Biomedical Engineering Reference
In-Depth Information
arterial aneurysm. We will focus only on studies of arterial wall disease that involve HG imaging in
conjunction with other imaging modalities.
13.7.1 Atherosclerosis
Atherosclerotic lesion progression is a complex process that invokes overlapping mechanisms that
lead to characteristic sequential remodeling of extracellular matrix components in the arterial wall.
A number of studies have investigated atherosclerotic disease, including several using multimodal
nonlinear optical imaging with CARS microscopy to identify lipids. Initial studies of atheroscle-
rotic lesions established that nonlinear optical imaging methods could detect cells, lipid deposits,
and matrical microstructures found in lesions by conventional histological methods [29,36,49-51].
Lilledahl et al. imaged vulnerable plaques in human aortic autopsy samples and found that the fibrous
cap emits primarily SHG due to collagen, in contrast to the necrotic core and healthy artery, which
emits primarily two-photon excited fluorescence from elastin [49]. Collagen in the cap appeared in
thick bundles, in a nondirectional structure. Le et al. imaged unstained components of the arterial
wall and atherosclerotic lesions including endothelial cells, extracellular lipid droplets, lipid-rich
cells, LDL aggregates, collagen, and elastin using multimodal nonlinear optical microscopy [29].
Collagen fibers in porcine iliac atheromas appeared disordered from a luminal view, and perpendicu-
lar to those in the arterial wall from a cross-sectional view. Based on integrated image intensities,
collagen density in the atheroma was increased as much as fourfold compared to the arterial wall.
Yu et al. imaged collagen (SHG), elastin (autofluorescence), leukocytes (EGFP), cell nuclei (Hoechst
33342), and neutral lipids (Nile Red) in immobilized carotid artery atherosclerotic plaques
in vivo
[36]. Ko et al. reported a loss of the internal elastic lamina and the appearance of scattered colla-
gen and lipid-rich structures (using CARS) in early WHHLMI rabbit atherosclerotic lesions [51]. In
advanced plaques, thicker, directional collagen fibers were seen along with increased lipid accumula-
tion. Megens et al. observed adhesion of inflammatory cells to the endothelium and increased inti-
mal collagen labeling with CNA in 15-week-old carotid arterial lesions in apo E−/− mice, consistent
with endothelial activation [50]. Parasassi et al. monitored endothelial adhesion and internaliza-
tion of lipid hydroxyperoxide-containing LDL particles labeled with the lipophilic fluorescent probe
2-dimethylamino-6-lauroyl-naphthalene in rat aorta preparations [52]. In these studies, oxidized
LDL was shown to induce fragmentation of autofluorescent matrix fibers in the arterial wall that
could be prevented by antioxidants. Although these studies have confirmed histological descriptions
of some stages of atherosclerotic lesion development, they have done little to further our understand-
ing of the underlying mechanisms.
Recently, investigators have provided new insights into atherosclerosis lesion progression using
multimodal nonlinear optical imaging to systematically assess the cellular and matrical changes that
occur during the different stages of the disease [31,53,54]. Atherosclerotic lesions are classified by the
American Heart Association as Type (I) initial lesion, (II) fatty streak, (III) intermediate lesion, (IV)
atheroma, (V) fibrous atheroma, and (VI) calcific atheroma [55,56]. Lim et al. characterized cellular
and structural changes in early stage II/III atherosclerotic plaques in apo E−/− mice using multimodal
optical imaging with CARS microscopy to identify lipids [53]. A high-fat, high-cholesterol Western
diet increased the intimal plaque area twofold, as defined by CARS signals of lipid-rich macrophages.
Quantitative analysis of the collagen SHG signal revealed a nearly fourfold decrease in collagen distri-
bution in the lipid-rich plaque regions (~13% versus ~4%, in standard versus Western diet). Collagen
content in the surrounding matrix decreased in a similar manner (~7% versus 3%, in standard ver-
sus Western diet). Wang et al. systematically analyzed diet-induced atherosclerotic lesion development
(Type I-VII) in porcine iliac arteries by CARS-based multimodal nonlinear microscopy [31]. Foam
cells, lipid droplets, collagen, elastin, and fibrous caps were visualized with 3D submicron resolution. All
stages of lesion development could be visualized by nonlinear microscopy and correlated with standard
histological analysis. Adaptive intimal thickening and foam cell lipid accumulation was seen in early
