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Superresolution image reconstruction method based on the structured illumination microscopy (SIM) principle with reduced and simplified pattern set is presented. The method described needs only 2 sinusoidal patterns shifted by half a period for each spatial direction of reconstruction, instead of the minimum of 3 for the previously known methods. The method is based on estimating redundant frequency components in the acquired set of modulated images. Digital processing is based on linear operations. When applied to several spatial orientations, the image set can be further reduced to a single pattern for each spatial orientation, complemented by a single non-modulated image for all the orientations. By utilizing this method for the case of two spatial orientations, the total input image set is reduced up to 3 images, providing up to 2-fold improvement in data acquisition time compared to the conventional 3-pattern SIM method. Using the simplified pattern design, the field of view can be doubled with the same number of spatial light modulator raster elements, resulting in a total 4-fold increase in the space-time product. The method requires precise knowledge of the optical transfer function (OTF). The key limitation is the thickness of object layer that scatters or emits light, which requires to be sufficiently small relatively to the lens depth of field. Numerical simulations and experimental results are presented. Experimental results are obtained on the SIM setup with the spatial light modulator based on the 1920x1080 digital micromirror device.

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 experime ntally blood fluidodynamics in microcapilla ries 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 V min z 0 and a maximum V max 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. Keywords : Fluorescence Correlation; velocimetry; microfluidics; fluorescence microscopy

The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones. © 2013 SPIE.