(73) Assignee: Honeywell International Inc., Katrin Kneip et al., Surface-enhanced Raman Spectroscopy in Morristown, NJ (US) Single Living Cells Using Gold Nanaparticles, 2002, Society for Applied Spectroscopy, vol. 56, No. 2, pp. 1-2.* (*) Notice: Subject to any disclaimer, the term of this Milton Kerker, Elastic and Inelastic Light Scattering in Flow patent is extended or adjusted under 35 Cytomery, Jan. 19, 1983, pp. 1-10.* U.S.C. 154(b) bV 295 davs. (b) by ayS (Continued)

Laser-induced fluorescence (LIF) and intrinsic fluorescence spectroscopy (IFS) have been used experimentally for diagnosing coronary atherosclerosis. In this study, we demonstrated the diagnostic superiority of IFS at 342-nm excitation (IFS342) versus LIF (LIF342) and described a protocol for head-to-head comparison of old (LIF) versus new (IFS) generations of similar diagnostic methods, labeled as “generational comparison model“. IFS342 and LIF342 were modeled with basis spectra of media, fibrous caps, and superficial foam cells and of their correspondent chemicals (elastin, collagen, and lipoproteins). The average accuracy and receiver operating characteristic area under the curve of IFS342 in single-, double-, and triple-parameter diagnostic algorithm iterations, geared toward identifying 84 atherosclerotic specimens from a group of 117 coronary segments, was 90% ± 1% and 0.87 ± 0.025, superior to LIF342 (84% ± 3% and 0.84 ± 0.016; P = 0.0002 and 0.02, respectively) in a generational comparison model.

Intracoronary Raman spectroscopy could open new avenues for the study and management of coronary artery disease due to its potential to measure the chemical and molecular composition of coronary atherosclerotic lesions. We have fabricated and tested a 1.5-mm-diameter (4.5 Fr) Raman catheter capable of collecting Raman spectra in both the fingerprint (400-1800 cm(-1)) and high-wavenumber (2400-3800 cm(-1)) regions. Spectra were acquired in vivo, using a human-swine xenograft model, in which diseased human coronary arteries are grafted onto a living swine heart, replicating the disease and dynamic environment of the human circulatory system, including pulsatile flow and motion. Results show that distinct spectral differences, corresponding to the morphology and chemical composition of the artery wall, can be identified by intracoronary Raman spectroscopy in vivo.

Spectrally-encoded miniature endoscopy uses a single optical fiber and wavelength division multiplexing to obtain macroscopic images through miniature, flexible probes. In turn, it has the potential to enable two- and three-dimensional imaging within the body at locations that are currently difficult to access with conventional endoscopes. Here we present a novel detection scheme for spectrally-encoded endoscopy using spectral-domain interferometry. Compared to previous time-domain configurations, this new detection method results in greater than 1000-fold increase in sensitivity (77 dB), a 6-fold increase in imaging speed (30 volumes per second), and a 2-fold increase in depth range (2.8 mm). We demonstrate spectrally-encoded, spectral-domain detection by conducting video-rate, three-dimensional imaging in a variety of specimens, including the paws of a mouse embryo and excised human ear bones. Our results show that this new technology enables video rate spectrally-encoded endoscopy and will therefore be useful for a variety of minimally invasive medical applications.

The chemical composition of vascular lesions, an important determinant of plaque progression and rupture, can not presently be determined in vivo. Prior studies have shown that Raman spectroscopy can accurately quantify the amounts of major lipid classes and calcium salts in homogenized coronary artery tissue. This study determines how the relative cholesterol content, which is calculated from Raman spectra collected at the luminal surface of an artery, is related to its depth in an intact arterial wall. Raman spectra of human atherosclerotic plaques were measured after thin tissue layers were successively placed on them. From these spectra, relative cholesterol contents were calculated and used to determine how cholesterol signal strength is attenuated by overlaying tissue. Then, intact artery samples (n=13) were examined spectroscopically, sectioned and stained specifically for cholesterol. Images of these sections were digitized, and image intensities were related to cholesterol content. These cholesterol amounts were weighed appropriately for depth into the tissue and area-integrated for comparison with spectroscopy results. A decaying exponential curve was fit to the layer study data (r2=0.97) and showed that ∼300 μm of tissue attenuates cholesterol signals by 50%. In intact plaques, the spectroscopically-determined cholesterol amounts correlated strongly and linearly with those determined by digital microscopy (r2=0.94). With Raman spectroscopy techniques, the cholesterol content of a lesion can be determined by properly accounting for its depth into an arterial wall. Our results suggest that chemical concentrations in an artery wall could be mapped throughout its thickness, possibly by combining Raman spectroscopy methods with other techniques.

We report a rapid and low cost Fourier transform spectrometer that uses a path length modulated Michelson interferometer to simultaneously measure excitation spectra and excitation wavelength-dependent emission lifetimes. Excitation spectra and lifetimes of excited tris(2,2'-bipyridyl) ruthenium(II) measured using this technique corresponded to values known in the literature. Excitation-dependent lifetimes of porous silicon measured with this technique suggest the influence of quantum confinement effects. This method may be useful for measuring mixtures of emitting species with closely spaced lifetimes as well as studying excitation wavelength-dependent emission phenomena.

Objective—Foam cells perform critical functions in atherosclerosis. We hypothesize that coronary segments with superficial foam cells (SFCs) situated in a region of interest with a depth of 200 &mgr;m can be identified using intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS). This is a key step in our ongoing program to develop a spectroscopic technique for real-time in vivo diagnosis of vulnerable atherosclerotic plaque. Methods and Results—We subjected 132 human coronary segments to in vitro IFS and DRS. We detected SFCs in 13 thick fibrous cap atheromas and 8 pathologic intimal thickening (PIT) lesions. SFCs colocalized with accumulations of smooth muscle cells and proteoglycans, including hyaluronan (P<0.001). Two spectroscopic parameters were generated from analysis of IFS at 480 nm excitation and DRS. A discriminatory algorithm using these parameters identified specimens with SFC area >40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively. Conclusion—Our combined IFS and DRS technique accurately detects SFCs in thick fibrous cap atheromas and PIT lesions. Because SFCs are associated with histological markers of plaque erosion, our spectroscopic technique could prove useful in identifying vulnerable plaques.

Spectral encoding is a recently developed, single optical fiber imaging approach that projects different wavelengths to distinct locations on a sample. In so doing, it enables a wide variety of reflectance and fluorescence medical imaging devices, including miniature endoscopes and handheld probes for two- and three-dimensional macroscopic imaging and microscopy.

Vulnerable plaques, which are responsible for most acute ischemic events, are presently invisible to x-ray angiography. Their primary morphological features include a thin or ulcerated fibrous cap, a large necrotic core, superficial foam cells, and intraplaque hemorrhage. We present evidence that multimodal spectroscopy (MMS), a novel method that combines diffuse reflectance spectroscopy (DRS), intrinsic fluorescence spectroscopy (IFS), and Raman spectroscopy (RS), can detect these markers of plaque vulnerability. To test this concept, we perform an MMS feasibility study on 17 human carotid artery specimens. Following the acquisition of spectra, each specimen is histologically evaluated. Two parameters from DRS, hemoglobin concentration and a scattering parameter, are used to detect intraplaque hemorrhage and foam cells; an IFS parameter that relates to the amount of collagen in the topmost layers of the tissue is used to detect the presence of a thin fibrous cap; and an RS parameter related to the amount of cholesterol and necrotic material is used to detect necrotic core. Taken together, these spectral parameters can generally identify the vulnerable plaques. The results indicate that MMS provides depth-sensitive and complementary morphological information about plaque composition. A prospective in vivo study will be conducted to validate these findings.

The rupture of vulnerable atherosclerotic plaque accounts for the majority of clinically significant acute cardiovascular events. Because stability of these culprit lesions is directly related to chemical and morphological composition, Raman spectroscopy may be a useful technique for their study. Recent developments in optical fiber probe technology have allowed for the real-time in vivo Raman spectroscopic characterization of human atherosclerotic plaque demonstrated in this work. We spectroscopically examine 74 sites during carotid endarterectomy and femoral artery bypass surgeries. Of these, 34 are surgically biopsied and examined histologically. Excellent signal-to-noise ratio spectra are obtained in only 1 s and fit with an established model, demonstrating accurate tissue characterization. We also report the first evidence that Raman spectroscopy has the potential to identify vulnerable plaque, achieving a sensitivity and specificity of 79 and 85%, respectively. These initial findings indicate that Raman spectroscopy has the potential to be a clinically relevant diagnostic tool for studying cardiovascular disease.

A single optical fibre acts as a flexible probe to transmit a superior image of an internal landscape. Microendoscopy, using devices a millimetre or so in diameter, is an accepted technique in ophthalmics, tumour diagnosis and other medical specialities. Submillimetre-diameter devices have been used in some clinical applications but have not been widely adopted because of their rigidity and poor image quality. A new type of endoscope has now been developed that can transmit video-rate, three-dimensional images from flexible probes that are the diameter of a single optical fibre, at 80–250 μm, comparable in size to a human hair.

Raman spectroscopy has been well established as a powerful in vitro method for studying biological tissue and diagnosing disease. The recent development of efficient, high-throughput, low-background optical fiber Raman probes provides, for the first time, the opportunity to obtain real-time performance in the clinic. We present an instrument for in vivo tissue analysis which is capable of collecting and processing Raman spectra in less than 2 s. This is the first demonstration that data acquisition, analysis, and diagnostics can be performed in clinically relevant times. The instrument is designed to work with the new Raman probes and includes custom written LabVIEW and Matlab programs to provide accurate spectral calibration, analysis, and diagnosis along with important safety features related to laser exposure. The real-time capabilities of the system were demonstrated in vivo during femoral bypass and breast lumpectomy surgeries. Such a system will greatly facilitate the adoption of Raman spectroscopy into clinical research and practice.

Background—A method capable of determining atherosclerotic plaque composition and measuring plaque viscoelasticity can provide valuable insight into intrinsic features associated with plaque rupture and can enable the identification of high-risk lesions. In this article, we describe a new optical technique, laser speckle imaging (LSI), that measures an index of plaque viscoelasticity. We evaluate the potential of LSI for characterizing atherosclerotic plaque. Methods and Results—Time-varying helium-neon laser speckle images were acquired from 118 aortic plaque specimens from 14 human cadavers under static and deforming conditions (0 to 200 &mgr;m/s). Temporal fluctuations in the speckle patterns were quantified by exponential fitting of the normalized cross-correlation of sequential frames in each image series of speckle patterns to obtain the exponential decay time constant, &tgr;. The decorrelation time constants of thin-cap fibroatheromas (TCFA) (&tgr;=47.5±19.2 ms) were significantly lower than those of other atherosclerotic lesions (P<0.001), and the sensitivity and specificity of the LSI technique for identifying TCFAs were >90%. Speckle decorrelation time constants demonstrated strong correlation with histological measurements of plaque collagen (R=0.73, P<0.0001), fibrous cap thickness (R=0.87, P<0.0001), and necrotic core area (R=−0.81, P<0.0001). Under deforming conditions (10 to 200 &mgr;m/s), &tgr; correlated well with cap thickness in necrotic core fibroatheromas (P>0.05). Conclusions—The measurement of speckle decorrelation time constant from laser speckle images provides an index of plaque viscoelasticity and facilitates the characterization of plaque type. Our results demonstrate that LSI is a highly sensitive technique for characterizing plaque and identifying thin-cap fibroatheromas.