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.

The combination of reflectance, fluorescence, and Raman spectroscopy-termed multimodal spectroscopy ͑MMS͒-provides complementary and depth-sensitive information about tissue composition. As such, MMS is a promising tool for disease diagnosis, particularly in atherosclerosis and breast cancer. We have developed an integrated MMS instrument and optical fiber spectral probe for simultaneous collection of all three modalities in a clinical setting. The MMS instrument multiplexes three excitation sources, a xenon flash lamp ͑370-740 nm͒, a nitrogen laser ͑337 nm͒, and a diode laser ͑830 nm͒, through the MMS probe to excite tissue and collect the spectra. The spectra are recorded on two spectrograph/charge-coupled device modules, one optimized for visible wavelengths ͑reflectance and fluorescence͒ and the other for the near-infrared ͑Raman͒, and processed to provide diagnostic parameters. We also describe the design and calibration of a unitary MMS optical fiber probe 2 mm in outer diameter, containing a single appropriately filtered excitation fiber and a ring of 15 collection fibers, with separate groups of appropriately filtered fibers for efficiently collecting reflectance, fluorescence, and Raman spectra from the same tissue location. A probe with this excitation/collection geometry has not been used previously to collect reflectance and fluorescence spectra, and thus physical tissue models ͑"phantoms"͒ are used to characterize the probe's spectroscopic response. This calibration provides probe-specific modeling parameters that enable accurate extraction of spectral parameters. This clinical MMS system has been used recently to analyze artery and breast tissue in vivo and ex vivo.

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 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 (PϽ0.001). 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 Ͼ40%, 20%, 10%, 5%, 2.5%, and 0% of the region of interest with 98%, 98%, 93%, 94%, 93%, and 90% accuracy, respectively. Key Words: foam cells Ⅲ spectroscopy Ⅲ atherosclerosis Ⅲ coronary artery disease Ⅲ human A s a continuation of our work of arterial fluorescence and Raman spectroscopy, 1 we developed an ongoing program targeting the diagnosis of vulnerable atherosclerotic plaques. The main objective of the current study was to demonstrate that a combination of intrinsic fluorescence spectroscopy (IFS) and diffuse reflectance spectroscopy (DRS) can accurately detect human coronary superficial foam cells (SFCs), a potential marker of vulnerable atherosclerotic plaques. Conclusion-Our combined IFS and DRS technique accurately detectsFibrous cap rupture and plaque erosion are the 2 main causes of coronary thrombosis. 2 Eroded plaques are responsible for at least a third of sudden cardiac deaths, with increased prevalence among young subjects. 2,3 Several studies have demonstrated the presence of macrophages and foam cells in ruptured and eroded vulnerable plaques. [3][4][5] Eroded plaques accumulate in their superficial layers macrophages, proteoglycans, smooth muscle cells (SMCs), and extracellular lipids. 2-7 However, whereas ruptured plaques feature a necrotic core and thin fibrous cap, these 2 structures are not necessarily present in eroded plaques. 2,3,6,7 Hence, an ideal technique for diagnosis of vulnerable plaques prone to rupture or erosion should be able to accurately identify macrophages and macrophage-derived foam cells.Although existing invasive diagnostic techniques such as intravascular ultrasound, elastography, and intravascular MRI can identify subsurface plaque features such as fibrous cap and necrotic core, it is only thermography and more recently optical coherence tomography that can identify areas of increased macrophage density. 8 However, none of these methods have focused specifically on foam cells, which, through their interaction with oxidized low-density lipoprotein (LDL) particles and complex inflammatory and plaque degrading functions, play a critical role in the vuln...

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.