Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM

Abstract

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

Keywords: atherosclerosis in rabbit models; cathodoluminescence; cholesterol crystals; correlative electron microscopy; lipid droplet.

Fig. 1.

Cryo-SEM and CL of freeze-fractured tissues of atherosclerotic lesions. (A–D) Schematic representations of lesion locations in the tissue. B and D are schematic representations of the samples of tissue slices examined in cryo-SEM-CL below. B refers to G and H and D to I and J and SI Appendix, Fig. S3. (A and B) Less-severe lesion. (C and D) More-severe lesion. (E and F) Cryo-SEM (E) and cryo-CL (F) images of the same region from the aorta of a control rabbit. Four cells are highlighted with different pseudocolors. e, elastic fibers; N, cell nucleus. (G and H) Cryo-SEM (G) and cryo-CL (H) images from the same region of a thin atherosclerotic lesion as in A and B, taken from the descending aorta ([b] in SI Appendix, Fig. S1A) of an affected rabbit. Four foam cells are highlighted with different pseudocolors. Arrowheads: white, plasma membrane; red, lipid droplet; yellow arrows, cholesterol crystals; N, cell nucleus. (I and J) cryo-SEM (I) and cryo-CL (J) images from the same region of a thick lesion as in C and D, taken from the ascending aorta of an affected rabbit ([a] in SI Appendix, Fig. S1A). Yellow arrows, cholesterol crystals. (Insets) SEM and CL of a synthetic crystal of cholesterol. A–D were created with BioRender.com.

Fig. 2.

Two-dimensional and 3D representation of the cryo-FIB-SEM stacks from M (region at the interface of the lesion with the tunica media of the blood vessel), C (core of lesion), and L (region of lesion bordering with the lumen). M, C, and L show representative slices of the three regions viewed in cryo-FIB-SEM. N, cell nucleus. Red arrows indicate heterogeneity in lipid droplet content. (Scale bar, 2 μm for M, C, and L.) M_vol, C_vol, and L_vol show segmented volume representations of the three regions presented to scale. N, cell nucleus; CC, cholesterol crystals. Dimensions of the displayed volumes are 25.6 × 10.3 × 3 μm³ for M, 29.7 × 14.2 × 3 μm³ for C, and 26.1 × 13.7 × 3 μm³ for L. Compare to volume representations following automatic segmentation in SI Appendix, Fig. S5. For further details on image analysis see Materials and Methods.

Fig. 3.

Cryo-FIB-SEM images of the media-lesion interface region. (A) Surface representation of the segmented image stack. Lipid droplets yellow, cholesterol crystals brown, cell nuclei light purple. Volume thickness = 5 μm. (B) One slice, featuring a cell containing intracellular thin crystals (arrows) and lipid droplets. The plasma membrane can be followed along the whole perimeter of the cell (see raw image in SI Appendix, Fig. S6A). The cell area is pseudocolored in pink and the cell nucleus in purple. The nucleus of the cell shown in B is in the top left quadrant of the volume in A, but the left side of the cell containing the crystals is not included in the segmented frame. (C) Individual crystals attached to a lipid droplet. The crystals that are in closest contact with the lipid droplet surfaces merge with the lipid droplet (arrows). (D) Surface representation of the segmented lipid droplet/crystal complex shown in C. Color code in D is the same as in A. The apparent striation in the segmented thin crystals derives from the reconstruction/segmentation procedure. [E(1)–(5)] Progressive slices through the volume of the same object, separated by 100 to 150 nm, demonstrating stacking of thin crystals on a lipid droplet. Red arrowhead in E5 indicates one of the vacuolar structures that is referred to as a hydrolysis pit (40).

Fig. 4.

Cryo-FIB-SEM images of the core region. (A) Surface representation of the segmented image stack. Color code as in Fig. 3. Volume dimensions = 30.92 × 13.58 × 5 μm³. (B) One slice, featuring thin crystals attached and merged with two lipid droplets. (C) Surface representation of the segmented lipid droplet/crystal complex in B. (Inset) Crystal segmentation without the lipid droplets, emphasizing the different orientations of the crystal growth on the surface of the lipid droplet. The apparent striation in the segmented thin crystals derives from the reconstruction/segmentation procedure. (D and E) Lamellar bodies (yellow arrows) also appear attached to thin crystals (white arrowheads) but are less frequent than the lipid droplets.

Fig. 5.

Cryo-FIB-SEM images of the intimal region bordering with the lumen. (A) Surface representation of the segmented image stack. The large crystals on the left side of the image appear in the first slice of the imaged box in an oblique cut, which makes them appear thicker than they are. Two thin crystals are in contact with a lipid droplet (red arrowheads). Color code as in Fig. 3. Volume thickness 3 μm. The apparent striation in the segmented thin crystals derives from the reconstruction/segmentation procedure. (B and C) Two slices, featuring two cells containing intracellular thin crystals and lipid droplets. The plasma membrane can be followed along the perimeter of the cells that are visible in the slice (compare with raw images in SI Appendix, Fig. S6 C and E). Cell areas are pseudocolored in pink and the cell nuclei in purple. The thin crystals are inside the cells (black arrows).

Fig. 6.

Cryo-FIB-SEM segmented images of representative cholesterol crystals. (A) Crystals segmented from L, M and C regions. (Top and Middle) Thin curved crystals wrap around lipid droplets. (Bottom) Thicker crystals gradually straighten up. (B–D) Crystals segmented from the core region. (B) Single crystals having regions delimited by well-defined crystal faces. (C) Thin crystal displaying growth steps (black arrow). (D) Conglomerate of intergrown crystals. For further image analysis details see Materials and Methods.