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Optical Microscopy has been applied to life science from its birth and reached widespread application due to its major advantages: limited perturbation of the biological tissue and the easy accessibility of the light sources. However, as the spatial and time resolution requirements and the time stability of the microscopes increase, researchers are struggling against some of its limitations: limited transparency and the refractivity of the living tissue to light and the field perturbations induced by the path in the tissue. We have developed a compact stand-Alone, completely scan-less, optical setup that allows to acquire non-linear excitation images and to measure the sample dynamics simultaneously on an ensemble of arbitrary chosen regions of interests. The image is obtained by shining a square array of spots on the sample obtained by a spatial light modulator and by shifting it (10 ms refresh time) on the sample. The final image is computed from the superposition of (100-1000) images. Filtering procedures can be applied to the raw images of the excitation array before building the image. We discuss results that show how this setup can be used for the correction of wave front aberrations induced by turbid samples (such as living tissues) and for the computation of space-Time cross-correlations in complex networks.

We have previously addressed experimentally blood fluidodynamics in microcapillaries by coupling optical microscopy to pixelated detection. By computing the Cross-Correlation Function (CCF) of signals coming from pixels at a distance along the flow we obtained information on the flow speed and direction. The extension of these experiments to more complex systems with high branching of capillaries and/or inverted flows needs a theoretical investigation that we present here. We focus first on straight capillaries and harmonic flows between a minimum Vmin ≠0 and a maximum Vmax flow speed. The CCF shows multiple peaks at lag times that correspond closely to the maximum and minimum flow speeds. The general analytical expression of the CCF is given, the position of its maxima are discussed by means of geometrical considerations and numerical analysis and an experimental validation are presented. The second case that we study is the flow in the branches of a y-shaped junction in a microcapillary. By simply modeling the branching in laminar flow (low Reynold numbers) and assuming a smooth transition of speeds along the branches we derive a simple numerical model to compute the trajectories of micro-beads. We estimate the flow speed in the branches by computing the CCFs between linear regions of interest set perpendicular to the axes of the branches.

The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.