Selective plane illumination microscope dedicated to volumetric imaging in microfluidic chambers

Abstract

In this article, we are presenting an original selective plane illumination fluorescence microscope dedicated to image "Organ-on-chip"-like biostructures in microfluidic chips. In order to be able to morphologically analyze volumetric samples in development at the cellular scale inside microfluidic chambers, the setup presents a compromise between relatively large field of view (∼ 200 µm) and moderate resolution (∼ 5 µm). The microscope is based on a simple design, built around the chip and its microfluidic environment to allow 3D imaging inside the chip. In particular, the sample remains horizontally avoiding to disturb the fluidics phenomena. The experimental setup, its optical characterization and the first volumetric images are reported.

Fig. 1.

Experimental set-up with L: laser, M: Mirror, DL: Diverging Lens, CL: Converging Lens, Cyl.L: Cylindrical Lens, MC: Microfluidic Chip, S: (manual & computed-controlled) micromertric stages, DO: Detection Objective, F: Filter, TL: Tube Lens, C: sCMOS Camera. A) Simplified sketch of the SPIM. B) Picture of the real set-up with in inserts: the yellow rectangle for the view of the laser source placed just behind the breadboard, and the blue rectangles for the view of the afocal system to enlarge the laser beam.

Fig. 2.

Scheme of the reference systems: the xyz system of the microfluidic chip (black basis) and the x’y’z’ system of the illuminated section (green basis).

Fig. 3.

A) z’y’ plane of the selected ROI and corresponding intensity profiles of the PSF along z’- and y’-direction with the fitted Gaussian curve associated for 500nm fluorescent bead embedded in hydrogel. B) z’x’ plane of the selected ROI and corresponding intensity profiles of the PSF along x’-direction.

Fig. 4.

Scheme of the U-cup shaped trap containing the organoid. Hydrogel is distributed on the edges of the microfluidic channels and in the trap, and the channels are filled with growth media to promote the development of the vascular network. The orientation axis defined in Fig. 2 are reported.

Fig. 5.

A) Inverted fluorescence microscope image that provides a 2D image of the projection of all fluorescent cells present on the chip forming a global vascular network after 8 days culture. B) 3D rendering of the same vascular network obtained from 170 planes images with the light sheet (z is the direction of the stage displacement). The orientation axis defined in Fig. 2 are reported. The images correspond to the Top / Bottom view of Fig. 4.

Fig. 6.

3D rendering of the same area of the same vascular network over time. A) After 1 day of culture (100 planes images). B) After 4 days of culture (crop, 247 planes images). C) After 6 days (152 planes images). D) After 8 days (170 planes images).