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Tilt-invariant scanned oblique plane illumination microscopy for large-scale volumetric imaging

Manish Kumar and Yevgenia Kozorovitskiy

Open Access Open Access

Abstract

This Letter presents the first demonstration of multi-tile stitching for large scale 3D imaging in single objective light-sheet microscopy. We show undistorted 3D imaging spanning complete zebrafish larvae and over 1  mm3 volumes for thick mouse brain sections. We use remote galvo scanning for light-sheet creation and develop a processing pipeline for 3D tiling across different axes. With the improved one photon (1p) tilt-invariant scanned oblique plane illumination (SOPi, /sōpī/) microscope presented here, we demonstrate cellular resolution imaging at depths exceeding 330 μm in optically scattering mouse brain samples and dendritic imaging in more superficial layers.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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Corrections

23 April 2019: A typographical correction was made to the figures.

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Supplementary Material (4)

NameDescription
Visualization 1       Image quality comparison of SOPi 1.0 (top row) and SOPi 2.0 (bottom row). The oblique plane illumination (x'y') view of same scanned region inside a thick mouse brain section is shown on the left.
Visualization 2       360° view of an entire zebrafish larva obtained by stitching 6 SOPi tiles acquired by translating the stage along the x-axis. GFP, RFP and combined view of the same zebrafish is shown. Total length of the zebrafish larva is ~4 mm.
Visualization 3       Oblique plane illumination (x'y') view of a large-scale stitched volume from a 1 mm thick, uncleared mouse brain section. The volume was obtained by stitching multiple tiles along the y-axis.
Visualization 4       Virtual depth scan through the stitched volume inside a 1 mm thick mouse brain section. The volume was obtained by stitching two overlapping tiles along the z-axis.

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Figures (6)
Fig. 1.
Fig. 1. (a) Comparison of OPM, SCAPE, and SOPi light-sheet scanning. (b) Schematics for the experimental setup of SOPi. (c) Role of G1 and G2. (d) Effective acceptance angle of the system and (e) extended schematics showing laser arrangement for two color imaging. MO: microscope objective, G: galvo scanner, L: lens, MDM: multiband dichroic mirror.

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Fig. 2.
Fig. 2. (a) Relative orientation of sample (light gray cuboid) and SOPi acquired tile (green sheared cuboid). (b) Geometrical transformations to reshape tile into correct 3D orientation. (c) Processing pipeline for acquiring, stitching, and 3D visualization of multiple SOPi tiles.

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Fig. 3.
Fig. 3. Comparison of SOPi 1.0 and 2.0. (a) Light-sheet generation in the original SOPi 1.0. A 3D perspective view (inverted gray LUT) of the same scanned volume using SOPi 1.0 in (b) and SOPi 2.0 in (c). Also see Visualization 1. View of an oblique ( x ′ y ′ ) section of the sample using SOPi 1.0 in (d) and SOPi 2.0 in (e). The arrows point to the cell body used for normalizing the intensity plot shown in (f). Vertical line segments, corresponding to the intensity plots, are marked in (d) and (e). Features shaded in gray illustrate higher signal to background ratio across depth (% increase as noted). LD: laser diode, SA: slit aperture, CL: cylindrical lens, DM: dichroic mirror. (Scale bar: 100 μm)

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Fig. 4.
Fig. 4. Stitching multiple tiles along the x -axis. (a) Schematics of tile arrangement along the length of the fish. 3D perspective side view in (b) and top view in (c) of green and red fluorescence. Also see Visualization 2. (Scale bar: 100 μm)

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Fig. 5.
Fig. 5. Stitching multiple tiles along the y -axis. (a) Tile arrangement (top view) along a 1 mm thick, uncleared mouse brain section. Scanned region highlighted by the dashed rectangle. (b) A virtual slice from the stitched dataset, at the depth of 100 μm, along with the inset showing an enlarged view. Also see Visualization 3. (Scale bar: 100 μm).

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Fig. 6.
Fig. 6. Stitching tiles along sample depth ( z -axis). (a) Placement and orientation of connected tiles along depth. The top view of the tile is highlighted by a rectangle. (b) Virtual x y slices along the depth of the stitched volume of a thick mouse brain section. Some neurons at > 350     μm depth (see Visualization 4) are resolved, with neuronal processes imaged at more superficial depths. (Scale bar: 100 μm).

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