Imaging and quantitative analysis of atherosclerotic lesions by CARS-based multimodal nonlinear optical microscopy

Abstract

Objective: The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee.

Methods and results: Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery.

Conclusions: We have demonstrated the capability of CARS-based multimodal NLO microscopy to interrogate different stages of lesion development with subcellular detail to permit quantitative analysis of lipid and collagen contents.

Figure 1

Representative images of a Type I lesion inspected by CARS (A), SFG (B), and TPEF (C). (D and E) Masson's trichrome staining. (F) H&E staining. Rectangular window in D indicates the relative area of NLO images in A–C. (G) Colocalized NLO image corresponding to the red square in A. Arrows indicate scattered lipid-laden cells. (H) Colocalized image corresponding to the white square in G. (I) Z-stack CARS image. L: lumen; TI: thickened intima; M: tunica media; IE: internal elastic lamina; gray for CARS; blue for SFG; green for TPEF. The same color code is used in other figures.

Figure 2

Representative images of a Type II lesion inspected by CARS (A), SFG (B), and TPEF (C). (D and E) H&E staining. Wide arrow: the relative location in A–C. (F) Masson's trichrome staining. (G and H) Colocalized NLO images from the red square in A. CARS reveals aggregated lipid-laden cells (arrows) with elongated shape (possibly smooth muscle cells) in the thickened intima. (I) Colocalized image relating to the white square in H.

Figure 3

Representative images of a Type III lesion inspected by CARS (A), SFG (B), and TPEF (C). LP: lipid pools. (D) Sub-gross image of H&E staining. Wide arrow: the relative location in A–C. (E and F) Zoomed-in H&E around the pathological intima-media interface. (G) Colocalized NLO image from the red square in A shows a cell-dominated distribution with no signal of collagen. (H and I) Colocalized NLO images from the yellow square in A.

Figure 4

Representative images of a Type IV lesion inspected by CARS (A), SFG (B), and TPEF (C). The lipid core (LC) was identified by CARS. (D and E) Masson's trichrome staining. Black rectangle: the corresponding area in A–C. (F) H&E staining. (G and H) Colocalized NLO images corresponding to the red and yellow squares in A, respectively. (I) Doxorubicin-labeled (red) image, around lipid core, colocalized with CARS and SFG signals.

Figure 5

Representative images of a Type V lesion inspected by CARS (A), SFG (B), and TPEF (C). FC: collagen fibrous cap around the lipid core (LC). (D) Masson's trichrome stained image. Black rectangle: the correlative area in A–C. (E and F) Zoomed-in images of Masson's trichrome and H&E staining around the lipid core. Colocalized NLO image in G and doxorubicin-labeled (red) image colocalized with SFG signal in H elucidate the relationship between elongated cells (arrows) and collagenous matrix. (I) Colocalized NLO image from the yellow square in A.