As lipid is a main component of atherosclerotic lesions, we first applied CARS microscopy to image lipid in the iliac arteries of Ossabaw pigs. To visualize lesions from arterial lumen view, we sliced iliac arteries along the longitudinal direction and placed flattened arteries on a glass surface, such that the lumen faced the 60× water immersion objective on an inverted microscope. The wave number difference between the pump and Stokes lasers, ωp
, was tuned to 2840 cm−1
, the peak of the CARS band for the symmetric CH2
stretch vibration. In arteries of an obese Ossabaw pig, strong CARS signals were detected at different depths (). Red blood cells and blood lipid deposited over a monolayer of endothelial cells were detected at the lumen surface . Lipid droplet enriched cells resembling foam cells were found at great depths . The concentration of lipid-rich cells varied from one affected area to another . The presence of lipid-rich cells were further confirmed by Oil Red O staining . It is worth mentioning that lipid-rich cells can be derived from either macrophage cells or smooth muscle cells.34,35
Being a sensitive probe of lipid bodies,23
CARS provides a way of imaging lipid-rich cells exclusively. Furthermore, CARS imaging allowed visualization of endothelial cells at the surface of atherosclerotic lesions . This detection sensitivity was probably due to an effective reflection of forward CARS signals from endothelial cells by the condensed plaque components. Such enhanced back refection of the forward signal was observed in CARS imaging of mouse skin tissues in vivo
Together, the observations shown in suggest that CARS microscopy is a highly sensitive and specific tool for the detection of atherosclerotic lesions.
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 (more ...)
In addition to lipid deposition and accumulation of lipid-rich cells, another feature of atheroma is the presence of a matrix rich in collagen fibrils.2,3
Hence, we applied SHG microscopy to image collagen fibrils in the arterial wall and atherosclerotic lesions. A femtosecond laser centered at 800 nm was used for excitation. Epi-reflected SHG signal was collected through a 375/50-nm bandpass filter. In normal arteries, collagen fibers were observed to be aligned parallel to one another from both cross sectional and luminal views of the vessel . On the contrary, collagen fibers in an atheroma appeared disordered from a luminal view  and perpendicular to those in the arterial wall from a cross sectional view . Based on integrated SHG intensities, the density of collagen in atheroma was higher than that in arterial walls by as much as four times. The density and orientation difference of collagen fibers between healthy artery wall and plaque was verified by histological evaluation of arterial sections treated with Masson’s trichrome stain . These observations showed that SHG can be used to identify atherosclerotic lesions based on collagen fibril density and orientation.
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 (more ...)
The elastin fibrils, a major component of the internal elastic membrane, were shown to give a strong autofluorescence signal.20,27
Using a femtosecond laser at 800 nm for excitation and a spectrometer attached to our NLO microscope (), we observed a relatively broad two-photon autofluorescence band that peaked at 502 nm20
and a sharp SHG band that peaked at 400 nm [, inset]. The autofluorescence signals were assigned for elastin and collagen by spectral analysis of Verhoeff-Van Gieson stained elastin bands () and Masson’s trichrome stained collagen fibrils , respectively. It should be noted that there are variations in the literature on elastin emission maxima, which includes 480 and 495 nm.20,27
There are two possible reasons for the variation between our reported value and those referenced. First, our elastin spectrum might have contributions from other arterial components. For instance, Zoumi et al. showed a red shift of the spectrum for elastin when mixed with smooth muscle cells.20
However, because we centered the focal volume within the internal elastic membrane, the contribution of other arterial components should be minimal. Second, there are intrinsic variations arising from different spectrometers used. The second explanation is most likely given the difference in equipment and experimental settings between our laboratory and those of referenced literature. Therefore, we would present 502 nm as the peak value of elastin under our experimental conditions.
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 (more ...)
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 (more ...)
With spectral selectivity of the collagen SHG signal and elastin autofluorescence signal, TPEF and SHG imaging of arteries from a cross sectional view was carried out to analyze distribution of elastin and collagen in an atheroma. The collagen and elastin contrasts were clearly distinguished from one another (). In a healthy artery, it was shown that collagen fibers were mostly located within the tunica media, delimited by an intact internal elastic membrane . In contrast, in a diseased artery, collagen fibers were found to extend beyond an interrupted internal elastic membrane . To further quantify the depth distribution of TPEF and SHG signals, we performed NLO imaging of arteries from a luminal view. Consistent with the described cross sectional view of a healthy artery, we observed that TPEF signal of elastin appeared first, then both collagen and elastin appeared at a greater depth from the lumen . The peak intensity for collagen appeared at approximately 7 μm deeper than that for elastin. On the contrary, TPEF and SHG signals appeared together from the lumen surface of an artery with atheroma . The different depth distribution of collagen and elastin between healthy artery and atheroma was clearly seen. These results show that multimodal SHG and TPEF imaging enables a clear demarcation between normal arterial wall and atheroma based on the order of appearance of their respective signals.
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 (more ...)
During multimodal imaging of atherosclerotic lesions, we observed strong TPEF and CARS signals from particles inside lipid-rich cells. Interestingly, although most TPEF signals colocalized with the CARS signals, far more particles were detected with CARS than TPEF . To have a better understanding of these two signals, we performed microspectroscopy analysis of single TPEF or CARS active particles using a spectrometer installed at the back port of our microscope and appropriate excitation laser sources (Sec. 2 Methods, ). shows a broad TPEF band that peaks at 525 nm and a narrow CARS band at 600 nm. The origin of the observed TPEF signal can be explained as follows. In a typical lipid-rich cell, cholesterol esters of oxidized LDL particles are hydrolyzed to free cholesterol and fatty acid.2
Free cholesterol molecules are then transported to the membrane and effluxed to extracellular receptors in a process described as reverse cholesterol transport. When extracellular cholesterol receptors are limiting, excess cellular cholesterol is re-esterified by acyl coenzyme A: acylcholesterol transferase, and deposited in the cytosol as insoluble lipid droplets.36
Because oxidized LDL particles have been shown to have a strong autofluorescence spectrum around 500 and 540 nm,11,37
they most likely give rise to the observed TPEF signals from lipid-rich cells, whereas CARS signals should arise from both cholesterol-rich LDL particles and lipid droplets, which are rich in CH2
groups. To verify this assignment, we recorded two-photon autofluorescence spectra of purified LDL particles (LP2-2MG, Chemicon) at different stages of oxidation. An emission maximum at 517 nm was observed after oxidation with CuSO4
for 24 h (). This value is close to our reported value of 525 nm for oxidized LDL in lipid-rich cells . However, lipid particles in lipid-rich cells showed a broader two-photon autofluorescence band . The difference in spectral maxima and shape is likely due to lipid particles comprising different stages of oxidized LDL. Another reason is that fluorescent components other than LDL are present in the excitation volume. Additionally, LDL purified from plasma and oxidized with CuSO4 might be different from oxidized LDL in lipid-rich cells that use different means of oxidation. In summary, shows the ability of our setup to monitor oxidized LDL particles and lipid droplets in the same lipid-rich cell by TPEF and CARS, respectively. As lipid-rich cells are becoming an important target for therapeutic intervention,36
this capability would be crucial to assay the functional activity of lipid-rich cells.
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 (more ...)
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) (more ...)
The rupture risk of an atheroma is associated with a thin collagen fibrous cap, increased extracellular lipid droplet deposits, and abundance of lipid-rich cells.4,6,7,38
To test whether multimodal NLO imaging is able to identify plaque rupture vulnerability, we performed SHG imaging of collagen, TPEF imaging of oxidized LDL, and CARS imaging of lipid-rich cells and extracellular lipid droplets within an atheroma. The results for a specific region are shown in . Based on the location of the internal elastic membrane detected with TPEF and the orientation of collagen fibrils detected with SHG, this atheroma was determined to be approximately 375 μm thick. Based on CARS and TPEF signal intensity as a function of the image field of view, the majority of extracellular lipid and lipid-rich cell CARS signals appeared within 200 μm of the arterial lumen . The density of extracellular lipid and lipid-rich cells in subendothelial space is further confirmed by histology of an atherosclerotic lesion stained for lipid with Oil Red O . In addition, a significant reduction of collagen density was observed in the area of the affected intima where lipid-rich cells were located . This observation is consistent with previous findings on lipid-rich cells’ secretion of proteases that degrade the extracellular matrix.2
In summary, showed that a region vulnerable to rupture risk could be clearly detected by NLO imaging of atheroma composition. In addition, as plaque rupture vulnerability is predicted based on the concentration of collagen fibrils and lipid-rich cells near the arterial lumen , NLO imaging should be capable of analyzing rupture risk, despite its inherent limitation in penetration depth.18,38
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 (more ...)
Further, SHG and TPEF imaging of an entire atherosclerotic artery was performed to determine the relative position of a vulnerable region within an atheroma (). Based on SHG imaging of collagen fibrils orientation in arterial wall and atheroma, the atheroma was found to occupy half of the artery (from 210 to 360 deg, ). Combined SHG imaging of collagen fibrils and TPEF imaging of oxidized LDL aggregates showed a significant increase of oxidized LDL density accompanying a marked reduction of collagen fibrils concentration at one shoulder region of this atheroma (, color panel 340 deg). This observation suggests that the rupture risk of this atheroma is highest at one shoulder region. The rupture risk of plaque at shoulder regions has been previously observed with histological analysis.2
Hence, multimodal NLO imaging of plaque composition permits identification of specific regions vulnerable to rupture within an atheroma without any labeling.
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 (more ...)