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. 2022 Jul 20;25(8):104805.
doi: 10.1016/j.isci.2022.104805. eCollection 2022 Aug 19.

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution

Lei Deng  1   2 , Jianwei Chen  1   2 , Yafeng Li  1   2 , Yutong Han  1   2 , Guoqing Fan  1   2 , Jie Yang  1   2 , Dongjian Cao  1   2 , Bolin Lu  1   2 , Kefu Ning  1   2 , Shuo Nie  1   2 , Zoutao Zhang  1   2 , Dan Shen  1   2 , Yunfei Zhang  1   2 , Wenbin Fu  3 , Wei Eric Wang  3 , Ying Wan  4   5 , Sha Li  6   7 , Yu-Qi Feng  6   7 , Qingming Luo  1   2   8 , Jing Yuan  1   2   9
Affiliations

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution

Lei Deng et al. iScience. .

Abstract

Optical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

Keywords: Biological sciences; Natural sciences; Neuroscience; Techniques in neuroscience.

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Conflict of interest statement

Q. L., J. Y., L. D., and J. C. have filed patent applications based on this work.

Figures

None
Graphical abstract
Figure 1
Figure 1
Cryo-fMOST (A) Schematic of the cryo-fMOST pipeline. (B) Resolution measurement. (C) Temperature measurement of the sample tank with the single- (red solid line), two- (blue dotted line), and three-stage (black dashed line) refrigeration. (n = 3, mean ± SD). (D) Real-time temperature measurement at the sleeve (red solid line) and base (blue dashed line) in the refrigeration process from 22 to −17°C. In addition, see Figures S1–S4. (n = 3, mean ± SD).
Figure 2
Figure 2
Imaging of the B6 ACTb-EGFP mouse tongue (A) 3D rendering of the whole tongue. (B and C) Reconstructed middle sagittal and transverse images; scale bars: 1 mm. (D) Acquired middle coronal image. The color-coded tensor image is on the left half; scale bar: 1 mm. (E) Reconstructed 3D data block corresponding to the white cube in (A) to show intricate muscle orientations. (F and G) Enlarged views of the corresponding white rectangles in (D); scale bars: 10 (F) and 20 (G) μm. SLM: superior longitudinal muscle; VM: vertical muscle; TM: transverse muscle; ILM: inferior longitudinal muscle; GM: genioglossus muscle; LA: lingual artery. D-V, dorsal-ventral; A-P, anterior-posterior; L-R, left-right. The experiment was repeated two times with similar results.
Figure 3
Figure 3
Imaging of the whole mT/mG mouse kidney tissue expressing tdTomato fluorescent protein (A) Typical frontal section image. OSOM: outer stripe of the outer medulla; ISOM: inner stripe of the outer medulla; IM: inner medulla; scale bar: 1 mm. (B–E) Enlarged views of the corresponding white squares in (A). GT: glomerular tuft; BS: Bowman’s space; RC: renal corpuscle; RT: renal tubule; MR: medullary ray; VB: vascular bundle; HL: Henle’s loop; CD: collecting duct; scale bar: 50 μm. (F) Reconstructed transverse image and an inset showing the enlarged segment of the cortex, corresponding to the white rectangle in the main image; scale bars: 1 mm and 100 μm, respectively. (G) 3D rendering of the whole kidney with a local microstructural network reconstructed along the artery indicated in the inset of (F). (H) Enlarged view of a partially reconstructed nephron at the starting position of the tracing in (G). PCT: proximal convoluted tubule. (I) Enlarged view of the corresponding data block in (G). The experiment was repeated three times with similar results.
Figure 4
Figure 4
Imaging of the mT/mG mice hearts (A) 3D rendering and sequential sagittal images at equal intervals of 1.2 mm; scale bar: 2 mm. LV: left ventricle, PM: papillary muscle. (B) Enlarged view of the corresponding white rectangle in (A). CM: cardiomyocytes; scale bar: 100 μm. (C–E) Enlarged views of the corresponding white rectangles in (B); scale bar: 10 μm. Arrows in (C) indicate cellular bridges. (F) Volumetric reconstruction of the healthy heart and major coronary vessels. Ao: aorta; RCA: right coronary artery; LCA: left coronary artery; SA: septal artery; LC: left circumflex branch; AI: anterior interventricular branch; LAD: left anterior descending; PC: proximal left collateral. The experiment was repeated three times with similar results. (G) Volumetric reconstruction of the MI heart and major coronary vessels. Insert shows the image of the section indicated by the white arrowheads in (G). Scale bar: 1 mm in the insert. (H) Enlarged 3D reconstruction view of the major vessels in the corresponding white cube in (G). CA: collateral artery. Yellow and purple vessels represent the infarcted and collateral arteries, respectively. (I–L) Enlarged views of the sections of the pre-ligation, ligated, post-ligation, and collateral arteries, respectively. The white arrows indicate the locations of the corresponding arteries. The dotted circle indicates the ligated artery. Scale bar: 50 μm.
Figure 5
Figure 5
Imaging results of a Thy1-GFP line M mouse brain (A) MIP of 300 μm brain sections at intervals of approximately 1 mm; scale bar: 1 mm. (B) Reconstructed DSM image corresponding to the white rectangle in (A); scale bar: 1 mm. (C) Enlarged views with different imaging modes of the corresponding white squares in (B); scale bar: 50 μm. (D) Normalized intensity profiles along the corresponding color lines in (C). CTX: cerebral cortex; cc: corpus callosum; fx: columns of the fornix; ac: anterior commissure; fi: fimbria; HPF: hippocampal formation; BLA: basolateral amygdalar nucleus. The experiment was repeated three times with similar results.
Figure 6
Figure 6
Ten targeted metabolic intermediates detection results of left and right brain slices with or without dealing with tissue clearing OSLB, original slices of the left brain; TCSLB, tissue clearing slices of the left brain; OSRB, original slices of the right brain; TCSRB, tissue clearing slices of the right brain. (n = 4, min to max).
Figure 7
Figure 7
FISH imaging of a brain slice (A) Image of the slice in situ image by cryo-imaging; scale bar: 1 mm. (B) Registration of the enlarged views of the corresponding squares in (A); scale bar: 500 μm (20 μm in the inset). (C) Three color images and their merged image of the slice hippocampal structure enlargement corresponding to the white rectangle in (A); scale bar: 500 μm. (D) Partial enlargement of the corresponding white rectangles in (C); scale bar: 100 μm (10 μm in the inset). The experiment was repeated three times with similar results.
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