Most of our knowledge about how the body functions comes from studies carried out on humans or medium to large animals. However, more than 90% of animal biomass is made up of animals weighing less than one gram, whose physiology remains largely unknown, as is that of the embryonic and larval forms of large animals. The difficulty in studying major physiological functions in small animals comes from the limitations of our measuring instruments. To gain insight into the cardiovascular physiology of fish embryos we use nanoindentation and spinning-disk confocal microscopy to obtain quantitative measurements of volume and force variations in zebrafish embryos and larvae. Nanoindentation is a technique designed to probe the elastic modulus of soft materials by pressing a spherical glass probe (6 to 200 µm in diameter) into the surface of the material. The device measures the indentation of the sphere and the compressive force exerted via the deflection of a rod of known stiffness. This equipment can reproducibly indent a wide range of materials with moduli ranging from 1 Pa to 10 GPa and measure forces ranging from 20 pN to 2 mN. We used a nanoindenter coupled with microscopy (Fig. 1) to measure the mechanical activity of the heart of a three-day-old zebrafish embryo. Figure 2 shows what we believe to be the mechanocardiogram of the smallest animal (3.5 mm, approximately 1 mg) ever recorded to date. We were also able to measure periodic pressure variations on a superficial capillary with a diameter smaller than that of a red blood cell.

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.