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.

In previous work [Opt. Lett. 44, 2827 (2019)], 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.

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.

A holographic microscopy method using several illuminations beams with a NA=0.5 aperture objective is proposed to image blood microcirculation in zebrafish larvae. Recent achievements in 3D imaging of blood flow are presented.

Une configuration de microscopie holographique qui utilise trois faisceaux d'illumination est proposée. Elle permet d'imager en 3D la positions des globules rouges en mouvement à partir d'un hologramme, ainsi que la structure 3D des vaisseaux sanguins perfusé à partir d'une séquence d'hologrammes.

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.

From the session Tomography and Computational Imaging (JSII_1) (invited) 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]. Fig. 1 (a) Holographic setup. (b, c and d) Images of blood flow within a zebrafish embryo: blood vessel (purple) and RBCs (green). The displayed 2D images correspond to the projections along z of the 3D images rotated along the y direction by-30° (b), 0° (c) and +30° (d). Bar is 50 m, The principle of the 4D DHM is illustrated by Fig. 1 (a). The setup is an upright microscope, which has been modified to perform Digital Holographic Microscopy (DHM) in transmission. The white light illumination is replaced by a laser, which is split in two beams of different directions. To perform holographic detection a reference arm (ELO field) is added on the detection side. The field E scattered by the object interferes with ELO , and a camera records sequences of frames. These frames are used to calculate the holograms, and to reconstruct in 3D the field scattered by the sample. By a proper analysis of this information, 3D images of the blood vessels, and 4D images of the moving red blood cells (RBCs) can be obtained see Fig,1 (b) to (d). To get these images, we have proceeded by the following way [2,3]. First, we calculate the holograms which differences of frames, and select by the way the signal from the moving components of the object (i.e.) the RBCs signal. We have then extract from the data, the holograms corresponding to the two illumination. For each RBC, and each illumination, the field that can be reconstructed by holography as a narrow cone of light (narrow because RCBs are much larger than the wavelength), which is angularly tilted like the illumination. The location of RBCs is then obtained by considering the intersection of the two cones. To prevent the signals of the different RBCs to mix together, the positions of the RBCs that give the largest signals are calculated first. The contributions of these RBCs are then removed from the hologram, and the positions of the following RBCs are calculated from the hologram thus cleaned. The reconstruction by cleaning is highly non-linear (unlike conventional holographic reconstructions), since it is the algorithm that decides to place a RBC in a plane and to remove the field diffused by this RBC from the other planes. In order to converge successively it requires to image sparses objects, that occupy only small part of the holographic reconstructed volume. 4D-DHM is thus capable of performing 3D reconstruction is made from a single hologram, but is restricted to sparse objects that occupy only small part of the volume.