Feature-driven whole-tissue imaging with subcellular resolution

Abstract

Existing microscopy approaches are often unable to identify and contextualize rare but biologically meaningful events due to limitations associated with simultaneously achieving both high-resolution imaging and a cm-scale field of view. Here, we present multiscale cleared tissue axially swept light-sheet microscopy (MCT-ASLM), a platform combining cm-scale imaging with targeted high-resolution interrogation of intact tissues in human-guided or autonomous modes. Capable of capturing fields of view up to 21 mm at micron-scale resolution, MCT-ASLM can seamlessly transition to a targeted imaging mode with an isotropic resolution that approaches ∼300 nm. This versatility enables detailed studies of hierarchical organization and spatially complex processes, including mapping neuronal circuits in rat brains, visualizing glomerular innervation in mouse kidneys, and examining metastatic tumor microenvironments. By bridging subcellular- to tissue-level scales, MCT-ASLM offers a powerful method for unraveling how local events contribute to global biological phenomena.

Keywords: CP: Imaging; LSFM; cancer; development; feature-driven cleared tissue imaging; multiscale ASLM; neuroscience.

Graphical abstract

Figure 1

Microscope design and multiscale biological imaging (A) Computer-aided design rendering of the MCT-ASLM system. (B) Close-up view of the sample chamber and illumination axes. (C) Mouse liver lobe stained for tyrosine hydroxylase, imaged at 1× and 38× magnifications. High magnification reveals detailed structures within the liver’s autonomic nervous system. (D) Mouse bladder stained for SMA (green), TUBB3 (cyan), and cytokeratin (magenta), imaged at 2× and 38× magnifications. Smooth muscle fibers and neuronal tracts are visible within the bladder wall and urethra. (E) Mouse kidney stained for cytokeratin (green), SIX2 (cyan), and TUBB3 (magenta), imaged at 4× and 38× magnifications. SIX2 labels nephron progenitors, while TUBB3 highlights neuronal components. (F) Mouse kidney stained for TUBB3 (green), synapsin (cyan), and CD31 (magenta), imaged at 5× and 38× magnifications. High-resolution imaging reveals interactions between neuronal synaptic structures and vasculature. (G) Mouse kidney stained for SMA (green), TUBB3 (cyan), and podocin (magenta), imaged at 3× and 38× magnifications. Subcellular details, including podocyte networks, are evident. Scale bars: (C) 1×, 1,000 μm and 38×, 50 μm; (D) 2×, 500 μm and 38×, 50 μm; (E) 4×, 250 μm and 38×, 50 μm; (F) 5×, 200 μm and 38×, 50 μm; and (G) 3×, 333 μm and 38×, 50 μm. See STAR Methods for additional details on labeling and clearing methods.

Figure 2

Multiscale imaging of neuronal architectures in the rat brain (A) Rat brain expressing an mScarlet reporter, imaged at 1× and 38× magnifications. Long-range axonal projections spanning over 13 mm are visualized, and bouton-like varicosities along axons are resolved at high magnification. Select presynaptic, bouton-like swellings are highlighted with white arrowheads. (B) Imaging sparse neurons in a rat brain. The entire brain was imaged at 1× magnification to provide a global overview, followed by 6× magnification for region-specific interrogation and fully automatic multi-position imaging at 38× magnification to capture high-resolution neuronal details. Scale bars: (A) 1×, 1,000 μm and 38×, 50 μm and (B) 1×, 1,000 μm, 6×, 170 μm, and 38×, 50 μm.

Figure 3

Imaging metastatic colonization with MCT-ASLM (A–C) Mouse bone stained for laminin (green) and luciferase (magenta), imaged at (A) 1×, (B) 6×, and (C) 38× magnifications. (D) A 3D rendering of the 38× magnification reveals a dense metastatic tumor mass and its proximity to the surrounding vasculature. Scale bars: (A) 1×, 1,000 μm; (B) 6×, 170 μm; and (C) 38×, 50 μm.

Figure 4

Feature-driven autonomous imaging of a mouse mammary tissue (A) Low-magnification image of mouse mammary tissue stained for nuclei (green), luciferase (cyan), and laminin (orange). The tissue boundary was automatically detected using image-based segmentation, and a tiling pattern was generated for high-resolution imaging with the nanoscale module. (B) High-resolution tiled acquisition of the same region using the defined tiling pattern. (C) Zoomed-in view of a selected tile acquired at 38× magnification, highlighting fine tissue architecture and subcellular detail. Scale bars: (A) 150 μm, (B) 150 μm, and (C) 25 μm.

Figure 5

Feature-driven autonomous imaging of glomeruli and innervation in a mouse kidney (A) Whole mouse kidney imaged at 6× magnification with the macroscale module, stained for podocin (magenta), which labels glomeruli, and TUBB3 (cyan), which labels nerves. On-the-fly segmentation was performed immediately after acquisition to identify glomeruli, enabling targeted high-resolution imaging with the nanoscale module. (B) Representative high-resolution images of glomeruli captured with the nanoscale module, highlighting the diversity of innervation across different regions. (C) Scatterplot showing the position of glomeruli in x, y, and z (color coded as per the lookup table) and their volumes (represented by the diameter of data points). (D) Histogram of distances between glomeruli and adjacent nerves. The data were fit to a mixture model of three Gaussian populations with mean distances of 7.17, 28.1, and 59.6 μm. Individual Gaussian populations are shown as solid lines and the cumulative mixture model as a dashed line. (E) Scatterplot showing glomeruli position in x and y, with color-coded distances to adjacent nerves (lookup table) and glomeruli volume (represented by data point diameter).