Fast and pure phase-shifting off-axis holographic microscopy with a digital micromirror device.

We present a phase-shifting digital holographic microscopy technique, where a digital micromirror device enables to perform a precise phase-only shift of the reference wave. By coupling the beam into a monomode fiber, we obtain a laser mode with a constant phase shift, equally acting on all pixels of the hologram. This method has the advantage of being relatively simple and compatible with high frame rate cameras, which makes it of great interest for the observation of fast phenomena. We demonstrate the validity of the technique in an off-axis configuration by imaging living paramecia caudata.

4D compressive sensing holographic imaging of small moving objects with multiple illuminations.

In previous work [Opt. Lett.44, 2827 (2019)OPLEDP0146-959210.1364/OL.44.002827], we presented a method based on digital holography and orthogonal matching pursuit, which is able to determine the 3D positions of small objects moving within a larger motionless object. Indeed, if the scattering density is sparse in direct 3D space, compressive sensing algorithms can be used. The method was validated by imaging red blood cell trajectories in the trunk vascular system of a zebrafish (Danio rerio) larva. We give here further details on the reconstruction technique and present a more robust version of the algorithm based on multiple illuminations.

High-speed quantitative 3D imaging by dual-illumination holographic microscopy.

A new blood flow imaging (BFI) technique using digital holography with double illumination of the sample is proposed. We imaged the moving red blood cells (RBCs) using a two microscope objective lenses setup. The setup consists in a larger angle of separation (90 °) between the two illumination beams, allowing a wider angular rotation at good z resolution. Moreover, the setup geometry allows an easier displacement of the sample in all directions. Results show that this technique is able to perform phase-shifting reconstruction for the two beams at the same time which is more suitable for the future implementation of live 3D holography. Experimental results are carried out for the verification of the effectiveness of the proposed technique on a zebrafish larvae sample. RESEARCH HIGHLIGHTS: Blood flow imaging techniques are often invasive and image analysis is time consuming. To alleviate this issue an imaging technique based on dual illumination in holographic domain is proposed. This method has been validated on zebrafish embryos.

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. © 2016 Wiley Periodicals, Inc.

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.