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.

We show that compressive sensing (CS) calculations are very ecient to reconstruct in 3D sparse objects whose 2D hologram has been recorded by digital holographic microscopy. The method is well adapted to image small scattering objects moving within a larger motionless object. This situation corresponds to red blood cells (RBCs) circulating in the vascular system of a zebrash (Danio rerio) larva. RBCs positions are imaged in 3D from a single hologram, while the RBCs trajectories, i.e. the perfused blood vessels, are imaged from a sequence of holograms. With respect to previous work (Donnarumma et al., Opt. express, 24, 26887, 2016), we get a gain of ∼ 500 in calculation speed.

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. 81:153-161, 2018. © 2016 Wiley Periodicals, Inc.