Biomedical Engineering Reference
In-Depth Information
lesions (types I and II). Intermediate lesions (type III) contained scattered interstitial lipid pools and
further intimal thickening. Disordered collagen fibrils around the lipid pools were detected. Type IV
lesions had a well-defined lipid core identified by a strong CARS signal starting at the shoulder of the
lesion. Abundant lipid-laden foam cells, identified by CARS and cellular doxorubicin signals, were seen
in the shoulder and in luminal regions of the atherosclerotic plaque. Extracellular lipid accumulation
was also observed (see Figure 13.10). Type V lesions were identified based on characteristic dense core of
lipid (CARS) and surrounding fibrous cap (SHG). Lipid and collagen content of the different lesion types
were quantified based on CARS and SHG signals, respectively. Lipid accumulation in thickened intima
culminated in Type IV whereas the highest collagen deposition was found in Type V lesions. Recently,
Kim et al. have been able to classify four different morphologies of atherosclerotic lipids using multiplex
CARS imaging; intracellular and extracellular lipid droplets as well as needle-shaped and plate-shaped
lipid crystals [54].
13.7.2 other Diseases
Human heart valve allografts from patients with cardiomyopathy are often harvested for human valve
replacement. Two-photon excitation microscopy and SHG have been used to assess the structural integ-
rity of heart valves harvested from human ischemic (ICM) and dilated cardiomyopthic (DCM) hearts,
and from model acute and chronic ischemic porcine hearts [57]. Degradation of collagen bundle struc-
tures was seen within the collagen-rich outflow side of human aortic and pulmonary ICM leaflets and
was even more pronounced on the inflow side of ICM leaflets. Depletion and disintegration of elastin-
containing structures was observed mainly within the outflow side of aortic and pulmonary leaflets of
DCM heart valves. Compared with normal porcine valve tissues, acute ICM specimens showed no sig-
nificant changes in extracellular matrix components. Similar to the changes seen in valves from human
ICM and DCM hearts, multiphoton imaging of chronic porcine ICM tissues demonstrated weak colla-
gen SHG and elastin autofluorescence, indicating marked ECM remodeling. Quantification of collagen
SHG signals revealed that normal leaflets were approximately six- to sevenfold higher in normal leaflets
compared to similar tissues from chronic ICM pigs. Since the total amount of collagen measured bio-
chemically was found to be unaltered in diseased tissues, these investigators suggest that the changes
in SHG signal may be due to abnormalities in extracellular matrix architecture. In a similar manner,
cryopreservation of heart valves also appears to diminish collagen SHG, suggesting this process causes
structural alterations in collagen [58]. Nonlinear imaging has also been used to assess changes in the
aortic arch in a rat model of dissecting aortic aneurysm [59]. Decreased SHG signal was seen from
medial and adventitial collagen of affected fetal and newborn pups, in the absence of any change in elas-
tin autofluorescence, suggesting that altered collagen structure plays a key role in this model of aortic
dissection.
13.8 Summary and Future Directions
Nonlinear optical imaging has provided important new insights into the structure and functional
interrelationships of macromolecular microstructures in the arterial vascular bed in both healthy and
diseased states. Initial studies led to the development of improved spectral separation of collagen SHG
and elastin autofluorescence, and to the application of multimodal nonlinear microscopy to allow imag-
ing of other arterial wall components, including lipid deposits. Currently, essentially all of the com-
ponents of the arterial wall can now be imaged, including cells, ECM components, and pathological
lipid deposits, using a combination of intrinsic signals (collagen SHG, elastin autofluorescence) and
exogenous fluorescent probes. Investigations of vessel mechanics have revealed the changes in distribu-
tion of extracellular matrix components that occur with changes in transluminal pressure as well as the
inherent artifacts in collagen and elastin distribution seen
in vitro
. Studies of LDL interactions with the
vascular wall have provided important new insights into the role that macromolecular microstructures
