Laser Raman spectroscopy of calcified atherosclerotic lesions in cardiovascular tissue Richard H. Clarke, Eugene B. Hanlon, Jeffrey M. isner, and Harris Brody
The use of lasers for the treatment of atherosclerosis by laser-initiated vaporization or ablation of the diseased tissue site requires knowledge of the composition of the site to be treated. The potential complexity of the lesions in terms of their heterogeneous physical and chemical makeup at a site to be treated means that laser conditions required for vaporization may have to be varied in accordance with the nature of the site undergoing treatment. An extreme example is the presence of calcific deposits, deposits which are far more refractory to laser ablation than is noncalcified soft plaque.1
Any laser treatment of a tissue will of necessity result in a degree of light scattering of the laser energy by both the surface and bulk at the tissue target. The Raman component of scattering carries with it information about the chemical composition of the irradiated tissue, information which may be of value in characterizing the tissue site for subsequent laser phototherapy.
We examined the Raman scattering of laser light from the surface of diseased and healthy tissue sites on postmortem specimens of aortic valve leaflets and coronary artery segments. The formalin-fixed tissue samples were studied as both intact segments and with the calcific sites exposed by dissection. In vitro radiographs of each specimen documented the presence and extent of tissue calcification. The samples were mounted in a freestanding configuration and were irradiated with the 514.5-nm output of a Spectra-Physics argon-ion laser. The interference-filtered laser line was limited to the 50-110-mW range as metered at the sample.
Orthogonally scattered light was collected into a Spex 1403 Raman spectrometer (1800-groove/mm holographic gratings). Spectra were dispersed to a bandpass of one to five wavenumbers and detected by a cooled Hamamatsu 956 photomultipher tube used with Spex DMl-B photon counting detection and electronics.
Mineralized plaque specimens for the Raman experiments were excised from the surrounding soft tissue, washed in distilled water, and dried at room temperature. Raman spectra were obtained from the intact surfaces of such samples as well as the bulk interior which had been exposed by cleaving. A total of fourteen Raman runs from three aortic valve samples and ten Raman spectra on two arterial segments were performed in the course of these experiments.
Fig. 1. Laser Raman scattering (514-nm laser excitation) observed over the 500-1200-cm-1 region from (top) a powdered sample of calcium hydroxyapatite; (middle) calcific region dissected out from a human aortic valve, background fluorescence subtracted out; (bottom) a calcified segment of human coronary artery, background
fluorescence subtracted out.
As a comparison of the spectral features from samples with extensive mineral deposition, a Raman spectrum of a standard sample of powdered calcium hyd roxyapa t i t e , Ca10(OH)2(PO4)6, was first obtained. A portion of this spectrum (from 500 to 1200 cm - 1) is displayed in Fig. 1. The dominant 960-cm-1 peak is characteristic of the presence of the phosphate group and is expected to provide the most easily detected feature indicative of calcific mineralization.
A typical Raman spectrum for an exposed calcific site on an aortic valve leaflet is also shown in Fig. 1. A broad featureless sloping background due to competitive fluorescence emission has been computer subtracted from these spectra. In general, the intensity of competing fluorescence varies with the coloration of the sample site exposed; in all cases, however, the same Raman features in Fig. 1 are ob-
served superimposed on the fluorescence background from every one of the four valve samples run in our experiments. When a comparison scattering spectrum was run on the tissue regions adjacent to the diseased site, regions with no visible evidence for any disease, no Raman features were observed, even in conditions of maximum gain.
Scattering experiments were also run on segments of coronary arteries which have been exposed at sites indicated to have extensive calcification by x-ray analysis. The bottom spectrum in Fig. 1 for the 500-1200-cm-1 region is typical of the results obtained for calcified regions of the coronary artery segments. Again, there is competitive fluorescence observable as a broad background, although not as extensive as in the case of the aortic valve leaflets. No similar Raman scattering was observed from the adjacent normal (disease-free) tissue sites.
The principal bands observed in the Raman spectra from the mineralized tissue samples are characterizable on the basis of the phosphate ion spectrum. The free phosphate ion displays four Raman-active modes 2 at 353, 515, 980, and 1082 cm-1 Taking into account small shifts in frequency imposed by the surrounding matrix, the prominent features of all the spectra observed in our experiments fall into those four frequency regions.
Previous Raman studies on biological apatites, including crystalline hydroxyapatite, have also observed this pattern, i.e., the dominant Raman features closely resembling those of the free phosphate ion.2-4 Splittings of these bands have been interpreted to indicate the local structure and symmetry of the solid-state system under observation.4 These previous studies also suggested that the splittings and frequency shifts could be correllated with substitutional changes in the apatite structure.3
In our experiments on both calcified arterial segements and aortic valve leaflets, the dominant 960-cm-1 band was an immediate indicator of the scattering from the mineralized plaque. Moving the laser to regions adjacent to the calcific site resulted in the direct disappearance of the 960-cm-1
band; in fact, the presence of the 960-cm-1 band alone served as a useful diagnostic discriminator regarding the presence or absence of a calcified lesion within the target tissue. All such diagnoses were verified by radiographs of the selected tissue sites.
The low frequency spectra observed below 300 cm-1 for both the calcified arteries and valves showed some evidence for new peaks. This region in the pure apatite materials has been assigned to collective lattice modes of the solid.3 In our experiments the low frequency structure was not sufficiently defined from sample to sample to determine whether the observed phonon modes were indicative of a similarity in solid-state structure or long-range organization from the various sources.
The Raman features from a series of aortic valve segments from different sources are reproducibly the same as that shown in Fig. 1. Although the physical appearance of the calcific regions differed appreciably from sample to sample, most notably in the hue of the samples (ranging from yellowish to almost completely white) which strongly affected the extent of competing fluorescence background, nonetheless, the set of phosphatelike Raman bands were reproducibly present in every sample examined.
Comparison of the aortic valve leaflet spectra with those obtained from calcified coronary arteries showed similar spectral features in the phosphate regions. Again, these features are reproducible among different artery segments, and the features are absent when adjacent healthy arterial wall sites are compared.
Although the main features of the valves and artery segments can be seen to be very similar, there are some small but noticeable differences in the structure of the observed bands, particularly in the υ3 phosphate region (1082 cm - 1) . In previous studies of the biological apatities, this region was considered to be especially sensitive to structural changes around the phosphate ion.3-5 Comparing the artery spectrum with that for the aortic valve in the 1000-cm-1 region, the two samples both show a group of three bands but with a distinct diffference in intensity distribution. The valve spectrum most closely resembles that of the hydroxyapatite standard spectrum in that region and may indicate a more structurally ordered solid than in the case of the artery. This would be consistent with the greater extent of calcific material generally present on the valves, presumably the result of a longer time of development of the diseased site. Alternatively, this difference may be due to a completely distinct mechanism of organization of mineralized deposits in the artery compared to the development of disease on the valve.
The significance of the Raman spectra to the field of laser angioplasty is worth noting. In all cases in which a significant mineralization was found at a tissue site, even in the earliest stages of data collection, the prominent 960-cm-1
phosphate band could be easily detected. Since the laser angioplasty procedure will, of necessity, be accompanied by light scattering, it seems reasonable to utilize the information contained in the scattered component of the laser light removing the diseased site collected by a suitable fiber-optic detection system. This provides the intriguing possibility of using Raman scattering as a real-time monitor of the chemical makeup of the laser target. As the laser removes diseased tissue, the scattered light from the target provides a diagnostic indicator of the nature of the diseased site and laser conditions, especially as heavily calcified sites are encountered—sites that are expected to provide the most stringent challenge to removal by laser energy6—could be appropriately adjusted. Raman scattering, combined with other detection techniques, might prove very useful in such a scheme.
This work was supported in part by grants HL 32747-01 and HL 36918-01 from the National Heart, Lung, and Blood Institute.
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2. G. Herzberg, Infrared and Raman Spectra of Polyatomic Molecules (Van Nostrand, New York, 1945).
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4. D. G. A. Nelson and B. E. Williamson, "Raman spectra of Phosphate and Monofluorophosphate Ion in Several Dentally-Relevant Materials," Caries Res. 19, 113 (1984).
5. D. C. O'Shea, M. L. Bartlett, and R. A. Young, "Compositional Analysis of Apetites with Laser Raman Spectroscopy," Arch. Oral Biol. 19, 995 (1974).
6. J. M. Isner, R. F. Donaldson, J. T. Funai, L. I. Deckelbaum, N. G. Pandian, R. H. Clarke, and J. S. Bernstein, "Factors Contributing to Perforation Resulting from Laser Coronary Angioplasty," Circulation 729, II, 191 (1985).
