the molecular brightness scales as the Strehl ratio squared.