We present a meso Oblique plane microscope. A microprism below the sample tilts the sheet, improving resolution and sectioning with a low magnification objectives. We analyzed blood flow in zebrafish embryos at 12Hz.

Here is a schematic of the principle: A single objective SPIM (soSPIM), which uses a micromirror cavity to reflect a sheet by 90 degrees (A). We adapt the soSPIM idea to an open top, OPM geometry, using total internal reflection in a micro-prism (C).

The system uses a “lens-less” scanning unit, and a transmission grating to “bend” the fluorescence light to the tertiary objective (diffractive OPM, @benjulab ). This creates a compact system, with ~2.3 microns spatial resolution and 3.7x1.5x1mm^3 volumetric coverage .

Here is the mandatory bead parade (500nm fluorescent nanospheres in Agarose). First one from a single volume, second one where six volumes were tiled in the third dimension, giving an imaging depth of 1mm with ~9 microns axial resolution.

We used the system to image the vasculature in Zebrafish embryos. The microscope resolved nuclei of endothelial cells (bright, localized spots) and the intricate branching patterns of zebrafish vasculature, including parachordal lymphiangioblasts.

Lastly we used optical flow implemented in OpenCV to estimate the direction and magnitude of the blood flow across the zebrafish vasculature. This also highlighted the need for rapid acquisition rates, here 12Hz.

The Olympus objectives are compensated to image into a 5mm column of water. With @TonmoyC59869398 we previously found that one can put a ~3.5mm glass plate in front of the objectives to compensate spherical aberrations when not imaging that deep. The prism is that plate.

The overall spherical aberrations in the system are balanced with an optical flat in front of O2, and we’re verified by imaging beads under widefield illumination (axial symmetry of blur rings).

Using the superposition principle, one can also leave the plate for O2 away and use a thicker prism for more scan range (2.5mm). Here is the glass prism. Light gets bend by total internal reflection.

Another fantastic work from your team!

*…with low magnification objectives…* Apologies for sloppy tweeting (“poasting”), it was early in the morning when the preprint got through.