Three-dimensional visualization of heart-wide myocardial architecture and vascular network simultaneously at single-cell resolution

Figure 1. Single-cell-level heart-wide data acquisition pipeline. (A) Whole-heart sample preparation. We produced vascular fluorescent-labeled transgenic mice (Tek-cre*Ai47) by crossing two transgenic mice (Tek-cre and Ai47), obtained their fresh heart tissues, and embedded them into HM20, a stiff resin. (B) System setup of HD-dfMOST. The system is composed of a line-illumination modulation (LiMo) microscope and a microtome. (Obj, objective lens; sCMOS, scientific complementary metal oxide semiconductors). (C) Cardiac fMOST data were processed and are shown in 2D and 3D.

Figure 2. Images of heart-wide myocardial architecture at single-cell resolution. (A) Sagittal section view of reconstructed whole-heart myocardial data. LA, left atrium; LV, left ventricle; RV, right ventricle; PM, papillary muscle. Scale bar: 1 μm. (B) Original image of an axial section of the heart. (B1–B3) display the cardiac muscle cells of the ventricular wall from the outside layer (epicardium), middle layer (myocardium), and inside layer (endocardium). Scale bars: 500 and 10 μm, respectively. (C) Enlarged view of the corresponding rectangle in (B). ID, intercalated disc. Scale bar: 10 μm. (D) A reconstructed myocardial data block with a single resolvable myocyte.

Figure 3. Images of the whole-heart vascular network at the capillary level. (A) The 3D rendering of whole-heart vascular data (top right) and the MIP generated from raw axial vascular images with a thickness of 300 μm. V, vein; RCA, right coronary artery; Ao, aorta; PA, pulmonary artery. Scale bar: 500 μm. (B,C) Scaled images corresponding to the white rectangles in (A), with an MIP of 100-μm-thick raw images, displaying endothelial cells in the vein and RCA, respectively. Scale bars: 10 μm. (D) An enlarged view of the area indicating a depth-coded 100-μm-thick MIP image (A), showing a partial capillary network with depth information. Scale bar: 10 μm.

Figure 4. Myocardial and vascular data of the entire heart within a given mouse. (A) The 3D rendering of heart-wide myocardial and vascular data. The myocardial structure is in fuchsia and the vascular structure in green. (B) A selected axial section in the middle of the heart. LV, left ventricle; RV, right ventricle. Scale bar: 500 μm. (C,D) Enlarged views of corresponding white boxes in (B) revealing the spatial correlation between cardiomyocytes and capillaries. Scale bars: 10 μm. (E,F) Enlarged image of the white square in (B). Scale bar: 100 μm.

Figure 5. Color-coded vascular network based on the curvature of the vascular wall. (A) The 3D rendering of the reconstructed vascular network. Blue arrows point out five branches along the LCA. Ao, aorta; LCA, left coronary artery; RCA, right coronary artery. (B–E) Endoscopic perspective of corresponding branches 1–4 in (A) with colors representing the curvature difference. (B) is the division of the Ao and LCA.