4D holographic microscopy of zebrafish larvae microcirculation

Abstract

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.

4D compressive sensing holographic microscopy imaging of small moving objects

“…The 3D calculation converge after 160 iterations (in about 2 minutes) using GPU (NVidia GTX Titan Xp) and CUDA libraries (cuFFT, cuBlas...). This is much faster than in [10], where convergence on a 512×512×128 voxels grid needed about 25000 iterations. Indeed, at each iteration, both methods perform roughly the same amount of Fourier calculations (2 × 128 or 2 × 160 FTs), whereas only one voxel is extracted in [10], and about 500 voxels are extracted here.…”

“…This is much faster than in [10], where convergence on a 512×512×128 voxels grid needed about 25000 iterations. Indeed, at each iteration, both methods perform roughly the same amount of Fourier calculations (2 × 128 or 2 × 160 FTs), whereas only one voxel is extracted in [10], and about 500 voxels are extracted here. This ∼ 500× improvement of the calculation speed is made possible by the orthogonal nature of the CS method.…”

“…x, y, z), the 3D scattering density of the moving objects is often sparse. This situation corresponds for example to the case of red blood cells (RBCs) circulating in a zebrash (Danio rerio) larva, imaged by digital holographic microscopy (DHM) [10]. By performing CS reconstruction from a single hologram, we obtained an instantaneous 3D image of the objects (the RBCs).…”

“…Unpigmented larvae were obtained from spontaneous spawning of casper young adult sh pairs and grown at 28.5 • C. They were anesthetized with tricaine at 5 days and mounted in lateral view in 1 % low melting point agarose in a 35 mm glass ibidi R bottom petri dish and the trunk blood ow was imaged immediately. The experimental setup was similar to the one of previous work [10]. To increase the angular diversity of illumination, the sample was imaged with a higher NA microscope objective (20x/NA=0.5 water immersion Zeiss microscope objective), and with three illumination beams (in place of NA=0.3 and 2 illuminations in [10]).…”

“…The experimental setup was similar to the one of previous work [10]. To increase the angular diversity of illumination, the sample was imaged with a higher NA microscope objective (20x/NA=0.5 water immersion Zeiss microscope objective), and with three illumination beams (in place of NA=0.3 and 2 illuminations in [10]). The holographic signal was acquired by an Andor Zyla sC-MOS camera (2560 × 2160 pixels, 12 Bits, 50 frames/s), and the frames I m recorded by the camera were stored on a 2560 × 2560 numerical grid.…”