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We have developed a portable, breast margin assessment probe leveraging diffuse optical spectroscopy to quantify the morphological landscape of breast tumor margins during breast conserving surgery. The approach presented here leverages a custom-made 16-channel annular photodiode imaging array (arranged in a 4 × 4 grid), a raster-scanning imaging platform with precision pressure control, and compressive sensing with an optimized set of eight wavelengths in the visible spectral range. A scalable Monte-Carlo-based inverse model is used to generate optical property [ ? s ? ( ? ) and ? a ( ? ) ] measures for each of the 16 simultaneously captured diffuse reflectance spectra. Subpixel sampling (0.75 mm) is achieved through incremental x , y raster scanning of the imaging probe, providing detailed optical parameter maps of breast margins over a 2 × 2 ?? cm 2 area in ? 9 ?? min . The morphological landscape of a tumor margin is characterized using optical surrogates for the fat to fibroglandular content ratio, which has demonstrated diagnostic utility in delineating tissue subtypes in the breast.

Diffuse reflectance spectroscopy with a fiber optic probe is a powerful tool for quantitative tissue characterization and disease diagnosis. Significant systematic errors can arise in the measured reflectance spectra and thus in the derived tissue physiological and morphological parameters due to real-time instrument fluctuations. We demonstrate a novel fiber optic probe with real-time, self-calibration capability that can be used for UV-visible diffuse reflectance spectroscopy in biological tissue in clinical settings. The probe is tested in a number of synthetic liquid phantoms over a wide range of tissue optical properties for significant variations in source intensity fluctuations caused by instrument warm up and day-to-day drift. While the accuracy for extraction of absorber concentrations is comparable to that achieved with the traditional calibration (with a reflectance standard), the accuracy for extraction of reduced scattering coefficients is significantly improved with the self-calibration probe compared to traditional calibration. This technology could be used to achieve instrument-independent diffuse reflectance spectroscopy in vivo and obviate the need for instrument warm up and post/premeasurement calibration, thus saving up to an hour of precious clinical time.

Noninvasive and longitudinal monitoring of tumor oxygen- ation status using quantitative diffuse reflectance spectroscopy is used to test whether a final treatment outcome could be estimated from early optical signatures in a murine model of head and neck cancer when treated with radiation. Implanted tumors in the flank of 23 nude mice are exposed to 39 Gy of radiation, while 11 animals exposed to sham irradiation serve as controls. Diffuse optical reflectance is mea- sured from the tumors at baseline prior to irradiation and then seri- ally until 17 days posttreatment. The fastest and greatest increase in baseline-corrected blood oxygen saturation levels are observed from the animals that show complete tumor regression with no recurrence 90 days postirradiation, relative to both untreated and treated animals with local recurrences. These increases in saturation are observed starting 5 days posttreatment and last up to 17 days posttreatment. This preclinical study demonstrates that diffuse reflectance spectros- copy could provide a practical method far more effective than the growth delay assay to prognosticate treatment outcome in solid tu- mors and may hold significant translational promise. © 2009 Society of

We explore the effects of the illumination and collection geometry on optical spectroscopic diagnosis of breast cancer. Fluorescence and diffuse reflectance spectroscopy in the UV-visible spectral range are made with a multiseparation probe at three illumination-collection separations of 735, 980, and 1225 microm, respectively, from 13 malignant and 34 nonmalignant breast tissues. Statistical analysis is carried out on two types of data inputs: (1) the fluorescence and diffuse reflectance spectra recorded at each of the three illumination-collection separations and (2) the integrated fluorescence (at each excitation wavelength) or diffuse reflectance over the entire spectrum at all three illumination-collection separations. The results show that using the integrated fluorescence intensities recorded at a single excitation wavelength at all three illumination-collection separations can discriminate malignant from nonmalignant breast tissues with similar classification accuracy to that using spectral data measured at several excitation wavelengths with a single illumination-collection separation. These findings have significant implications with respect to the design of an optical system for breast cancer diagnosis. Examining the intensity attenuation at a single wavelength rather than spectral intensities at multiple wavelengths can significantly reduce the measurement and data processing time in a clinical setting as well as the cost and complexity of the optical system.

The goal of the work is to experimentally verify Monte Carlo modeling of fluorescence and diffuse reflectance measurements in turbid, tissue phantom models. In particular, two series of simulations and experiments, in which one optical parameter (absorption or scattering coefficient) is varied while the other is fixed, are carried out to assess the effect of the absorption coefficient ( μ a ) and scattering coefficient ( μ s ) on the fluorescence and diffuse reflectance measured from a turbid medium. Moreover, simulations and experiments are carried out for several fiber optic probe geometries that are designed to sample small tissue volumes. Additionally, a group of conversion expressions are derived to convert the optical properties and fluorescence quantum yield measured from tissue phantoms for use in Monte Carlo simulations. The conversions account for the differences between the definitions of the absorption coefficient and fluorescence quantum yield of fluorophores in a tissue phantom model and those in a Monte Carlo simulation. The results indicate that there is good agreement between the simulated and experimentally measured results in most cases. This dataset can serve as a systematic validation of Monte Carlo modeling of fluorescent light propagation in tissues. The simulations are carried out for a wide range of absorption and scattering coefficients as well as ratios of scattering coefficient to absorption coefficient, and thus would be applicable to tissue optical properties over a wide wavelength range (UV-visible/near infrared). The fiber optic probe geometries that are modeled in this study include those commonly used for measuring fluorescence from tissues in practice.

Developing fiber optic probe geometries to selectively measure fluorescence spectra from different sublayers within human epithelial tissues will potentially improve the endogenous fluorescence contrast between neoplastic and nonneoplastic tissues. In this study, two basic fiber optic probe geometries, which are called the variable aperture (VA) and multidistance (MD) approaches, are compared for depth-resolved fluorescence measurements from human cervical epithelial tissues. The VA probe has completely overlapping illumination and collection areas with variable diameters, while the MD probe employs separate illumination and collection fibers with a fixed separation between them. Monte Carlo simulation results show that the total fluorescence detected is significantly higher for the VA probe geometry, while the probing depth is significantly greater for the MD probe geometry. An important observation is that the VA probe is more sensitive to the epithelial layer, while the MD probe is more sensitive to the stromal layer. The effect of other factors, including numerical aperture (NA) and tissue optical properties on the fluorescence measurements with VA and MD probe geometries, are also evaluated. The total fluorescence detected with both probe geometries significantly increases when the fiber NA is changed from 0.22 to 0.37. The sensitivity to different sublayers is found to be strongly dependent on the tissue optical properties. The simulation results are used to design a simple fiber optic probe that combines both the VA and MD geometries to enable fluorescence measurements from the different sublayers within human epithelial tissues.

The possibility of optical oximetry of the blood in the fetal brain measured across the maternal abdomen just prior to birth is under investigation. Such measurements could detect fetal distress prior to birth and aid in the clinical decision regarding Cesarean section. This paper uses a perturbation method to model photon transport through an 8-cm-diam fetal brain located at a constant 2.5 cm below a curved maternal abdominal surface with an air/tissue boundary. In the simulation, a near-infrared light source delivers light to the abdomen and a detector is positioned up to 10 cm from the source along the arc of the abdominal surface. The light transport [W/cm2 fluence rate per W incident power] collected at the 10 cm position is Tm=2.2×10−6 cm−2 if the fetal brain has the same optical properties as the mother and Tf=1.0×10−6 cm−2 for an optically perturbing fetal brain with typical brain optical properties. The perturbation P=(Tf−Tm)/Tm is −53% due to the fetal brain. The model illustrates the challenge and feasibility of transabdominal oximetry of the fetal brain. © 2000 Society of Photo-Optical Instrumentation Engineers.

In this work experimental tissue phantoms and numerical models were developed to estimate photon migration through the fetal head in utero. The tissue phantoms incorporate a fetal head within an amniotic fluid sac surrounded by a maternal tissue layer. A continuous wave, dual-wavelength (lambda = 760 and 850 nm) spectrometer was employed to make near-infrared measurements on the tissue phantoms for various source-detector separations, fetal-head positions, and fetal-head optical properties. In addition, numerical simulations of photon propagation were performed with finite-difference algorithms that provide solutions to the equation of radiative transfer as well as the diffusion equation. The simulations were compared with measurements on tissue phantoms to determine the best numerical model to describe photon migration through the fetal head in utero. Evaluation of the results indicates that tissue phantoms in which the contact between fetal head and uterine wall is uniform best simulates the fetal head in utero for near-term pregnancies. Furthermore, we found that maximum sensitivity to the head can be achieved if the source of the probe is positioned directly above the fetal head. By optimizing the source-detector separation, the signal originating from photons that have traveled through the fetal head can drastically be increased.

Near infrared (NIR) measurements were made from the ma- ternal abdomen (clinical studies) and laboratory tissue phantoms (ex- perimental studies) to gain insight into photon migration through the fetal head in utero. Specifically, a continuous wave spectrometer was modified and employed to make NIR measurements at 760 and 850 nm, at a large (10 cm) and small (2.5/4 cm) source-detector separa- tion, simultaneously, on the maternal abdomen, directly above the fetal head. A total of 19 patients were evaluated, whose average ges- tational age and fetal head depth, were 37 weeks 63 and 2.25 cm 60.7, respectively. At the large source-detector separation, the pho- tons are expected to migrate through both the underlying maternal and fetal tissues before being detected at the surface, while at the short source-detector separation, the photons are expected to migrate primarily through the superficial maternal tissues before being de- tected. Second, similar NIR measurements were made on laboratory tissue phantoms, with variable optical properties and physical geom- etries. The variable optical properties were obtained using different concentrations of India ink and Intralipid in water, while the variable physical geometries were realized by employing glass containers of different shapes and sizes. Third, the NIR measurements, which were made on the laboratory tissue phantoms, were compared to the NIR measurements made on the maternal abdomen to determine which tissue phantom best simulates the photon migration path through the fetal head in utero. The results of the comparison were used to pro- vide insight into the optical properties and physical geometry of the maternal and fetal tissues in the photon migration path. © 2000 Society of Photo-Optical Instrumentation Engineers. (S1083-3668(00)00702-4)

Preface David A. Boas, Constantinos Pitris, Nimmi Ramanujam I. Background Geometrical Optics Ting-Chung Poon Diffraction Optics Colin Sheppard Optics: Basic Physics Raghuveer Parthasarathy Light Sources, Detectors, and Irradiation Guidelines Carlo Amadeo Alonzo, Malte C. Gather, Jeon Woong Kang, Giuliano Scarcelli, Seok-Hyun Yun Tissue Optical Properties Alexey N. Bashkatov, Elina A. Genina, Valery V. Tuchin II. Spectroscopy and Spectral Imaging Reflectance Spectroscopy Sasha McGee, Jelena Mirkovic, Michael Feld Multi/Hyper-Spectral Imaging Costas Balas, Christos Pappas, George Epitropou Light Scattering Spectroscopy Le Qiu, Irving Itzkan, Lev T. Perelman Broadband Diffuse Optical Spectroscopic Imaging Bruce J. Tromberg, Albert E. Cerussi, So-Hyun Chung, Wendy Tanamai, Amanda Durkin Near Infrared Diffuse Correlation Spectroscopy for Assessment of Tissue Blood Flow Guoqiang Yu, Turgut Durduran, Chao Zhou, Ran Cheng, Arjun G. Yodh Fluorescence Spectroscopy Darren Roblyer, Richard A. Schwarz, Rebecca Richards-Kortum Raman, SERS and FTIR Spectroscopy Andrew J. Berger III. Tomographic Imaging Optical Coherence Tomography: Introduction and Theory Yu Chen, Evgenia Bousie, Constantinos Pitris, James G. Fujimoto Functional Optical Coherence Tomography in Preclinical Models Melissa C. Skala, Yuankai K. Tao, Anjul M. Davis, Joseph A. Izatt Optical Coherence Tomography: Clinical Applications Brian D. Goldberg, Melissa J. Suter, Guillermo J. Tearney, Brett E. Bouma Forward Models of Light Transport in Biological Tissue Andreas H. Hielscher, Hyun Keol Kim, Alexander K. Klose Inverse Models of Light Transport Simon Arridge, Martin Schweiger, John C. Schotland Laminar Optical Tomography Sean A. Burgess, Elizabeth M. C. Hillman Diffuse Optical Tomography using Continuous Wave and Frequency Domain Imaging Systems Subhadra Srinivasan, Scott C. Davis, Colin M. Carpenter Diffuse Optical Tomography: Time Domain Juliette Selb, Adam Gibson Photoacoustic Tomography and Ultrasound-Modulated Optical Tomography Changhui Li, Chulhong Kim, Lihong V. Wang Optical and Photoacoustic Molecular Tomography of Small Animals Vasilis Ntziachristos IV. Microscopic Imaging Assesing Microscopic Structural Features Using Fourier-Domain Low Coherence Interferometry Robert N. Graf, Francisco E. Robles, Adam Wax Phase Imaging Microscopy: Beyond Darkfield, Phase and Differential Interference Contrast Microscopy Chrysanthe Preza, Sharon V. King, Nicoleta M. Dragomir, Carol J. Cogswell Confocal Microscopy William C. Warger II, Charles A. DiMarzio, Milind Rajadhyaksha Fluorescence Microscopy with Structured Excitation Illumination Alexander Brunner, Gerrit Best, Roman Amberger, Paul Lemmer, Thomas Ach, Stefan Dithmar, Rainer Heintzmann, Christoph Cremer Nonlinear Optical Microscopy for Biology and Medicine Daekeun Kim, Heejin Choi, Jae Won Cha, Peter T. C. So Fluorescence Lifetime Imaging Microscopy, Endoscopy and Tomography James McGinty, Clifford Talbot, Dylan Owen, David Grant, Sunil Kumar, Neil Galletly, Bebhinn Treanor, Gordon Kennedy, Peter M. P. Lanigan, Ian Munro, Daniel S. Elson, Anthony Magee, Dan Davis, Gordon Stamp, Mark Neil, Christopher Dunsby, Paul W. M. French Application of Digital Holographic Microscopy in Biomedicine Christian Depeursinge, Pierre Marquet, Nicolas Pavillon Polarized Light Imaging of Biological Tissues Steven L. Jacques V. Molecular Probe Development Molecular Reporter Systems for Optical Imaging Walter J. Akers, Samuel Achilefu Nanoparticles for Targeted Therapeutics and Diagnostics Timothy Larson, Kort Travis, Pratixa Joshi, Konstantin Sokolov Plasmonic Nanoprobes for Biomolecular Diagnostics of DNA Targets Tuan Vo-Dinh, Hsin-Neng Wang VI. Phototherapy Photodynamic Therapy Jarod C. Finlay, Keith Cengel, Theresa M. Busch, Timothy C. Zhu Low Level Laser and Light Therapy Ying-Ying Huang, Aaron C-H Chen, Michael R. Hamblin

Neovascularization in cervical intraepithelial neoplasia (CIN) is studied because it is the precursor to the third most common female cancer worldwide. Diffuse reflectance from 450-600 nm was collected from 46 patients (76 sites) undergoing colposcopy at Duke University Medical Center. Total hemoglobin, derived using an inverse Monte Carlo model, significantly increased in CIN 2+ (N=12) versus CIN 1 (N=16) and normal tissues (N=48) combined with P

This study demonstrates the use of optical spectroscopy for monitoring tumor oxygenation and metabolism in response to hyperoxic gas breathing. Hemoglobin saturation and redox ratio were quantified for a set of 14 and 9 mice, respectively, measured at baseline and during carbogen breathing (95% O(2), 5% CO(2)). In particular, significant increases in hemoglobin saturation and fluorescence redox ratio were observed upon carbogen breathing. These data were compared with data obtained concurrently using an established invasive technique, the OxyLite partial oxygen pressure (pO(2)) system, which also showed a significant increase in pO(2). It was found that the direction of changes were generally the same between all of the methods, but that the OxyLite system was much more variable in general, suggesting that optical techniques may provide a better assessment of global tumor physiology. Optical spectroscopy measurements are demonstrated to provide a reliable, reproducible indication of changes in tumor physiology in response to physiologic manipulation.

A hybrid optical device that uses a multimode fiber coupled to a tunable light source for illumination and a 2.4-mm photodiode for detection in contact with the tissue surface is developed as a first step toward our goal of developing a cost-effective, miniature spectral imaging device to map tissue optical properties in vivo. This device coupled with an inverse Monte Carlo model of reflectance is demonstrated to accurately quantify tissue absorption and scattering in tissue-like turbid synthetic phantoms with a wide range of optical properties. The overall errors for quantifying the absorption and scattering coefficients are 6.0±5.6 and 6.1±4.7%, respectively. Compared with fiber-based detection, having the detector right at the tis- sue surface can significantly improve light collection effi- ciency, thus reducing the requirement for sophisticated detectors with high sensitivity, and this design can be eas- ily expanded into a quantitative spectral imaging system for mapping tissue optical properties in vivo. © 2008 Society

We explore the use of Monte-Carlo-model-based approaches for the analysis of fluorescence and diffuse reflectance spectra measured ex vivo from breast tissues. These models are used to extract the absorption, scattering, and fluorescence properties of malignant and nonmalignant tissues and to diagnose breast cancer based on these intrinsic tissue properties. Absorption and scattering properties, including beta-carotene concentration, total hemoglobin concentration, hemoglobin saturation, and the mean reduced scattering coefficient are derived from diffuse reflectance spectra using a previously developed Monte Carlo model of diffuse reflectance. A Monte Carlo model of fluorescence described in an earlier manuscript was employed to retrieve the intrinsic fluorescence spectra. The intrinsic fluorescence spectra were decomposed into several contributing components, which we attribute to endogenous fluorophores that may present in breast tissues including collagen, NADH, and retinol/vitamin A. The model-based approaches removes any dependency on the instrument and probe geometry. The relative fluorescence contributions of individual fluorescing components, as well as beta-carotene concentration, hemoglobin saturation, and the mean reduced scattering coefficient display statistically significant differences between malignant and adipose breast tissues. The hemoglobin saturation and the reduced scattering coefficient display statistically significant differences between malignant and fibrous/benign breast tissues. A linear support vector machine classification using (1) fluorescence properties alone, (2) absorption and scattering properties alone, and (3) the combination of all tissue properties achieves comparable classification accuracies of 81 to 84% in sensitivity and 75 to 89% in specificity for discriminating malignant from nonmalignant breast tissues, suggesting each set of tissue properties are diagnostically useful for the discrimination of breast malignancy.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, three-dimensional functional imaging technique. This study investigates the potential for in vivo pre-cancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic co-factor, NADH. The dimethylbenz[α]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers were imaged using multiphoton FLIM at 780 nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells had short (0.29 ± 0.03 ns) and long lifetime components (2.03 ± 0.06 ns), attributed to free and protein-bound NADH, respectively. Low grade pre-cancers (mild to moderate dysplasia) and high grade pre-cancers (severe dysplasia and carcinoma in situ) were discriminated from normal tissues by their decreased protein-bound NADH lifetime (p

In this work experimental tissue phantoms and numerical models were developed to estimate photon migration through the fe- tal head in utero. The tissue phantoms incorporate a fetal head within an amniotic fluid sac surrounded by a maternal tissue layer. A con- tinuous wave, dual-wavelength (l5760 and 850 nm) spectrometer was employed to make near-infrared measurements on the tissue phantoms for various source-detector separations, fetal-head posi- tions, and fetal-head optical properties. In addition, numerical simu- lations of photon propagation were performed with finite-difference algorithms that provide solutions to the equation of radiative transfer as well as the diffusion equation. The simulations were compared with measurements on tissue phantoms to determine the best numeri- cal model to describe photon migration through the fetal head in utero. Evaluation of the results indicates that tissue phantoms in which the contact between fetal head and uterine wall is uniform best simu- lates the fetal head in utero for near-term pregnancies. Furthermore, we found that maximum sensitivity to the head can be achieved if the source of the probe is positioned directly above the fetal head. By optimizing the source-detector separation, the signal originating from photons that have traveled through the fetal head can drastically be increased. © 2000 Society of Photo-Optical Instrumentation Engineers. (S1083-3668(00)01402-7)