Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy

Abstract

Arterial tissues collected from Ossabaw swine bearing metabolic syndrome-induced cardiovascular plaques are characterized by multimodal nonlinear optical microscopy that allows coherent anti-Stokes Raman scattering, second-harmonic generation, and two-photon excitation fluorescence imaging on the same platform. Significant components of arterial walls and atherosclerotic lesions, including endothelial cells, extracellular lipid droplets, lipid-rich cells, low-density lipoprotein aggregates, collagen, and elastin are imaged without any labeling. Emission spectra of these components are obtained by nonlinear optical microspectrometry. The nonlinear optical contrast is compared with histology of the same sample. Multimodal nonlinear optical imaging of plaque composition also allows identification of atherosclerotic regions that are vulnerable to rupture risk. The demonstrated capability of nonlinear optical microscopy for label-free molecular imaging of atherosclerotic lesions with 3-D submicrometric resolution suggests its potential application to the diagnosis of atherosclerotic plaques, determination of their rupture risk, and design of individualized drug therapy based on plaque composition.

Fig. 1

A multimodal multiphoton microscope that allows CARS, SHG, TPEF imaging and spectral analysis on the same platform. C: combiner. DM: dichroic mirror. FM: flip mirror. M: mirror. PMT: photomultiplier tube.

Fig. 2

CARS imaging of an atherosclerotic iliac artery from an obese Ossabaw pig. (a), (b), and (c) Depth imaging of red blood cells (RBC), blood lipid deposits (BL), endothelial cells (EC), and lipid-rich cells (LC) in an affected area. (d) CARS imaging of lipid-rich cells in another affected area. (e) and (f) A lipid-rich cell stained with Oil Red O and imaged with (e) CARS and (f) TPEF. Images were acquired from luminal views with a 60 × water immersion objective. Scale bars: 5 μm.

Fig. 3

SHG imaging and histology of healthy and atherosclerotic iliac arteries from Ossabaw pigs. (a) Cross sectional and (b) luminal views of collagen in a healthy arterial wall. (c) Histology of a healthy iliac artery stained for collagen (blue) using Masson’s trichrome stain. (d) Cross sectional and (e) luminal views of collagen in an atherosclerotic artery. (f) Histology of an atherosclerotic artery stained for collagen using Masson’s trichrome stain (blue). SHG images were acquired with a 20× air objective. Histology images were acquired with a 40× air objective. Scale bars: 75μm. (Color online only.)

Fig. 4

SHG and TPEF cross sectional images of healthy and atherosclerotic iliac arteries from Ossabaw pigs. (a) SHG image of collagen and (b) TPEF image of elastin in a healthy artery. Inset: Emission spectra of SHG signals from collagen (green) and TPEF signals from elastin (red). (c) Overlaid image of (a) and (b) with collagen (green) and elastin (red). (d) SHG image of collagen and (e) TPEF image of elastin in an atherosclerotic artery. (f) Overlaid image of (d) and (e) with collagen (green) and elastin (red). Images were acquired with a 20× air objective. Scale bars: 75 μm. (Color online only.)

Fig. 5

Verhoeff-Van Gieson (VVG) stained elastin fibers of an iliac arterial wall. Histology image was acquired with a 40× air objective. Scale bar: 75 μm. TPEF spectrum of a VVG stained elastin band and SHG spectrum of Masson’s trichrome stained collagen fibrils acquired with microspectrometry are shown in the inset of Fig. 4(b). Stained elastin and collagen yielded the same spectra as those unstained in fresh tissues (data not shown).

Fig. 6

Depth characterizations of healthy and atherosclerotic iliac arteries from Ossabaw pigs. (a) and (b) Luminal view of elastin (red) and collagen (green) in a healthy artery at two different depths from the lumen. (c) Depth intensity profiles of elastin (red) and collagen (green) in a healthy artery. (d) and (e) Luminal view of elastin (red) and collagen (green) in an atherosclerotic lesion at two different depths from the lumen. (f) Depth intensity profile of elastin (red) and collagen (green) in an atherosclerotic lesion. Images were acquired with a 20× air objective. Scale bars: 75 μm. (Color online only.)

Fig. 7

Characterization of oxidized LDL aggregates and lipid droplets in lipid-rich cells of an atherosclerotic plaque by TPEF and CARS microscopy. (a) TPEF image of oxidized LDL aggregates and (b) CARS image of lipid droplets in an atherosclerotic lesion. Images were acquired with a 20× air objective. Scale bars: 25 μm. (c) Overlay of the TPEF (green) and CARS (red) images. (d) Emission spectra of TPEF signals from oxidized LDL aggregates (green) and CARS signals from lipid droplets (red). (Color online only.)

Fig. 8

Two-photon autofluorescence spectra of purified native LDL (blue) and LDL oxidized with CuSO₄ for 3 h (green) and 24 h (red). Purified human LDL purchased from Chemicon (LP2-2MG) was diluted to a final concentration of 1 mg/ml with PBS buffer (native) or 10μM of CuSO₄ in PBS buffer for oxidation. LDL samples were incubated at 37°C. LDL spectra were acquired over the oxidation process using a microspectrometer described in Fig. 1. Oxidation of LDL red shifted the emission spectra. Complete LDL oxidation was observed at 24 h after CuSO₄ addition. (Color online only.)

Fig. 9

Characterization of atherosclerotic plaques vulnerable to rupture by multimodal NLO microscopy. (a), (b), and (c) Cross sectional images of (a) collagen (SHG), (b) lipid-rich cells, and extracellular lipid droplets (CARS), and (c) elastin and oxidized LDL aggregates (TPEF). (d) Overlay of CARS (red), SHG (green), and TPEF (blue) images. (e) Histology of an atherosclerotic lesion stained with Oil Red O. (f) Average SHG (collagen, green), TPEF (oxidized LDL aggregates and elastin, blue), and CARS (lipid-rich cells and extracellular lipid droplets, red) intensity as a function of atheroma thickness. Images were acquired with a 20× air objective. Scale bars: 75 μm. (Color online only.)

Fig. 10

Imaging rupture vulnerability of an atheroma. Gray panels: SHG imaging of a whole artery to determine degree atheroma. The artery was rotated at approximately a 30-deg angle for each frame. The degree atheroma of this artery is 180 deg (from 210 to 360 deg). Color panels: SHG (green) and TPEF (red) evaluation of an affected area vulnerable to rupture. These images showed that lipid-rich cells were concentrating toward the lumen and one shoulder region of an atheroma (340 deg). Collagen density at this shoulder area (340 deg) was also drastically reduced. Cross sectioned images acquired with a 20× air objective. Scale bars: 75 μm. (Color online only.)