An holographic microscopy setup using two illumination beams is proposed to image blood microcirculation in preclinical models. Greedy algorithm reconstruction of the two resulting holograms leads to a 3D reconstruction of the sample over time.

A holographic microscopy algorithm is proposed enabling to deal with high numerical aperture holograms. Hologram reconstruction kernel performs under afocal conditions , which allows keeping the full NA over wide defocus range.

Microvessel blood flow imaging techniques are widely used in biomedical research and clinical diagnostics where many diseases have a vascular etiology or involvement. For testing purposes, zebrafish embryo provides an ideal animal model to achieve high-resolution imaging of superficial and deeply localized vessels. Moreover, the study of the formation of a closed circulatory system in vertebrates is a topic of recent interest in biophysics. However, most of the existing techniques are invasive due to the use of a contrast agent for imaging purposes. Recent developments in Digital Holography and Laser Doppler Holography techniques can be considered to alleviate this issue. Laser Doppler holography and transmission microscopy can be coupled to analyze blood flow in fish embryos by adapting a laser Doppler holographic setup to a standard bio-microscope: the two beams of the holographic interferometer (illumination of the object and reference), whose frequency offset is controlled, were addressed to the microscope by optical fibers. Multimodal acquisition and analysis of the data is made by acting on the frequency offset of the two beams, and on the location of the Fourier space filtered zone. In this work, we show that it is possible to select the signal of moving scatterers, and to image Red Blood Cells (RBCs) and blood vessels. Individual RBCs are imaged, and movies showing the RBC motion are obtained. Microsc. Res. Tech., 2016. © 2016 Wiley Periodicals, Inc.

An original technique that combines digital holography, dual illumination of the sample and cleaning algorithm 3D reconstruction is proposed. It uses a standard transmission microscopy setup coupled with a digital holography detection. The technique is 4D, since it allows to determine, at each time step, the 3D locations (x, y, z) of many moving objects that scatter the dual illumination beam. The technique has been validated by imaging the microcirculation of blood in a fish larvae sample (the moving objects are thus red blood cells RBCs). Videos showing in 4D the moving RBCs superimposed with the perfused blood vessels are obtained.

An holographic laser Doppler scheme using two illumination beams is proposed to image blood vessel in fish embryo. The coincidence of the reconstructed images made for each illumination orientation leads to an accurate z sectioning.

Le couplage d'une méthode d'imagerie laser Doppler par holographie hétérodyne à un microscope commercial est proposé. Cette méthode permet d'obtenir, sans étape de calibration préalable, des images Doppler résolues à la fois en norme des vecteurs vitesses qu'en direction, ainsi que des contrastes d'amplitude de la chair ou de la vascularisation du zebrafish.

A laser Doppler imaging scheme combined to an upright microscope is proposed. Quantitative Doppler imaging in both velocity norm and direction, as well as amplitude contrast of either zebrafish flesh or vasculature is demonstrated.