CC BY
25B-I-14
The Fastest High-Resolution 3D Imaging of Sperm Cells
during Free Swim
Natan T. Shaked
Department of Biomedical Engineering, Tel Aviv University, Israel.
Email: nshaked@tau.ac.il. Website: www.eng.tau.ac.il/~omni
I review our latest advances [1], presenting a new approach for dynamic three-dimensional (3D) sperm imaging without using cell staining. The selection of sperm cells possessing normal morphology and motility is crucial for intracytoplasmic sperm injection, a very common type of in vitro fertilization (IVF), in which a clinician chooses a single sperm cell using a micropipette and injects it into the female egg in a dish. Since stains cannot be used in human IVF, as they may damage sperm viability, clinicians examine sperm cells using imaging methods that only provide gross two-dimensional morphology, leading to a lack of consistency in sperm selection. Without staining, sperm cells are nearly transparent under bright-field microscopy. A contrast mechanism that can be used when imaging cells without staining is their refractive index, which can be recorded by tomographic phase microscopy in three dimensions. For tomography, the cell needs to be recorded from various angles. There are two approaches for obtaining these angular projections: rotating the entire sample and scanning the illumination. However, none of these approaches are fast enough for recording sperm cells during free swim. Even when using sperm staining in confocal fluorescence microscopy (which is not allowed in human IVF), it is very challenging to obtain the entire sperm head and tail 3D reconstruction during rapid free swim, due to the need to scan over time and the low amount of fluorescent emission in each temporal frame. In Ref. [1], we presented the first high-resolution tomographic phase microscopy method for acquisition of the entire sperm cell (head with organelles and tail) during free swim and without cell staining, in 2000 frames per second and spatial resolution of 0.5 micron. This method is based on the natural repetitive rotation of the sperm head during free swim, which gives access to its angular projections, and a novel set of reconstruction algorithms. By modeling the sperm head as an ellipsoid, we are able to predict the viewing angle based on the minor and major radii extracted from the segmented quantitative phase maps of the head. All off-axis holograms, experimentally acquired during the sperm free swim, are then processed into the complex-wavefront projections, which are positioned in the 3D Fourier spectrum, allowing reconstruction of the 3D refractive-index distribution of the sperm head through the optical diffraction tomography theory. The tail location in 3D is reconstructed from each frame, taking into consideration the fact that using off-axis holography we acquire the full complex wavefront, so that we can digitally refocus to various z planes for sparse objects like the sperm tail. See results in Fig. 1. The method has a great potential for revealing the mechanism that connects sperm movement, morphology and fertilization potential, thus increasing IVF success rates.
(a)
□ 1.355 SRI < 1.37
u 1.37 S RI <1.425
1.425 S RI < 1.465
■ RI > 1.465
Flagellum
Nucleus Acrosomal
Midpiece I
f "
Centriole region
(b)
— Cell membrane
Fig. 1. High-resolution dynamic 3D imaging of a sperm cell swimming freely. (a) A single frame from the 3D motion in Movie S3 in Ref. [1], revealing the internal structure of the sperm cell. RI, Refractive index. (b) Overlay of four frames from the 3D motion in Movie S3 in Ref. [1].
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[1] G. Dardikman-Yoffe, S. K. Mirsky, I. Barnea, N. T. Shaked, High-resolution 4-D acquisition of freely swimming human sperm cells without staining, Science Advances, vol. 6, no. 15, eaay7619 (2020).
