In 4D-DHM, a modified microscope first records in vivo microcirculation holograms without using any fluorescence marker. Our setup enables single-shot localization of flowing RBCs in 3D through scattering tissue. The three insets highlight the changing in 3D structure of the microcirculation in consequence of a 30⁰ rotation. The method is quantitative and allows assessing physiological values in zebrafish embryos. Dimensional bar: 100 μm.

Blood flow imaging (BFI) techniques that allow to investigate the behaviorof the red blood cells (RBCs) in microcirculation have been widely usedin bio-medical applications. Between various techniques that have been proposed, Doppler scanning [1] and Laser speckle contrast analysis [2] are the conventional and most frequently used imaging techniques to detect RBCs.

Yet these techniques relied on elaborate scanning steps and time-consuming process, other major enhancements were carried out such as exposure time optimization [3], or intensity fluctuation [4] but all of the above techniques lack the structural reconstruction of the blood flow in 3D.

This limits the BFI applications to go further in depth by using 3D information. We have recently developed a 4D Digital holographic microscopy (4D-DHM) technique to image sparse moving objects and validated it for RBCs in a living fish larvae [5, 6, 7].

To understand the zebrafish microcirculation development, it is vital to identify the exact position of RBCs in space. For that, we have carried out several steps to achieve the 4D reconstruction, i.e. 3D identification of the object position (x,y,z) in time, firstly by generating the holograms from frame differences of the movingobjects.

For each sparsely moving object, the scattered field of each one of the two-illumination beams can be reconstructed as an angularly tilted cone of light. Secondly, the exact location of the object is given from the intersection of the two cones. Once the position is identified, the contribution of this object is removed from the hologram with the help of a cleaning algorithm; thus, the position of the next object is calculated from the new hologram by iterating the same procedure.

The algorithm calculates first the position of the strongest signals so to prevent mixing between the signals from different objects. Our techniques tackles the issue of reconstructing the moving RBCs of the sample in 3D through bidirectional illumination in digital holography.

This technique can provide biologists with a more accurate 4D reconstruction of the sample. We believe that our work gives significant advances in identifying the efficacy of moving RBCs in 3D live imaging.

Researchers
Dario Donnarumma, Alexey Brodoline, Nitin Rawat, Daniel Alexandre and Michel Gross
Laboratorie Charles Coulomb UMR5221CNRS-UM, Place Eugène Bataillon 34095 Montpellier, France

References

1. Y. Yeh et al. Appl. Phys. Lett. 4, 178 (1964)
2. J. D. Briers et al. J. Biomed. Opt. 1, 179 (1996).
3. S. Yuan et al. Appl. Opt. 44, 1830 (2005).
4. Y. Zeng et al.Opt. Lett. 38, 1315(2013).
5. D. Donnarumma et al. Opt. Exp. 24, 26900 (2016). 
6. D. Donnarumma et al. MRT 81,2 153-161 (2018)
7. D.Donnarumma et al. MRT 81.12 1361-1365 (2018)

The answer to the ultimate question to life the universe and everything... is love!

The first France quidditch cup has been an amazing sport event organized by the FQF (France Quidditch Federation). I have the chance to write my impressions about the two days event on an international online journal, the Quidditch Post. So if you want to know more about quidditch, or if you want to be updated about quidditch events all around the world, follow the link.

A special thanks to Andy Mermer that reviewed my article.