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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 3, Iss. 1 — Jan. 29, 2008
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Ex vivo Characterization of Atherosclerosis using Intravascular Photoacoustic Imaging

Shriram Sethuraman, James H. Amirian, Silvio H. Litovsky, Richard W. Smalling, and Stanislav Y. Emelianov  »View Author Affiliations


Optics Express, Vol. 15, Issue 25, pp. 16657-16666 (2007)
http://dx.doi.org/10.1364/OE.15.016657


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Abstract

The assessment of plaque composition is one of the important steps in the interventional management of atherosclerosis. The difference in the optical absorption between the arterial wall and plaque constituents could be utilized to obtain high resolution photoacoustic images. Therefore, intravascular photoacoustic (IVPA) imaging has the potential to play a major role in the detection and differentiation of atherosclerotic lesions. Using a rabbit model of atherosclerosis, we performed ex vivo imaging studies to evaluate the ability of IVPA imaging to detect the presence of inflammation in the plaque. Specifically, the difference in the magnitude of the photoacoustic response from the free lipids, macrophage foam cells, blood and the rest of the arterial wall were used in detecting the fibro-cellular inflammatory plaque. The constituents identified in the IVPA images were confirmed by the results from histology.

© 2007 Optical Society of America

1. Introduction

Generally, during hypercholesterolemic conditions, atherosclerotic plaques build up on the wall of the arteries and cause a stricture in the lumen. The resulting stenosis is visualized on an angiogram through radiographic images. However, angiographic studies have also demonstrated the ambiguity in the relationship between the culprit lesion and stenosis causing fatal outcomes [1

1. J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J. Leavy, M. Weiss, S. Borrico, R. Gorlin, and V. Fuster, “Angiographic progression of coronary artery disease and development of myocardial infarction,” J. Am. Col. Cardiol. 12, 56–62 (1998). [CrossRef]

, 2

2. W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows, F. R. Kahl, and W. P. Santamore, “Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease?,” Circulation 78, 1157–1166 (1988). [CrossRef] [PubMed]

]. Indeed, plaque rupture leading to acute coronary syndrome (ACS) could also occur in arteries not revealing significant stenosis in an angiogram. These high risk plaques, also called vulnerable plaques are usually characterized by the endothelial dysfunction, lipid deposition, angiogenesis, fragile fibrous cap and the accumulation of inflammatory cells [3

3. M. Naghavi, P. Libby, E. Falk, S. W. Casscells, S. Litovsky, J. Rumberger, J. J. Badimon, C. Stefanadis, P. Moreno, G. Pasterkamp, Z. Fayad, P. H. Stone, S. Waxman, P. Raggi, M. Madjid, A. Zarrabi, A. Burke, C. Yuan, P. J. Fitzgerald, D. S. Siscovick, C. L. de Korte, M. Aikawa, K. E. Juhani Airaksinen, G. Assmann, C. R. Becker, J. H. Chesebro, A. Farb, Z. S. Galis, C. Jackson, I. K. Jang, W. Koenig, R. A. Lodder, K. March, J. Demirovic, M. Navab, S. G. Priori, M. D. Rekhter, R. Bahr, S. M. Grundy, R. Mehran, A. Colombo, E. Boerwinkle, C. Ballantyne, W. Insull Jr., R. S. Schwartz, R. Vogel, P. W. Serruys, G. K. Hansson, D. P. Faxon, S. Kaul, H. Drexler, P. Greenland, J. E. Muller, R. Virmani, P. M. Ridker, D. P. Zipes, P. K. Shah, and J. T. Willerson, “From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I,” Circulation 108, 1664–1672 (2003). [CrossRef] [PubMed]

, 4

4. R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz, “Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions,” Arterioscler. Thromb. Vasc. Biol. 20, 1262–1275 (2000). [CrossRef] [PubMed]

].

The complex pathological mechanisms in the plaque progression instigate changes in the content of the plaque [5

5. P. Libby and R. Ross, Cytokines and growth regulatory molecules (Lippincott-Raven, Philadelphia, 1996).

, 6

6. R. Ross, “Atherosclerosis--an inflammatory disease,” N. Engl. J. Med. 340, 115–126 (1999). [CrossRef] [PubMed]

]. For example, the vulnerable inflammatory lesion infested with macrophage foam cells may also be accompanied by angiogenesis and intra-plaque hemorrhage. In such a scenario, apart from structural (stenosis/intimal thickening) and mechanical (soft/fibrous) changes, there is a definite change in the composition of the plaque (lipid, collagen and blood). Therefore, imaging the morphology and activity in the arterial vessel wall is essential to detect and further characterize the vulnerable plaque [3

3. M. Naghavi, P. Libby, E. Falk, S. W. Casscells, S. Litovsky, J. Rumberger, J. J. Badimon, C. Stefanadis, P. Moreno, G. Pasterkamp, Z. Fayad, P. H. Stone, S. Waxman, P. Raggi, M. Madjid, A. Zarrabi, A. Burke, C. Yuan, P. J. Fitzgerald, D. S. Siscovick, C. L. de Korte, M. Aikawa, K. E. Juhani Airaksinen, G. Assmann, C. R. Becker, J. H. Chesebro, A. Farb, Z. S. Galis, C. Jackson, I. K. Jang, W. Koenig, R. A. Lodder, K. March, J. Demirovic, M. Navab, S. G. Priori, M. D. Rekhter, R. Bahr, S. M. Grundy, R. Mehran, A. Colombo, E. Boerwinkle, C. Ballantyne, W. Insull Jr., R. S. Schwartz, R. Vogel, P. W. Serruys, G. K. Hansson, D. P. Faxon, S. Kaul, H. Drexler, P. Greenland, J. E. Muller, R. Virmani, P. M. Ridker, D. P. Zipes, P. K. Shah, and J. T. Willerson, “From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I,” Circulation 108, 1664–1672 (2003). [CrossRef] [PubMed]

, 7

7. Z. A. Fayad and V. Fuster, “Clinical imaging of the high-risk or vulnerable atherosclerotic plaque,” Circ. Res. 89, 305–316 (2001). [CrossRef] [PubMed]

].

Currently several imaging techniques including optical coherence tomography, magnetic resonance imaging, ultrafast computed tomography, thermography, angioscopy, Raman spectroscopy and intravascular palpography are under development to study the high risk plaques and each one has advantages and limitations [7

7. Z. A. Fayad and V. Fuster, “Clinical imaging of the high-risk or vulnerable atherosclerotic plaque,” Circ. Res. 89, 305–316 (2001). [CrossRef] [PubMed]

]. However, none of these techniques have yet made a clinical impact and angiography continues to be the gold standard.

Catheter-based ultrasound is a relatively new approach to the arterial vascular wall imaging. Intravascular ultrasound (IVUS) is a well developed technology that is routinely used in many interventional laboratories to guide procedures [8]. IVUS is an invasive modality that permits direct and real-time imaging of atheroma and provides high-quality cross-sectional or even volumetric views of the vessel and atherosclerotic disease with spatial resolution from 60 to 150 µm. Diagnostic applications of IVUS include detection of angiographically unrecognized disease, detection of lesions of uncertain severity, and risk stratification of atherosclerotic lesions in interventional practice. IVUS can delineate the thickness and echogenicity of vessel wall structures and may be used to select the most appropriate option of transcatheter therapy (rotational atherectomy, stents, etc.). However, histopathological information obtained with IVUS imaging is limited [8

8. S. E. Nissen and P. Yock, “Intravascular ultrasound: novel pathophysiological insights and current clinical applications,” Circulation 103, 604–616 (2001). [PubMed]

]. Angioscopy and histological studies generally report low sensitivity of IVUS in detection of thrombus and lipid-rich lesions. To overcome the limitations of IVUS imaging, intravascular photoacoustic (IVPA) imaging, integrated with intravascular ultrasound (IVUS) was introduced [9

9. S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, “Development of a combined intravascular ultrasound and photoacoustic imaging system,” Proceedings of the 2006 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing 6086, F1–F10 (2006).

, 10

10. S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, “Intravascular photoacoustic imaging using an IVUS imaging catheter,” IEEE Trans. Ultrason. Ferroelectr. Freq. Control 54, 978–986 (2007). [CrossRef] [PubMed]

].

The photoacoustic effect [11

11. A. G. Bell, “On the production and reproduction of sound by light,” Am. J. Sci. 20, 305–324 (1880).

] — the generation of acoustic waves by absorbed photons — can potentially be employed to interrogate the structure and composition of biological tissues. The deposition of optical energy by short-pulsed laser irradiation leads to thermoelastic expansion in the tissues and the subsequent generation of photoacoustic transients. Photoacoustic (or optoacoustic) image is obtained by ultrasonically detecting the laser induced acoustic waves [12

12. A. A. Oraevsky and A. A. Karabutov, Optoacoustic tomography (CRC Press, Boca Raton, Florida, 2003).

16

16. S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott “Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging,” in Proceedings of the IEEE International Ultrasonics Symposium(Vancouver, Canada, 2006), pp. 405–415.

]. The optical absorption map presented in such an image is often indicative of the physiological and pathological state of the tissue. For example, the elevated absorption of light by hemoglobin in the proliferative neovasculature compared to the adjacent tissues may be useful in detecting tumors [17

17. R. I. Siphanto, K. K. Thumma, R. G. M. Kolkman, T. G. van Leeuwen, d. M. F.F.M., J. W. van Neck, L. N. A. van Adrichem, and W. Steenbergen, “Serial noninvasive photoacoustic imaging of neovascularization in tumor angiogenesis,” Opt. Express 13, 89–95 (2004). [CrossRef]

]. The high resolution localization of tissue structures by the photoacoustic technique could also find useful application in the imaging of vulnerable atherosclerotic plaques.

2. Materials and methods

2.1 Animal model of atherosclerosis

The IVPA imaging experiments were performed on a well-characterized animal model of atherosclerosis. Cholesterol-fed rabbits are classical models for the study of lipoproteins and atherosclerosis [18

18. M. Overturf and D. Loose-Mitchell, “In vivo model system: the choice of experimental model for analysis of lipoproteins and atherosclerosis,” Curr. Opin. Lipidology 3, 179–185 (1992). [CrossRef]

]. Generally, the variation in the dietary regimen will induce lesions with varying severity. In this study, we chose a mild dietary regimen where a 1 year old New Zealand rabbit was placed on a low cholesterol diet (0.15%) over a long period of time (12 months) to produce proliferative fibrocellular lesions [19

19. M. E. Rosenfeld, A. Chait, E. L. Bierman, W. King, P. Goodwin, C. E. Walden, and R. Ross, “Lipid composition of aorta of Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Plasma lipid composition determines aortic lipid composition of hypercholesterolemic rabbits,” Arteriosclerosis 8, 338–347 (1988). [CrossRef] [PubMed]

23

23. M. Overturf, H. Sybers, J. Schapers, and H. Taegtmeyer, “Hypertension and atherosclerosis in cholesterol-fed rabbits. Part 1. Mild, two kidney one-clip goldblatt hypertension treated with enlapril,” Atherosclerosis 59, 283–299 (1986). [CrossRef] [PubMed]

]. The advanced lesions resembling human plaque development have extracellular matrix, cholesterol crystals and macrophage enriched foamy lesions [19

19. M. E. Rosenfeld, A. Chait, E. L. Bierman, W. King, P. Goodwin, C. E. Walden, and R. Ross, “Lipid composition of aorta of Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Plasma lipid composition determines aortic lipid composition of hypercholesterolemic rabbits,” Arteriosclerosis 8, 338–347 (1988). [CrossRef] [PubMed]

21

21. S. J. Daley, E. Herderick, J. F. Cornhill, and K. A. Rogers, “Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 2: Differing lesion area of volume despite equal plasma cholesterol levels,” Arteroscler. Thromb. 14, 105–114 (1994). [CrossRef]

, 24

24. J. R. Guyton and K. F. Klemp, “Early extracellular lipid deposits in aorta of cholesterol-fed rabbits,” Am. J. Pathol. 141, 925–936 (1992). [PubMed]

]. For comparison, another rabbit was placed on a normal diet for the same period of time — this rabbit served as a control animal.

Prior to ex-vivo imaging experiments, an in-vivo IVUS pull-back imaging studies (Galaxy-2, Boston Scientific, Inc.) were performed from the thoracic to the renal region either to locate and confirm the presence of suspected plaque deposition in atherosclerotic rabbit or to confirm the absence of the plaques in control animal. The rabbits were pre-anesthetized, placed on a small animal laboratory ventilator, and imaged using a 2.5F IVUS imaging catheter (Atlantis SR PLUS, Boston Scientific, Inc.). In the atherosclerotic animal, the areas suspected with the presence of plaques were identified and the locations were noted using anatomical landmarks. The rabbits were then euthanized using approved protocol and the 5 cm long normal arterial sections or vessels containing suspected plaques were stored in saline for about five hours before the ex-vivo IVUS/IVPA imaging experiments were performed.

2.2 IVPA imaging of excised rabbit aorta

The experimental setup for the ex vivo imaging of atherosclerotic plaques is illustrated in Fig. 1. The custom-designed IVUS/IVPA imaging system is comprised of five major components, namely, the optical excitation, ultrasonic excitation, ultrasound/photoacoustic detection, motion control and signal/image acquisition systems.

The arterial tissue sample was immersed in a water tank to ensure acoustic coupling. A Q-switched Nd:YAG laser (Polaris II, New Wave Research, Inc.) provided pulsed (3–5 ns) optical illumination at a wavelength of 532 nm. The laser was capable of providing a maximum energy of 24 mJ per pulse and operating at a maximum repetition frequency of 20 Hz. The atherosclerotic tissue sample was imaged using pulse energy of approximately 14 mJ and the control aorta was imaged using optical pulse energy of about 20 mJ. Optical pulses for photoacoustic imaging were delivered to the artery specimen using a prism and thereby illuminating the sample from outside (Fig. 1). Light was further broadened using a ground glass diffuser to limit the energy fluence incident on the tissue to approximately 1–2 mJ/cm2. The photoacoustic transients generated by the absorption of laser pulses were detected using a 2.5F IVUS imaging catheter (Atlantis SR PLUS, Boston Scientific Inc.) containing a 40 MHz, 0.5 mm diameter, single element ultrasound transducer. The IVUS imaging catheter, inserted in the lumen of the arterial sample, served as a common detector for both IVUS and IVPA imaging.

Fig. 1. Experimental setup of the intravascular ultrasound (IVUS) and intravascular photoacoustic (IVPA) imaging system

The IVUS and IVPA radiofrequency signals were acquired and stored for further offline processing. Multiple (typically, 16–20) A-lines were obtained and averaged at each azimuthal position to improve the signal to noise ratio (SNR). The averaged signals were filtered using a finite impulse response digital bandpass filter. Pass band cut-off frequencies (25 MHz — 45 MHz) were selected based on the detection bandwidth of the transducer element. Further, the signals were envelope detected by creating the analytical signal of the input using a Hilbert transform and then taking the absolute value of the analytical signal. The absolute value of the complex analytical signal was utilized to display both the IVUS and IVPA images. Therefore, both images were obtained without tomographic or any other type of reconstruction and speckles are expected in these images.

The scattering and absorption of light following optical illumination from the outer surface of the vessel reduces the amount of light reaching the deeper (inner) regions in the aorta. As a result, the magnitude of photoacoustic signal was small at these spatial locations in the arterial wall. Therefore, in our particular experimental setup, the optical attenuation reduced the magnitude of photoacoustic signals from the intima. We used an exponential time gain compensation factor calculated based on the effective optical attenuation coefficient (µeff). The exponential compensation curve with a coefficient of 20.6 cm-1 was utilized based on the absorption (µa) and reduced scattering coefficient (µs′) reported for an advanced fibrous atheroma [25

25. A. A. Oraevsky, S. L. Jacques, and F. K. Tittel, “Measurement of tissue optical properties by time-resolved detection of laser-induced transient stress,” Appl. Opt. 36, 402–415 (1997). [CrossRef] [PubMed]

]. Finally, the image in the polar (R-θ) system of coordinates was scan converted to the Cartesian (X-Y) system of coordinates using a cubic interpolation method. Since the IVUS and IVPA images were inherently coregistered during the experimental procedure, the IVPA image was overlaid on the IVUS image to evaluate the photoacoustic response from the plaque in the context of the structure of the aortic tissue.

2.3 Histological analysis

After the imaging experiments, the tissue samples were marked at the location of the imaged cross-section, fixed in 10% buffered formaldehyde solution and dissected. The arterial segments were subsequently processed for routine paraffin embedding and sections of the artery were sliced at 5 µm intervals close to the imaged spot. For each segment, cross-sections were stained for collagen with Picrosirius red stain, for macrophage foam cells with RAM-11 and general morphology based on the intimal-medial thickness with Hematoxylin & Eosin (H&E). The lipids encapsulated in macrophage cells were identified in the sections as positively stained brown by RAM-11, a marker for macrophages. The H&E and RAM-11 stained sections were observed under the bright field microscope. The collagen is naturally birefringent due to the arrangement of the fibers and this property is enhanced by the Picrosirius Red dye. Under a linearly polarized light, the fibers (collagen type I and III) can be distinguished based on the colored appearance [26

26. T. Neumann, A. Vollmer, T. Schaffner, O. M. Hess, and G. Heusch, “Diastolic dysfunction and collagen structure in canine pacing-induced heart failure,” J. Mol. Cell. Cardiol. 31, 179–192 (1999). [CrossRef] [PubMed]

]. As fiber thickness increases, the color changes from green to yellow to orange. The histological photomicrographs were correlated with the visual assessment of the IVUS and IVPA images.

3. Results

Representative IVUS, IVPA and histological cross-sectional images of the aorta excised from cholesterol fed rabbit are shown in Fig. 2. The 6.75 mm diameter grayscale IVUS image in Fig. 2(a) is presented using 40 dB display dynamic range. The IVUS image suggests the presence of a diffused plaque all along the inner layer of the aorta (marked by the arrow). The contrast in the IVUS B-Scan is not sufficient to clearly demarcate the lesion. However, the thickening of the intimal layer combined with a definite presence of hypoechoic ultrasonic signals may suggest the presence of a lipid filled plaque. The mean thickness of the vessel wall in the IVUS image is 1.2 mm and the diameter of the lumen measures about 3 mm.

The attenuation-compensated IVPA image in Fig. 2(b) represents the photoacoustic response from the atherosclerotic rabbit aorta. This color image is displayed using 37 dB dynamic range. An important feature in the IVPA image is the dark region in deeper regions of the plaque. The low magnitude of IVPA response could be attributed to the presence of lipids — a weak light absorber at 532 nm. Furthermore, the IVPA image also indicates relatively strong photoacoustic response from the media, medial-adventitial boundary and the inner layer of the intimal plaque as marked by arrows in Fig. 2(b). The combined IVUS/IVPA image in Fig. 2(c) allows the analysis of the photoacoustic response from tissue within the structural content of the vessel wall provided by IVUS image. The IVUS/IVPA image shows ultrasound echo and photoacoustic signal correspondence in the fibrous regions of the aorta. The low-amplitude photoacoustic signal matches the plaque-media boundary containing hypoechoic IVUS signals. The concentric rings in the center of image in Figs. 2(b) and 2(c) are the strong photoacoustic response generated on the surface of the transducer element.

The contribution of the plaque components to the photoacoustic response was evaluated by analyzing the tissue histology. The high-resolution photographs of the cross-sections stained with H&E, RAM-11 and Picrosirius red are presented in Fig. 2(d–f). The H&E stained image in Fig. 2(d) indicated the general morphology of the aorta and plaque. The increase in the thickness of the intima is clearly seen in the image. The migration of the smooth-muscle cells from the media into the intima in addition to the accumulation of lipids cause an increase in the size of the intima whereas a normal intimal layer is usually a thin endothelial layer lining the arterial wall. The result of progressive cellular events involving monocyte migration into the intima, accumulation of macrophages and subsequent encapsulation of lipids into the macrophage cells is seen as dense clusters of macrophage foam cells in Fig. 2(e). These macrophages filled with lipids are stained brown by RAM-11 and are accumulated all over the plaque. Furthermore, the smooth muscle cells migrating into the plaque is responsible for the synthesis of fibrous collagen. The result of this process is manifested as thick type I collagen fibers. The polarization photomicrograph (Fig. 2(f)) of the tissue section stained with Picrosirius red suggests the presence of normal and thin Type III collagen all along the vessel and focally dense deposits of the thick Type I collagen in the plaque. The histological report analysis also suggests that the plaque appears to be more cellular (lipid filled macrophage cells) than fibrous (collagen).

Fig. 2. Ex vivo IVUS/IVPA images (6.75 mm diameter) and histology of an advanced atherosclerotic lesion. (a) The IVUS image of the arterial cross-section shows a hypoechoic concentric plaque with significant structural thickening of the intima. (b) The IVPA image at 532 nm optical excitation with lipids in the plaque indicated by low photoacoustic signals. The higher photoacoustic response from the rest of the plaque corresponds to the presence of the fibrous collagenous cap infiltrated with macrophages. (c) Coregistered IVUS and IVPA image. (d) H&E stained histology image. (e) RAM11 stained image showing the highly expressed RAM11 antigen with intense macrophages embedded in the plaque. (f) Polarized image of the Picrosirius red stained cross-section showing the presence of focally dense collagen in the plaque.

In contrast, both the IVUS and IVPA images from the control aorta presented in Fig. 3 have a homogeneous appearance. The IVUS image in Fig. 3(a) accurately represents the anatomical features of the aorta. This grayscale ultrasound image is shown using 40 dB display dynamic range. The field of view of this image and all other images in Fig. 3 is 8.25 mm in diameter. The IVUS images presented in Figs. 2 and 3 were obtained from approximately similar regions in the aorta from the respective rabbits — 48 mm from the aortic arch for the control animal compared to 53 mm for the atherosclerotic rabbit. Therefore, it is reasonable to compare the thickness of the wall and the size of the lumen. The thickness of the tissue wall in the normal aorta was smaller compared to the plaque laden aorta and the lumen was intact with no significant constriction. The luminal diameter measured from the IVUS image was approximately 4.5 mm and the mean wall thickness measured 0.9 mm. The thin wall of the aorta made it difficult to mechanically rotate and perform the scan and therefore artifacts resulting from irregular motion are evident in the IVUS B-scan at several azimuthal locations (e.g., 11 o’clock and 5 o’clock).

Fig. 3. IVUS and IVPA images (8.25 mm diameter) and the histology of the cross-section of the aorta from the control rabbit. (a) The IVUS image of the normal rabbit aorta with no plaques. The lumen of the normal aorta is larger than the plaque-filled aorta. (b) The IVPA image details uniform photoacoustic signals from the fibrous tissue (elastin and collagen) layers. (c) The coregistered IVUS and IVPA image. (d–f) The histological images stained with Hematoxylin-Eosin (H&E), RAM-11 and Picrosirius red. The histology confirms the absence of the plaque

The IVPA image of the normal aorta is presented in Fig. 3(b) using 45 dB display dynamic range. The photoacoustic response is relatively homogeneous from all parts of the tissue in the IVPA image. Indeed, the IVPA images of the normal aorta obtained from the control rabbit does not indicate the layered appearance. Overall, these photoacoustic signals in Fig. 3(b) are similar to the photoacoustic response obtained at the fibrous medial-adventitial boundary regions in atherosclerotic aorta in Fig. 2(b). The combined image in Fig. 3(c) shows the IVPA image overlaid on the IVUS B-scan. The normal aortic section stained by H&E in Fig. 3(d) was characterized by a thin intima composed of an endothelial cell layer with an underlying media composed of elastic fibers and smooth muscle cells. The absence of macrophages is clearly indicated by the RAM-11 stained image in Fig. 3(e). The polarization microscopy image of the tissue section stained with Picrosirius red in Fig. 3(f) demonstrates the presence of the normal thin collagen. The histology images show a cut in the media; this is an artifact that resulted from the frozen sectioning. Further, the decrease in the size of the aorta in the histological images may be a result of the tissue shrinkage during the histological analysis.

4. Discussion

The ex-vivo IVPA imaging studies on excised samples of an atherosclerotic rabbit aorta was primarily aimed at understanding the photoacoustic response from an advanced fibro-cellular plaque. The IVUS image indicated the presence of a diffused plaque but could not clearly demarcate the lesion. There was a morphological indication of the presence of the lesion as observed by a thickening of the intima and deterioration of the ultrasound speckle in the plaque. In contrast, the difference in the magnitude of photoacoustic response helped in spatially localizing the lipid and fibrous regions of the plaque. The normal aorta comprising thin fibrillar collagen, however, produced homogeneous photoacoustic response from the vessel wall.

The IVPA images in Fig. 2 and Fig. 3 cannot be compared quantitatively. Indeed, the energies employed to obtain the IVPA images were different and, therefore, the optical fluence was also slightly different in two measurements. Furthermore, the images are shown using different display dynamic range. However, the IVPA images can be compared qualitatively. Generally, the energy delivered to the vessel wall and atherosclerotic plaques will depend on several factors including size of the lumen, proximity of the integrated IVUS/IVPA probe to the vessel wall, etc.

Although both IVUS and IVPA imaging techniques are based on acoustic detection, the process of signal generation is different. The photoacoustic response results from the wavelength dependent optical absorption characteristics of the heterogeneous tissues and plaque. Therefore the magnitude of signals in the IVPA image depends in part on the wavelength of the laser used for excitation. For example, as is evident from the image in Fig. 2(b), the laser-induced acoustic response from the lipid filled plaque was generally low in comparison to the signal from the rest of the fibrous tissue. Indeed, the mammalian fat has low absorption (µa=0.01 cm-1) at 532 nm [27

27. R. L. P. van Veen, H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, and R. Cubeddu, “Determination of visible near-IR absorption coefficients of mammalian fat using time- and spatially resolved diffuse reflectance and transmission spectroscopy,” J. Biomed. Opt. 10, 0540041–0540046 (2005). [CrossRef]

] while the maximum absorption coefficient (µa=0.1 cm-1) occurs at 920 nm [27

27. R. L. P. van Veen, H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, and R. Cubeddu, “Determination of visible near-IR absorption coefficients of mammalian fat using time- and spatially resolved diffuse reflectance and transmission spectroscopy,” J. Biomed. Opt. 10, 0540041–0540046 (2005). [CrossRef]

, 28

28. B. J. Tromberg, N. Shah, R. Lanning, A. Cerussi, J. Espinoza, T. Pham, L. Svaasand, and J. Butler, “Noninvasive in vivo characterization of breast tumors using photon migration spectroscopy,” Neoplasia 2, 26–40 (2000). [CrossRef] [PubMed]

]. Nevertheless, the photoacoustic regions of low photoacoustic signal provided the contrast to distinguish the plaque from the adjoining tissue.

Another component of the vulnerable plaque that has significant implications on the plaque stability is the monocyte derived macrophage cells [29

29. P. R. Moreno, E. Falk, I. F. Palacios, J. B. Newell, V. Fuster, and J. T. Fallon, “Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture,” Circulation 90, 775–778 (1994). [PubMed]

]. Dense macrophage cells encapsulating the lipids were observed throughout the plaque as indicated by the positive expression of RAM-11 antigen. Indeed, the lipids in the macrophage cells are expected to render optical characteristics similar to the free lipids in the plaque. Therefore, the macrophages and the free lipids may contribute to the overall photoacoustic signal from the plaque. However, these signals are lower in magnitude compared to the surrounding fibrous tissue. In addition to these cellular components, the plaque also contains some fibrous regions comprised of collagen types I and III. Generally, we observed high signal from these fibrous regions. Although the optical absorption by collagen may not be significant, these fibrous regions are highly scattering leading to an increase in optical fluence and therefore contributing to the overall photoacoustic response.

A significantly higher magnitude of photoacoustic signal was noticable from the portion of the intimal layer close to the lumen. Although these signals observed in the IVPA image in Fig. 2(b) were enhanced by the exponential time gain compensation, but even without such compensation the response from these fibrous regions was significant. The possibility of high sub-surface laser fluence causing a higher magnitude of photoacoustic response is limited to the outer boundary of the vessel facing laser incidence. In the boundary close to the lumen and further away from the laser incidence, optical absorption is the most likely cause for the IVPA signals. Indeed, inflammation in the plaque is often accompanied by the development of a network of micro-vessels from the tunica adventitia and the lumen that serve to nourish the plaque [30

30. P. R. Moreno, K. R. Purushothaman, V. Fuster, D. Echeverri, H. Truszczynska, S. K. Sharma, J. J. Badimon, and W. N. O’Connor, “Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability,” Circulation 110, 2032–2038 (2004). [CrossRef] [PubMed]

, 31

31. M. Jeziorska and D. E. Woolley, “Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development,” Hum. Pathol. 30, 919–925 (1999). [CrossRef] [PubMed]

]. Therefore, blood from these vessels could be responsible for the high magnitude signal from the thick fibrous cap. These angiogenic vessels may serve as valuable optical absorbers to identify inflammation during plaque progression. Possibilities that include artifacts arising from residual blood staining the lumen cannot be completely eliminated. However, the aorta was flushed with saline several times before the experiment to minimize the effect of luminal wall staining by blood. Therefore, we suspect that a combination of a network of micro-vessels, water and modified thick collagen contribute to the overall high photoacoustic response.

The ex vivo experiments were performed in saline to eliminate the absorption effects of blood at the laser excitation wavelength of 532 nm. Indeed, the absence of blood helped us to analyze the photoacoustic signal contribution from the other constituents of the plaque and aorta. During in vivo IVPA imaging, it is anticipated that blood absorption will produce photoacoustic signals and also decrease the laser fluence incident on the artery and plaque. Therefore, to maximize light penetration and obtain sufficient image contrast, IVPA imaging could be performed in the optical wavelength range of 700–900 nm. Furthermore, the photoacoustic signals from the luminal blood can be identified and filtered out by using the boundary of the arterial lumen as the reference from the IVUS image. Nevertheless, in-vivo IVPA imaging at 532 nm accompanied by a temporary flushing of blood may be useful in imaging the angiogenesis and vasa vasorum in the plaque.

The optical absorption properties of the aortic plaque and tissue components change over a wide range of wavelengths in the near-infrared spectrum. Therefore, limiting the optical excitation wavelength to a single value may not result in an IVPA image with the best contrast. To improve differentiation of the plaque and enhance image contrast, an analysis of the trend of spectral variation of absorption obtained from the change in the magnitude of the photoacoustic response can be utilized.

5. Conclusions

The results of the ex vivo plaque characterization studies utilizing IVPA imaging at 532 nm laser excitation indicated the potential to detect a mixed fibro-cellular advanced atherosclerotic plaque. The varying magnitudes of photoacoustic response provided the contrast to demarcate the inflammatory lesion. Our results indicating low photoacoustic response from the accumulated lipids and foam cells, high signals from the angiogenic vessels in the inflammatory site and homogeneous IVPA response in the fibrous regions of the plaque was validated by the analysis from the histology images. The significance of these studies is immense and a combined IVUS and IVPA imaging technique has the potential to serve as a comprehensive imaging utility in interventional studies. However, several studies and significant understanding of the imaging technique are required to confirm the ability to detect and differentiate a vulnerable plaque. Therefore, it is too early to draw definite conclusions about the clinical translation and utility of a combined IVUS/IVPA imaging to identify composition of vulnerable atherosclerotic plaques.

Acknowledgments

This work was partially supported by the American Heart Association under grant 0655033Y and National Institutes of Health under grants EB004963 and HL084076. The authors would like to acknowledge the technical support from Boston Scientific, Inc., and Mrs. Srivalleesha Mallidi and Mrs. Patty Richards for help with the animal experiments.

References and links

1.

J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J. Leavy, M. Weiss, S. Borrico, R. Gorlin, and V. Fuster, “Angiographic progression of coronary artery disease and development of myocardial infarction,” J. Am. Col. Cardiol. 12, 56–62 (1998). [CrossRef]

2.

W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows, F. R. Kahl, and W. P. Santamore, “Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease?,” Circulation 78, 1157–1166 (1988). [CrossRef] [PubMed]

3.

M. Naghavi, P. Libby, E. Falk, S. W. Casscells, S. Litovsky, J. Rumberger, J. J. Badimon, C. Stefanadis, P. Moreno, G. Pasterkamp, Z. Fayad, P. H. Stone, S. Waxman, P. Raggi, M. Madjid, A. Zarrabi, A. Burke, C. Yuan, P. J. Fitzgerald, D. S. Siscovick, C. L. de Korte, M. Aikawa, K. E. Juhani Airaksinen, G. Assmann, C. R. Becker, J. H. Chesebro, A. Farb, Z. S. Galis, C. Jackson, I. K. Jang, W. Koenig, R. A. Lodder, K. March, J. Demirovic, M. Navab, S. G. Priori, M. D. Rekhter, R. Bahr, S. M. Grundy, R. Mehran, A. Colombo, E. Boerwinkle, C. Ballantyne, W. Insull Jr., R. S. Schwartz, R. Vogel, P. W. Serruys, G. K. Hansson, D. P. Faxon, S. Kaul, H. Drexler, P. Greenland, J. E. Muller, R. Virmani, P. M. Ridker, D. P. Zipes, P. K. Shah, and J. T. Willerson, “From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I,” Circulation 108, 1664–1672 (2003). [CrossRef] [PubMed]

4.

R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz, “Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions,” Arterioscler. Thromb. Vasc. Biol. 20, 1262–1275 (2000). [CrossRef] [PubMed]

5.

P. Libby and R. Ross, Cytokines and growth regulatory molecules (Lippincott-Raven, Philadelphia, 1996).

6.

R. Ross, “Atherosclerosis--an inflammatory disease,” N. Engl. J. Med. 340, 115–126 (1999). [CrossRef] [PubMed]

7.

Z. A. Fayad and V. Fuster, “Clinical imaging of the high-risk or vulnerable atherosclerotic plaque,” Circ. Res. 89, 305–316 (2001). [CrossRef] [PubMed]

8.

S. E. Nissen and P. Yock, “Intravascular ultrasound: novel pathophysiological insights and current clinical applications,” Circulation 103, 604–616 (2001). [PubMed]

9.

S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, “Development of a combined intravascular ultrasound and photoacoustic imaging system,” Proceedings of the 2006 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing 6086, F1–F10 (2006).

10.

S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, “Intravascular photoacoustic imaging using an IVUS imaging catheter,” IEEE Trans. Ultrason. Ferroelectr. Freq. Control 54, 978–986 (2007). [CrossRef] [PubMed]

11.

A. G. Bell, “On the production and reproduction of sound by light,” Am. J. Sci. 20, 305–324 (1880).

12.

A. A. Oraevsky and A. A. Karabutov, Optoacoustic tomography (CRC Press, Boca Raton, Florida, 2003).

13.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol. 21, 803–806 (2003). [CrossRef] [PubMed]

14.

S. Y. Emelianov, S. R. Aglyamov, J. Shah, S. Sethuraman, W. G. Scott, R. Schmitt, M. Motamedi, A. Karpiouk, and A. Oraevsky, “Combined ultrasound, optoacoustic and elasticity imaging,” Proceedings of the 2004 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing 5320, 101–112 (2004).

15.

P. C. Beard and T. N. Mills, “Characterization of post mortem arterial tissue using time-resolved photoacoustic spectroscopy at 436, 461 and 532 nm,” Phys Med Biol 42, 177–198 (1997). [CrossRef] [PubMed]

16.

S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott “Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging,” in Proceedings of the IEEE International Ultrasonics Symposium(Vancouver, Canada, 2006), pp. 405–415.

17.

R. I. Siphanto, K. K. Thumma, R. G. M. Kolkman, T. G. van Leeuwen, d. M. F.F.M., J. W. van Neck, L. N. A. van Adrichem, and W. Steenbergen, “Serial noninvasive photoacoustic imaging of neovascularization in tumor angiogenesis,” Opt. Express 13, 89–95 (2004). [CrossRef]

18.

M. Overturf and D. Loose-Mitchell, “In vivo model system: the choice of experimental model for analysis of lipoproteins and atherosclerosis,” Curr. Opin. Lipidology 3, 179–185 (1992). [CrossRef]

19.

M. E. Rosenfeld, A. Chait, E. L. Bierman, W. King, P. Goodwin, C. E. Walden, and R. Ross, “Lipid composition of aorta of Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Plasma lipid composition determines aortic lipid composition of hypercholesterolemic rabbits,” Arteriosclerosis 8, 338–347 (1988). [CrossRef] [PubMed]

20.

S. J. Daley, E. Herderick, J. F. Cornhill, and K. A. Rogers, “Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 1: Differing lesion area of volume despite equal plasma cholesterol levels,” Arteroscler. Thromb. 14, 95–104 (1994). [CrossRef]

21.

S. J. Daley, E. Herderick, J. F. Cornhill, and K. A. Rogers, “Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 2: Differing lesion area of volume despite equal plasma cholesterol levels,” Arteroscler. Thromb. 14, 105–114 (1994). [CrossRef]

22.

J. B. Atkinson, R. L. Hoover, K. K. Berry, and L. L. Swift, “Cholesterol-fed heterozygous watanabe heritable hyperlipidemic rabbit: A new model for Atherosclerosis,” Atherosclerosis 78, 123–126 (1989). [CrossRef] [PubMed]

23.

M. Overturf, H. Sybers, J. Schapers, and H. Taegtmeyer, “Hypertension and atherosclerosis in cholesterol-fed rabbits. Part 1. Mild, two kidney one-clip goldblatt hypertension treated with enlapril,” Atherosclerosis 59, 283–299 (1986). [CrossRef] [PubMed]

24.

J. R. Guyton and K. F. Klemp, “Early extracellular lipid deposits in aorta of cholesterol-fed rabbits,” Am. J. Pathol. 141, 925–936 (1992). [PubMed]

25.

A. A. Oraevsky, S. L. Jacques, and F. K. Tittel, “Measurement of tissue optical properties by time-resolved detection of laser-induced transient stress,” Appl. Opt. 36, 402–415 (1997). [CrossRef] [PubMed]

26.

T. Neumann, A. Vollmer, T. Schaffner, O. M. Hess, and G. Heusch, “Diastolic dysfunction and collagen structure in canine pacing-induced heart failure,” J. Mol. Cell. Cardiol. 31, 179–192 (1999). [CrossRef] [PubMed]

27.

R. L. P. van Veen, H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, and R. Cubeddu, “Determination of visible near-IR absorption coefficients of mammalian fat using time- and spatially resolved diffuse reflectance and transmission spectroscopy,” J. Biomed. Opt. 10, 0540041–0540046 (2005). [CrossRef]

28.

B. J. Tromberg, N. Shah, R. Lanning, A. Cerussi, J. Espinoza, T. Pham, L. Svaasand, and J. Butler, “Noninvasive in vivo characterization of breast tumors using photon migration spectroscopy,” Neoplasia 2, 26–40 (2000). [CrossRef] [PubMed]

29.

P. R. Moreno, E. Falk, I. F. Palacios, J. B. Newell, V. Fuster, and J. T. Fallon, “Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture,” Circulation 90, 775–778 (1994). [PubMed]

30.

P. R. Moreno, K. R. Purushothaman, V. Fuster, D. Echeverri, H. Truszczynska, S. K. Sharma, J. J. Badimon, and W. N. O’Connor, “Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability,” Circulation 110, 2032–2038 (2004). [CrossRef] [PubMed]

31.

M. Jeziorska and D. E. Woolley, “Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development,” Hum. Pathol. 30, 919–925 (1999). [CrossRef] [PubMed]

OCIS Codes
(110.5120) Imaging systems : Photoacoustic imaging
(110.7170) Imaging systems : Ultrasound
(170.2150) Medical optics and biotechnology : Endoscopic imaging
(170.3880) Medical optics and biotechnology : Medical and biological imaging

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: September 4, 2007
Revised Manuscript: November 28, 2007
Manuscript Accepted: November 29, 2007
Published: November 30, 2007

Virtual Issues
Vol. 3, Iss. 1 Virtual Journal for Biomedical Optics

Citation
Shriram Sethuraman, James H. Amirian, Silvio H. Litovsky, Richard W. Smalling, and Stanislav Y. Emelianov, "Ex vivo Characterization of Atherosclerosis using Intravascular Photoacoustic Imaging," Opt. Express 15, 16657-16666 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-15-25-16657


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References

  1. J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J. Leavy, M. Weiss, S. Borrico, R. Gorlin, and V. Fuster, "Angiographic progression of coronary artery disease and development of myocardial infarction," J. Am. Col. Cardiol. 12, 56-62 (1998). [CrossRef]
  2. W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows, F. R. Kahl, and W. P. Santamore, "Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease?," Circulation 78, 1157-1166 (1988). [CrossRef] [PubMed]
  3. M. Naghavi, P. Libby, E. Falk, S. W. Casscells, S. Litovsky, J. Rumberger, J. J. Badimon, C. Stefanadis, P. Moreno, G. Pasterkamp, Z. Fayad, P. H. Stone, S. Waxman, P. Raggi, M. Madjid, A. Zarrabi, A. Burke, C. Yuan, P. J. Fitzgerald, D. S. Siscovick, C. L. de Korte, M. Aikawa, K. E. Juhani Airaksinen, G. Assmann, C. R. Becker, J. H. Chesebro, A. Farb, Z. S. Galis, C. Jackson, I. K. Jang, W. Koenig, R. A. Lodder, K. March, J. Demirovic, M. Navab, S. G. Priori, M. D. Rekhter, R. Bahr, S. M. Grundy, R. Mehran, A. Colombo, E. Boerwinkle, C. Ballantyne, W. Insull, Jr., R. S. Schwartz, R. Vogel, P. W. Serruys, G. K. Hansson, D. P. Faxon, S. Kaul, H. Drexler, P. Greenland, J. E. Muller, R. Virmani, P. M. Ridker, D. P. Zipes, P. K. Shah, and J. T. Willerson, "From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I," Circulation 108, 1664-1672 (2003). [CrossRef] [PubMed]
  4. R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz, "Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions," Arterioscler. Thromb. Vasc. Biol. 20, 1262-1275 (2000). [CrossRef] [PubMed]
  5. P. Libby, and R. Ross, Cytokines and growth regulatory molecules (Lippincott-Raven, Philadelphia, 1996).
  6. R. Ross, "Atherosclerosis--an inflammatory disease," N. Engl. J. Med. 340, 115-126 (1999). [CrossRef] [PubMed]
  7. Z. A. Fayad, and V. Fuster, "Clinical imaging of the high-risk or vulnerable atherosclerotic plaque," Circ. Res. 89, 305-316 (2001). [CrossRef] [PubMed]
  8. S. E. Nissen, and P. Yock, "Intravascular ultrasound: novel pathophysiological insights and current clinical applications," Circulation 103, 604-616 (2001). [PubMed]
  9. S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, "Development of a combined intravascular ultrasound and photoacoustic imaging system," Proceedings of the 2006 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing 6086, F1-F10 (2006).
  10. S. Sethuraman, S. R. Aglyamov, J. H. Amirian, R. W. Smalling, and S. Y. Emelianov, "Intravascular photoacoustic imaging using an IVUS imaging catheter," IEEE Trans. Ultrason. Ferroelectr. Freq. Control 54, 978-986 (2007). [CrossRef] [PubMed]
  11. A. G. Bell, "On the production and reproduction of sound by light," Am. J. Sci. 20, 305-324 (1880).
  12. A. A. Oraevsky, and A. A. Karabutov, Optoacoustic tomography (CRC Press, Boca Raton, Florida, 2003).
  13. X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, "Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain," Nat. Biotechnol. 21, 803-806 (2003). [CrossRef] [PubMed]
  14. S. Y. Emelianov, S. R. Aglyamov, J. Shah, S. Sethuraman, W. G. Scott, R. Schmitt, M. Motamedi, A. Karpiouk, and A. Oraevsky, "Combined ultrasound, optoacoustic and elasticity imaging," Proceedings of the 2004 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing 5320, 101-112 (2004).
  15. P. C. Beard, and T. N. Mills, "Characterization of post mortem arterial tissue using time-resolved photoacoustic spectroscopy at 436, 461 and 532 nm," Phys Med Biol 42, 177-198 (1997). [CrossRef] [PubMed]
  16. S. Y. Emelianov, S. R. Aglyamov, A. B. Karpiouk, S. Mallidi, S. Park, S. Sethuraman, J. Shah, R. W. Smalling, J. M. Rubin, and W. G. Scott, "Synergy and applications of combined ultrasound, elasticity, and photoacoustic imaging," in Proceedings of the IEEE International Ultrasonics Symposium(Vancouver, Canada, 2006), pp. 405-415.
  17. R. I. Siphanto, K. K. Thumma, R. G. M. Kolkman, T. G. van Leeuwen, d. M. F.F.M., J. W. van Neck, L. N. A. van Adrichem, and W. Steenbergen, "Serial noninvasive photoacoustic imaging of neovascularization in tumor angiogenesis," Opt. Express 13, 89-95 (2004). [CrossRef]
  18. M. Overturf, and D. Loose-Mitchell, "In vivo model system: the choice of experimental model for analysis of lipoproteins and atherosclerosis," Curr. Opin. Lipidology 3, 179-185 (1992). [CrossRef]
  19. M. E. Rosenfeld, A. Chait, E. L. Bierman, W. King, P. Goodwin, C. E. Walden, and R. Ross, "Lipid composition of aorta of Watanabe heritable hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Plasma lipid composition determines aortic lipid composition of hypercholesterolemic rabbits," Arteriosclerosis 8, 338-347 (1988). [CrossRef] [PubMed]
  20. S. J. Daley, E. Herderick, J. F. Cornhill, and K. A. Rogers, "Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 1: Differing lesion area of volume despite equal plasma cholesterol levels," Arteroscler. Thromb. 14, 95-104 (1994). [CrossRef]
  21. S. J. Daley, E. Herderick, J. F. Cornhill, and K. A. Rogers, "Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 2: Differing lesion area of volume despite equal plasma cholesterol levels," Arteroscler. Thromb. 14, 105-114 (1994). [CrossRef]
  22. J. B. Atkinson, R. L. Hoover, K. K. Berry, and L. L. Swift, "Cholesterol-fed heterozygous watanabe heritable hyperlipidemic rabbit: A new model for Atherosclerosis," Atherosclerosis 78, 123-126 (1989). [CrossRef] [PubMed]
  23. M. Overturf, H. Sybers, J. Schapers, and H. Taegtmeyer, "Hypertension and atherosclerosis in cholesterol-fed rabbits. Part 1. Mild, two kidney one-clip goldblatt hypertension treated with enlapril," Atherosclerosis 59, 283-299 (1986). [CrossRef] [PubMed]
  24. J. R. Guyton, and K. F. Klemp, "Early extracellular lipid deposits in aorta of cholesterol-fed rabbits," Am. J. Pathol. 141, 925-936 (1992). [PubMed]
  25. A. A. Oraevsky, S. L. Jacques, and F. K. Tittel, "Measurement of tissue optical properties by time-resolved detection of laser-induced transient stress," Appl. Opt. 36, 402-415 (1997). [CrossRef] [PubMed]
  26. T. Neumann, A. Vollmer, T. Schaffner, O. M. Hess, and G. Heusch, "Diastolic dysfunction and collagen structure in canine pacing-induced heart failure," J. Mol. Cell. Cardiol. 31, 179-192 (1999). [CrossRef] [PubMed]
  27. R. L. P. van Veen, H. J. C. M. Sterenborg, A. Pifferi, A. Torricelli, and R. Cubeddu, "Determination of visible near-IR absorption coefficients of mammalian fat using time- and spatially resolved diffuse reflectance and transmission spectroscopy," J. Biomed. Opt. 10, 0540041-0540046 (2005). [CrossRef]
  28. B. J. Tromberg, N. Shah, R. Lanning, A. Cerussi, J. Espinoza, T. Pham, L. Svaasand, and J. Butler, "Non-invasive in vivo characterization of breast tumors using photon migration spectroscopy," Neoplasia 2, 26-40 (2000). [CrossRef] [PubMed]
  29. P. R. Moreno, E. Falk, I. F. Palacios, J. B. Newell, V. Fuster, and J. T. Fallon, "Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture," Circulation 90, 775-778 (1994). [PubMed]
  30. P. R. Moreno, K. R. Purushothaman, V. Fuster, D. Echeverri, H. Truszczynska, S. K. Sharma, J. J. Badimon, and W. N. O'Connor, "Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability," Circulation 110, 2032-2038 (2004). [CrossRef] [PubMed]
  31. M. Jeziorska, and D. E. Woolley, "Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development," Hum. Pathol. 30, 919-925 (1999). [CrossRef] [PubMed]

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