Expansion tomography for large volume tissue imaging with nanoscale resolution

Abstract

Expansion microscopy enables conventional diffraction limit microscopy to achieve super-resolution imaging. However, the enlarged tissue lacks an objective lens with sufficient working distance that can image tissues with whole-brain-scale coverage. Here, we present expansion tomography (ExT) to solve this problem. We have established a modified super-absorbent hydrogel (ExT gel) that possesses high mechanical strength and enables serial sectioning. ExT gel enables tissue and cell imaging and is compatible with various fluorescent labeling strategies. Combining with the high-throughput light-sheet tomography (HLTP) system, we have shown the capability of large volume imaging with nanoscale resolution of mouse brain intact neuronal circuits. The ExT method would allow image samples to support super-resolution imaging of intact tissues with virtually unlimited axial extensions.

Fig. 1.

Expansion tomography (ExT) strategy.

Fig. 2.

The establishment of ExT gel. (a) The schematic representation of synthesis of ExT hydrogel. (b) Possible hydrogen-bonding structures in the swollen ExT hydrogel. (c) The elastic modulus and expansion factor of expansion hydrogels with different AMPS-Na concentration (mean ± s.d., n = 3 samples each). (d) The elastic modulus and expansion factor of expansion hydrogels with different MBA concentration (mean ± s.d., n = 3 samples each). (e) The compression modulus of the expansion hydrogels with various expansion recipes (ExM [22,25], MAP [24], U-ExM [46,47], ExM-2 is defined as an expansion recipe which has the same molar concentration as the corresponding monomer component in ExT gel solution) (mean ± s.d., n = 3 samples each). (f) Measurements of surface evenness of ExT hydrogel after continuous sectioning with a vibratome. X is the direction of sectioning and Y is perpendicular to the direction of sectioning. The Root Mean Square (RMS) values in X direction and Y direction are (948.11 ± 35.62) nm and (953.51 ± 51.74) nm respectively (n = 3).

Fig. 3.

ExT gel enables biological tissue and cells imaging. (a) Confocal images of the immunostained GFP mouse brain slice before and after expansion treatment. (b) An exemplary measurement for the expansion factor. A line scan was drawn over corresponding regions before and after expansion, as indicated in panel (a) and (b) by the colored lines. The expansion factor in this example is 4.46-fold. (c) An analysis of the root mean square error (RMSE) of the distortions between aligned pre- and post-expansion images. (d) Imaging of immunostained α-tubulin in U-2 OS cells with Alexa 546 after ExT gel expansion (F=4.34). (e) The full width at half maximum (FWHM) measurement of the red region in (d) as an indication of resolution. FWHM is obtained by a best Gaussian curve fitting and its value is 54.66 nm in this example. (f-g) Numbers of statistical measurements of FWHM in microtubules. The FWHM average value is (63.36 ± 14.46) nm (mean ± s.d., n = 63 microtubules). (h) Representative images of various conventional fluorescent secondary antibody immunostained brain slides before and after ExT hydrogel expansion. (i) Fluorescence retention for these dyes after ExT hydrogel workflow treatment in immunolabeled mouse brain slices (mean ± s.d., n = 30 somas from four brain slices of each group). (j) Image of dendritic spines in the GFP brain slice without immunostaining, streptavidin conjugated Alexa 647(SA-Alexa647) staining after expansion, overlay pre-ExT and post-ExT images using similarity transformation in a local region, registration pre-ExT and post-ExT image used B-spline registration (F=4.42). (k-l) Synaptic structure resolved with a post-synaptic marker, homer1, and dendritic spines in a cortical region. Homer1 was stained with Alexa 568, and dendritic spines used GFP-positive signal as the reporter which stained with Alexa 488 (F=4.45). (m-n) Intensity plot along the axis of the synapse in (n) and (o). The lines represent the Gaussian curve fitting. (o-q) Images of blood vessels stained with lectin-DyLight 594 after ExT processing (F=4.29). All distances and scale bars correspond to pre-expansion dimensions. Scale bar: (a) 10 μm, (d) 5 μm, (f) 2 μm, (h) 20 μm, (j) 2 μm, (k-l) 500 nm, (o) 500 μm, (p-q) 20 μm.

Fig. 4.

Scalable 3D super-resolution imaging of mouse brain tissue with HLTP system. (a) Volume rendering of a half coronal plane containing cortex and hippocampus. (b) Volume rendering of granule cells and fibers in dentate gyrus (DG). (c) Enlargements of insets (white boxes) from (b). (d) Magnified segmented views of the morphology of axonal boutons. (e) Volume rendering of a group of integral layer V pyramidal neurons in cortex. (f) Neural tracing and dendritic spine reconstruction across the mouse primary visual cortex. (g-h) Magnified segmented views of the morphology of a basal dendrite and its spines. All the scale bar was on a biological scale. Scale bars: (e-f) 2 μm.