Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution

Abstract

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Figure 1 -. ctASLM enables isotropic, sub-micron imaging over large field of views.

(a) Working principle of axially swept light-sheet microscopy: a thin light-sheet is scanned in its propagation direction. The rolling shutter readout of an sCMOS camera, adjusted to the size of the beam waist, is tightly synchronized to the light sheet scan. (b) Experimentally measured PSF of Airy scan confocal microscopy, conventional light-sheet fluorescence microscopy (LSFM), and ctASLM. LSFM and ctASLM PSFs are shown for NA 0.4 (indicated with “1”) and 0.7 objectives (indicated with “2”). (c-d) Maximum intensity projections of CLARITY cleared cortical Thy1-eYFP neurons as imaged by LSFM and ctASLM, respectively. (e) An XY view of a section 2.5mm from the top surface of a Thy1-eGFP PEGASOS cleared brain. (f) Zoom-in of the red box with a rendered neuron. Insets provide magnified views of synaptic spines. (g) Volume rendering of a mouse kidney labeled with Flk1-GFP. Inset shows a 3D rendering of endothelial cells in one selected glomerulus. (h) Maximum intensity projection of a mouse tibia bone marrow plug. (i) Zoom-in of magenta box shows alpha-catulin-GFP+ cells (green, Alexa Fluor 488), Lepr+ niche cells (red, tdTomato) and cKit+ cells (grey, Alexa Fluor 647). Alpha-catulin-GFP+/cKit+ hematopoietic stem cells (HSC) are labeled with cyan spheres. (j&k) are the volume-rendering of boxed region in (h), showing progenitor cells in grey and niche cells in red. All biological data acquired with NA 0.4 objectives. Scale bars are: (b) 3 μm, (c,d) 20 μm, (e) 1 mm, (g) 500 μm, (h) 1 mm, (i) 200 μm, (j) 40×40×40 μm³, (k) 5 μm.

Figure 2 -. ctASLM allows detection of cellular and subcellular features in large tissues.

(a) Machine learning based supervised detection and unsupervised clustering of spines in a Clarity cleared mouse brain. (b) The shape of the median spine in each cluster. (c) Principal component analysis of the spines. (d) automatic detection of glomeruli in the mouse kidney data set shown in Fig 2 (e). (e) Lateral maximum intensity projection of one glomerulus and detection of individual endothelial cells (yellow circles). Data acquired with NA 0.4 objectives. Scale bar: (a) 20 μm, (d) 200 μm, (e) 50 μm

Figure 3 -. ctASLM allows visualization of subcellular interactions amongst cells in large tissues.

(a) Maximum intensity projection of a mouse bone marrow specimen. (b) Zoom-in of yellow box shows c-kit+ stem/progenitor cells (magenta, Alexa 647), nerve fibers (green, Alexa 488) and blood-vessels (gray, tdTomato). (c) 3D segmented and rendered view of (b). Orange depicts arterioles. (d) Zoomed in view (segmented & rendered) view of the green box in (b) showing c-kit+ cells in contact with a nerve fiber. (e) XZ cross-section of cKit+ cells residing on the sinusoids. (f) c-kit+ cells are positioned along fenestrations in the walls of the sinusoids. (g&h) 3D rendering of cell highlighted in (f). (i) Nerve fibers with punctate staining adjacent to cKit+ positive cells. (j) Zoom in view shows a possible interaction. (k) YZ view along a plane indicated by white triangles in (j) showing an interaction between a nerve fiber and a c-kit+ cell. (l) Volume rendering of (j). Data acquired with NA 0.7 objectives. Scale bar: (a) 500 μm (b&c) 10 μm (d) 2.5 μm (e&f) 5 μm (i) 10 μm (j&k) 2.5 μm