An integrated probe has been developed containing the essential optoelectronic components necessary for monitoring tissue perfusion using the laser Doppler principle. The device includes a VCSEL for illumination, and a chip containing photodetectors, analog amplifiers for signal enhancement, and digital circuitry for external probe control. VCSEL and detector chip are mounted on a common ceramic platform, which also includes an integrated temperature sensor. A Peltier element may be included for temperature stabilization, or thermal cycling for physiological purposes. Two special chips have been developed: one containing an array of five detectors, at various distances from the laser, which will allow for some degree of depth discrimination, and a single detector chip. In this paper the multidetector chip and the probe design are presented.

The Surface Plasmon Resonance (SPR) technique is considered by many researchers as a very attractive approach for immunosensor development. By proper chemical modification of the gold surface, this technique avoids the use of a tracer material, such as a radioisotope, fluorophore, or enzyme, to identify the specific analyte, thus allowing kinetic analysis of biointeracting systems. However, the technique has found limited applications for the direct assay of complex biological samples due to the variable degree of non-specific binding that may occur along with the primary antigen-antibody reaction. This work describes a possible approach for the development of a new class of SPR based assays where non-specific binding effects could be minimized. The principle relies on selective UV inactivation of a gold surface coated with either antibody or antigen molecules. It is shown that under proper conditions it is possible to synthesize a surface with a pre-defined 2D variation of immunoactivity. Following exposure to a positive sample, image contrast under SPR illumination of the immunoactivated surface would be indicative of a positive reaction. The degree of SPR image contrast can be quantified and is a measure of the analyte concentration in solution. The approach minimizes non-specific binding effects, and the principle can be extended for the development of immunoassays for large scale testing of complex biological samples.

The goal of this pilot study was to determine whether in vivo laser-induced fluorescence intensity may be used to distinguish normal skin from malignant lesions.

Elastic light scattering spectroscopy (ESS) has the potential to provide spectra that contain both morphological and chromophore information from tissue. We report on a preliminary study of this technique, with the hope of developing a method for diagnosis of highly-pigmented skin lesions, commonly associated with skin cancer. Four opossums were treated with dimethylbenz(a)anthracene to induce both malignant melanoma and benign pigmented lesions. Skin lesions were examined in vivo using both UV-visible and near infrared ESS, with wavelength ranges of 330 - 900 nm and 900 - 1700 nm, respectively. Both portable systems used identical fiber-optic probe geometry throughout all of the measurements. The core diameters for illuminating and collecting fibers were 400 and 200 micrometers , respectively, with center-to-center separation of 350 micrometers . The probe was placed in optical contact with the tissue under investigation. Biopsies from lesions were analyzed spectral correlation for 11/13 lesions. The NIR-ESS correlated well with 12/13 lesions correctly.

In this work, we examine the role of diffraction in the design of ring-shaped ultrasonic receivers for photoacoustic probes. The sensitivity patterns of ultrasonic receiving rings are calculated at 1 MHz using the angular spectrum technique. Three separate series of simulations are considered: flat ring with a fixed outer diameter and a variable inner diameter, flat ring with a fixed inner diameter and variable outer diameter, and a spherically focused ring with a variable inner diameter. From these results the focal zone characteristic and sensitivities are calculated. For the planar rings, we find that the spatial sensitivity varies markedly with ring width but the overall sensitivities are comparable. For the focused (spherically curved) rings, the focal distances and beam volumes were similar while the sensitivity grew with ring thickness.

Stereotactic radiofrequency (RF)-lesioning in the central part of the brain is performed on patients that, for instance, have severe movement or psychiatric disorders. The size of the generated lesion can to some extent be controlled by RF-generator settings such as temperature and time as well as the electrode configuration. Today, MR- imaging and CT are the essential diagnostic methods to confirm the lesion size in vivo. The aim of this study was to investigate whether it is possible to use changes in the reflected light intensity and laser Doppler flowmetry as a marker for size estimation during RF-lesioning.

Interstitial laser thermotherapy is a minimally invasive surgical procedure that utilizes laser to coagulate and treat benign prostatic hyperplasia. This study explores the use of a computer-assisted interstitial laser coagulation system to aid surgeons in performing this procedure.

An optical spectroscopy-based system for intraoperative guidance of brain tumor resection is currently under development at Vanderbilt University and Vanderbilt University Medical Center. The effectiveness of brain tumor margin detection, especially the infiltrating type, using combined in vivo autofluorescence and diffuse reflectance spectroscopy was successfully demonstrated in a preliminary clinical trial (on a total of 26 brain tumor patients). In this trial, a two-step empirical discrimination algorithm based on autofluorescence and diffuse reflectance intensity at 460 nm and 625 nm yielded 100% sensitivity and 76% specificity in differentiating infiltrating tumor margins from normal brain tissues.

An elevated serum bilirubin concentration in the newborn infant presents a therapeutic as well as a diagnostic problem to the physician. It has long been recognized that high levels of bilirubin cause irreversible brain damage and even death. The authors introduce the use of semiconductor diode lasers and diode-pumped solid-state lasers that can be used for solving such diagnostic and therapeutic problems. These new laser sources can improve the ergonomics of using laser, enhance performance capabilities and reduce the cost of employing laser energy to pump bilirubin out of an infant's body. The choice of laser wavelengths follows the principles of bilirubinometry and phototherapy of neonatal jaundice. The wide spread use of these new laser sources for clinical monitoring and treatment of neonatal hyperbilirubinemia will be made possible as each incremental or quantum jump cost reduction is achieved. Our leading clinical experience as well as the selection rules of laser wavelengths will be presented.

During deep brain stimulation, a neurosurgical procedure to relieve tremors, a thin electrode is inserted into deep brain regions to provide stimulation. Accurate electrode placement is crucial to provide tremor suppression without damaging adjacent optical and motor regions. A portable, real-time display fiber optic reflectance probe is used to obtain reflected signals from living, human brain tissues. The optical results are compared to pre-operative MRI scans to confirm anatomical structures and verify electrode placement. In addition to reflectance, tissue oxygen saturation may assist brain tissue identification.

A clinical study has been initiated to compare an experimental IR device, the Vein Contrast Enhancer (VCE), with standard techniques for finding veins for venipuncture. The aims of this proposal are (1) to evaluate the performance of the VCE in a clinical setting, specifically by comparing its sensitivity of detection with existing vein-finding techniques used by experienced nurses or phlebotomists, (2) to study its usefulness in subjects who are obese, who have difficult venous access or thrombosed veins, or whose veins are not visible or difficult to palpate, and (3) to show that it performs as well on subjects with darkly pigmented skin as on subjects with lightly pigmented skin. The VCE will first be studied in adult subjects, and then in pediatric subjects.

We have recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. New clinical data from individuals and groups completely reproduce and extend all the earlier results. These new data reveal how certain small differences between individuals result in some variability in their noninvasive quantitation. We show the origin of this variability and how to obtain quantitative corrections based entirely on the individual measurement and tabulated data.

The high efficacy of laser phototherapy combined with transcutaneous monitoring of serum bilirubin provides optimum safety for jaundiced infants from the risk of bilirubin encephalopathy. In this paper the authors introduce the design and operating principles of a new laser system that can provide simultaneous monitoring and treatment of several jaundiced babies at one time. The new system incorporates diode-based laser sources oscillating at selected wavelengths to achieve both transcutaneous differential absorption measurements of bilirubin concentration in addition to the computer controlled intermittent laser therapy through a network of optical fibers. The detailed description and operating characteristics of this system are presented.

Near-Infrared Spectroscopy (NIRS) can be employed to monitor local changes in haemodynamics and oxygenation of human tissues. A preliminary study has been performed in order to evaluate the NIRS transmittance response to induced forearm ischaemia in patients with coronary artery disease (CAD). The population consists in 40 patients with cardiovascular risk factors and angiographically documented CAD, compared to a group of 13 normal subjects. By inflating and subsequently deflating a cuff placed around the patient arm, an ischaemia has been induced and released, and the patients have been observed until recovery of the basal conditions. A custom NIRS spectrometer has been used to collect the backscattered light intensities from the patient forearm throughout the ischaemic and the recovery phase. The time dependence of the near-infrared transmittance on the control group is consistent with the available literature. On the contrary, the magnitude and dynamics of the NIRS signal on the CAD patients show deviations from the documented normal behavior, which can be tentatively attributed to abnormal vessel stiffness. These preliminary results, while validating the performance of the IRIS spectrometer, are strongly conducive towards the applicability of the NIRS technique to ischaemia analysis and to the endothelial dysfunction characterization in CAD patients with cardiovascular risk factors.

In this work, we examine the orientation-dependent scattering and attenuation properties of white mater from mammalian brain tissue. We find that both the backscatter and attenuation of ultrasound in these tissues exhibit anisotropy. Furthermore, when extrapolated down to 1 MHz, it appears that the attenuation differences will be small but the backscatter differences are potentially resolvable. From a tissue characterization context, this means that the impact of changes due to the rotation of overlying tissues will be small compared to the changes in the strength of the backscatter signals from the regions of interest.

Endoscopy is a versatile medical device, but with deficiency of severe image distortion from a super-wide angle lens CCD- camera installed. We designed a planar circular pattern target mounted on a XYZ transnational stage and developed a calibration method to correct the distorted image. Through the image shaping similarity and symmetric properties of a circular pad posing in the specific spatial coordinates, an optimized algorithm is to adjust the orientation of the gastroscopic head and the coordinates of the target until the accurate system alignment obtained. The profiles of image mapping and the calibration object are to derive both the image distortion and calibration function as single variable polynomial equations. Using the calibration function, the distorted gastroscopic image could transfer to comply with an ideal pinhole mapping. The experimental results validated that a gastroscopic image could be corrected and depict better geometrical information. Concurrently displaying with the traditional screen, this technique could aid the physician in metering a tumor or lesion size.

Oral squamous cell carcinoma is a disease which progresses through a number of well-defined morphological and biochemical changes. Optical coherence tomography (OCT) is a rapidly-evolving, non-invasive imaging modality which allows detailed probing of subsurface tissue structures with resolution on the order of microns. While this technique offers tremendous potential as a diagnostic tool for detection and characterization of oral cancer, OCT imaging is presently associated with a field of view on the order of millimeters, and acquisition time on the order of seconds. Thus, OCT's utility as a rapid cancer screening technique is presently limited. On the other hand, imaging of tissue autofluorescence provides a very rapid, high-throughput method for cancer screening. However, while autofluorescence measures may be sensitive to cancer, they are often non- specific and lead to a large number of false positives. In the present work, we have developed a fluorescence image guided optical coherence tomographic (FIG-OCT) probe in which tissue autofluorescence images are simultaneously used to guide OCT image acquisition of suspicious regions in real time. We have begun pre-clinical pilot studies with this instrument in a DMBA-induced model of oral cancer in the hamster cheek pouch. Initial results indicate that the FIG- OCT approach shows promise as a rapid and effective tool for screening of oral cancer.

Optical coherence tomography (OCT) is a novel biomedical imaging technique that uses low-coherence optical interferometry to obtain micron-scale resolution cross- sectional images of tissue microstructure noninvasively. OCT fills a valuable niche in imaging of tissue structure, providing subsurface imaging with high spatial resolution (on the order of 10 micrometers) and penetration depths of 1 - 2 mm with no contact or matching medium needed between the probe and the tissue. An OCT system for gastrointestinal (GI) endoscopy has been developed using a small-diameter rotary-scanning probe compatible with standard GI endoscopes and capable of imaging in real-time. To date more than 100 volunteers have been imaged during routine upper and lower endoscopic procedures. Results of imaging in normal organs have demonstrated visualization of morphological layers (epithelium, lamina propria, muscularis mucosa, submucosa, muscularis propria) and microscopic structures (glands, villi, crypts, vessels) in all endoscopically accessible GI organs. It has been observed in more than 30 patients that the EOCT appearance of Barrett's mucosa is clearly differentiable from normal gastric or esophageal mucosa. Furthermore, the EOCT appearance of dysplasia and neoplastic lesions, including adenocarcenoma in Barrett's and villous tumor in colon have been observed and are under investigation. Preliminary data indicate the potential of EOCT for routine clinical diagnostics in GI tissues, including early cancer detection and staging and detection of tumor margins.

Submicron fiber-optic biosensors have been developed and used to measure toxic chemicals within single cells. These sensors are fabricated by pulling the distal-end of an optical fiber to a diameter of less than one micron and coating them with antibodies to selectively bind the species of interest. This paper describes the use of these fibers to selectively measure the concentration of benzo[a]pyrene tetrol, a metabolite of benzo[a]pyrene, within individual cells of two different cell lines, human mammary carcinoma cells and rat liver epithelial cells. The results from these measurements have been used to evaluate the analytical figures of merit of these sensors such as detection limits, which were found to be 0.64 +/- 0.17 X 10^-11 M for BPT. In addition, measurements were performed both extracellularly and intracellularly, and an increase in variation of approximately 4% was determined for the intracellular measurements, relative to the extracellular measurements.

A confocal microendoscope for in vivo imaging at the cellular level has been developed. The system consists of a slit-scanning confocal microscope coupled to a fiber optic imaging bundle with a miniature objective lens and focusing mechanism at the distal tip of the catheter. The system is used primarily for imaging tissue fluorescence--either autofluorescence or fluorescence from an exogenous compound. The microendoscope has a lateral resolution of 2 micrometers allowing visualization of structures at the cellular level, and because it is confocal, it can image at selected depths below the tissue surface. Excellent in vivo imaging results have been obtained in animal models and encouraging ex vivo results have been achieved with human tissue.

Laser Doppler perfusion imaging (LDPI) has successfully been used to map the myocardial perfusion on patients undergoing coronary bypass surgery on the arrested heart. The need for intra-operative evaluation of graft function is obvious in routine surgery but even more imperative when adapting new surgical techniques where the procedure is performed on the beating heart. When using LDPI on the beating heart, artifacts originating from the movement of the heart are superimposed on the Doppler signal. We have investigated a method to reduce these artifacts by controlling the sampling sequence with ECG-triggering. The method has been assessed in an animal model on the beating calf heart. After sternotomy, an area covering 1 cm² was imaged at the anterior wall of the left ventricle. In this area, six perfusion images were captured each of them recorded at fixed, but different time intervals in the cardiac cycle. In addition continuous measurements at one spot was done during 1 - 2 minutes. The signal recorded during pumping action was high compared to measurements performed in the same muscle area during infusion of blood with a syringe pump. Repeated measurements captured at a fixed delay time from the R-peak in the same areas at the same heart frequency showed reproducibility. ECG-triggering of the laser Doppler signal is the first step in our attempts to adapt LDPI to enabling assessment of myocardial perfusion on the beating heart. Further technical achievements and in-vivo investigations are, however, needed and will be performed by our research team in future studies.

We demonstrate in vivo optical coherence tomography imaging of neoplasia in patients using an integrated OCT colposcope. OCT images of epithelial structure were correlated with histological findings in patients with cervical intraepithelial neoplasia and cancer.

We observed differences between the mid-infrared spectra of sera originating from healthy volunteers and from patients with diabetes mellitus or rheumatoid arthritis. These differences were found to be significant in terms of the Fisher criterion, the t-test, and the Kolmogorov-Smirnov test. The significance allows for a classification of the spectra and a probability (`DPR-score') of belonging to the class `healthy' can be computed. In comparing the samples from 80 diabetes patients with samples from 40 healthy volunteers we are able to achieve a sensitivity and a specificity of 80% and above. The DPR-score correlates better with the actual status of health than the glucose concentration alone. In a study on rheumatoid arthritis we compared the spectral signatures of sera taken from 188 rheumatoid arthritis patients and sera from 196 healthy volunteers. By applying linear discriminant analysis to 2/3 of the samples we are able to classify the remaining third of the samples (independent validation) with a sensitivity of 84% and a specificity of 88%.

While many macroscopic approaches have been used to study bone's mechanical properties, little is known about the effect of mechanical stresses on bone tissue at the microstructural and submicrostructural levels. Raman microspectroscopy has been found to be an extremely sensitive technique for examining the effects of mechanical loading on polymer structures and thin films, as well as for the study of protein conformations. We present the first application of this technique to bone tissue. The organic component of bone is a highly ordered matrix composed mainly of collagen fibrils. Stress upon the bone tissue creates disorder in this structure; this disorder can easily be detected by Raman spectroscopy. Small changes in the matrix structure manifest themselves as band shifts in the Raman spectra.

The composition of the bone tissue initially formed during the early mineralization of calvarial bone is poorly understood. Calvarial de novo mineral deposition occurs rapidly; however, whether the mineral is first deposited as an amorphous calcium phosphate or some other calcium phosphate lattice is unclear. Raman microscopy offers the ability to distinguish differences in the mineral lattice through the positions and shifts in the bands of the bone tissue mineral constituents, particularly in the phosphate v₁ stretch vibration (950 - 963 cm^-1). The ratios of the mineral and organic matrix constituents throughout the sampled region can also elucidate the type of bone tissue deposited. The ability to examine intact specimens at high spatial resolution, without interference from water, is an important feature of Raman microscopy. Using postnatal murine calvaria, we show that the earliest mineral detected is a carbonated hydroxyapatite with other phosphate environments present at lower levels as well. We discuss the mineral composition changes with respect to the age of the mouse over the time period of 2 weeks using a sequence of calvarial sections: postnatal days 3, 7 and 14. We use two different data analysis techniques, factor analysis and center of gravity calculations, to elucidate these discrete changes.

Coherent anti-Stokes Raman spectroscopy is evaluated for the purpose of real-time noninvasive microscopic imaging. We find optimal parameters for optical system design and demonstrate effective suppression of non-resonant background. High-speed microscopic imaging is demonstrated as well.

We recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and then normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. A more careful comparison of tissue modulated spectra with static in vitro and invasive in vivo spectra suggests that there are small microcirculation differences between individuals resulting in some variability in their noninvasive quantitation. This variability is based on the mechanism for blood volume normalization and various means for obtaining necessary corrections are suggested. We present new clinical data from individuals and groups supporting this mechanism and suggesting how such measurements might also be used to quantify various microcirculation abnormalities.

This paper analyzes the short pulse laser propagation through tissues for development of a time-resolved optical tomography system for detection of tumors and inhomogeneities in tissues. Traditional method for analyzing optical transport in tissues is the parabolic or diffusion approximation in which the energy flux is assumed proportional to the fluence (intensity averaged over all solid angles) gradients. The inherent drawback in this model is that it predicts infinite speed of propagation of the optical signal. In this paper accurate hyperbolic or wave nature of transient radiative transfer formulation is used to overcome such drawbacks. The transmitted and reflected intensity distributions are obtained using hyperbolic P₁ and discrete ordinates method and the results are compared with the parabolic diffusion P₁ approximation. Parametric study of tissue thickness, pulse width, scattering and absorption coefficients of tissues, tumor location, size and properties, and scattering phase function distribution is also performed to analyze their effect on the transmitted and reflected optical signals.