Daniel Alexandre

Laser Doppler holographic microscopy in transmission: application to fish embryo imaging

We have extended Laser Doppler holographic microscopy to transmission geometry. The technique is validated with living fish embryos imaged by a modified upright bio-microcope. By varying the frequency of the holographic reference beam, and the combination of frames used to calculate the hologram, multimodal imaging has been performed. Doppler images of the blood vessels for different Doppler shifts, images where the flow direction is coded in RGB colors or movies showing blood cells individual motion have been obtained as well. The ability to select the Fourier space zone that is used to calculate the signal, makes the method quantitative.

4D holographic microscopy of zebrafish larvae microcirculation.

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.

Blood flow imaging in zebrafish by laser doppler digital holography

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.

Improved 3D Imaging of Zebrafish Larvae Microcirculation by Digital Holography

A microscopic technique based on digital holography is proposed to investigate blood microcirculation and vascular development in model organisms such as zebrafish larvae. Recent achievements in 3D imaging of blood flow in vessels are presented.

4D digital holographic microscopy

4D Digital Holographic Microscopy (4D-DHM) is a new technique capable of performing 3D imaging of moving objects. 3D reconstruction is made from a single hologram, but is restricted to sparse objects that occupy only small part of the volume. For a sequence of holograms, 4D-DHM therefore performs 4D (x; y; z and t) imaging. 4D-DHM is well suited to image micro circulation, and has been validated imaging red blood cells circulating in a zebrafish embryo in vivo [1-3].