Gregory M. Palmer

Monte Carlo-based inverse model for calculating tissue optical properties. Part I: Theory and validation on synthetic phantoms

A flexible and fast Monte Carlo-based model of diffuse reflectance has been developed for the extraction of the absorption and scattering properties of turbid media, such as human tissues. This method is valid for a wide range of optical properties and is easily adaptable to existing probe geometries, provided a single phantom calibration measurement is made. A condensed Monte Carlo method was used to speed up the forward simulations. This model was validated by use of two sets of liquid-tissue phantoms containing Nigrosin or hemoglobin as absorbers and polystyrene spheres as scatterers. The phantoms had a wide range of absorption (0-20 cm(-1)) and reduced scattering coefficients (7-33 cm(-1)). Mie theory and a spectrophotometer were used to determine the absorption and reduced scattering coefficients of the phantoms. The diffuse reflectance spectra of the phantoms were measured over a wavelength range of 350-850 nm. It was found that optical properties could be extracted from the experimentally measured diffuse reflectance spectra with an average error of 3% or less for phantoms containing hemoglobin and 12% or less for phantoms containing Nigrosin.

Comparison of multiexcitation fluorescence and diffuse reflectance spectroscopy for the diagnosis of breast cancer (March 2003)

Nonmalignant (n = 36) and malignant (n = 20) tissue samples were obtained from breast cancer and breast reduction surgeries. These tissues were characterized using multiple excitation wavelength fluorescence spectroscopy and diffuse reflectance spectroscopy in the ultraviolet-visible wavelength range, immediately after excision. Spectra were then analyzed using principal component analysis (PCA) as a data reduction technique. PCA was performed on each fluorescence spectrum, as well as on the diffuse reflectance spectrum individually, to establish a set of principal components for each spectrum. A Wilcoxon rank-sum test was used to determine which principal components show statistically significant differences between malignant and nonmalignant tissues. Finally, a support vector machine (SVM) algorithm was utilized to classify the samples based on the diagnostically useful principal components. Cross-validation of this nonparametric algorithm was carried out to determine its classification accuracy in an unbiased manner. Multiexcitation fluorescence spectroscopy was successful in discriminating malignant and nonmalignant tissues, with a sensitivity and specificity of 70% and 92%, respectively. The sensitivity (30%) and specificity (78%) of diffuse reflectance spectroscopy alone was significantly lower. Combining fluorescence and diffuse reflectance spectra did not improve the classification accuracy of an algorithm based on fluorescence spectra alone. The fluorescence excitation-emission wavelengths identified as being diagnostic from the PCA-SVM algorithm suggest that the important fluorophores for breast cancer diagnosis are most likely tryptophan, NAD(P)H and flavoproteins.

In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters

Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ∼2 h to complete, plus up to several weeks for tumor growth and treatment procedures.

Monte Carlo-based inverse model for calculating tissue optical properties. Part II: Application to breast cancer diagnosis

The Monte Carlo-based inverse model of diffuse reflectance described in part I of this pair of companion papers was applied to the diffuse reflectance spectra of a set of 17 malignant and 24 normal-benign ex vivo human breast tissue samples. This model allows extraction of physically meaningful tissue parameters, which include the concentration of absorbers and the size and density of scatterers present in tissue. It was assumed that intrinsic absorption could be attributed to oxygenated and deoxygenated hemoglobin and beta-carotene, that scattering could be modeled by spheres of a uniform size distribution, and that the refractive indices of the spheres and the surrounding medium are known. The tissue diffuse reflectance spectra were evaluated over a wavelength range of 400-600 nm. The extracted parameters that showed the statistically most significant differences between malignant and nonmalignant breast tissues were hemoglobin saturation and the mean reduced scattering coefficient. Malignant tissues showed decreased hemoglobin saturation and an increased mean reduced scattering coefficient compared with nonmalignant tissues. A support vector machine classification algorithm was then used to classify a sample as malignant or nonmalignant based on these two extracted parameters and produced a cross-validated sensitivity and specificity of 82% and 92%, respectively.

Quantitative Optical Spectroscopy: A Robust Tool for Direct Measurement of Breast Cancer Vascular Oxygenation and Total Hemoglobin Content In vivo

We propose the use of a robust, biopsy needle-based, fiber-optic tool for routine clinical quantification of tumor oxygenation at the time of diagnostic biopsy for breast cancer. The purpose of this study was to show diffuse reflectance spectroscopy as a quantitative tool to measure oxygenation levels in the vascular compartment of breast cancers in vivo via an optical biopsy technique. Thirty-five patients undergoing surgical treatment for breast cancer were recruited for the study at Duke University Medical Center. Diffuse reflectance spectroscopy was performed on the tumors in situ before surgical resection, followed by needle-core biopsy of the optically measured tissue. Hemoglobin saturation and total hemoglobin content were quantified from 76 optical spectra-tissue biopsy pairs, consisting of 20 malignant, 23 benign, and 33 adipose tissues. Hemoglobin saturation in malignant tissues was significantly lower than nonmalignant tissues (P<0.002) and was negatively correlated with tumor size and pathologic tumor category (P<0.05). Hemoglobin saturation was positively correlated with total hemoglobin content in malignant tissues (P<0.02). HER2/neu-amplified tumors exhibited significantly higher total hemoglobin content (P<0.05) and significantly higher hemoglobin saturation (P<0.02), which is consistent with a model of increased angiogenesis and tumor perfusion promoted by HER2/neu amplification. Diffuse reflectance spectroscopy could aid in prognosis and prediction in breast cancer via quantitative assessment of tumor physiology at the time of diagnostic biopsy.

Advances in Quantitative UV-Visible Spectroscopy for Clinical and Pre-clinical Application in Cancer

Methods of optical spectroscopy that provide quantitative, physically or physiologically meaningful measures of tissue properties are an attractive tool for the study, diagnosis, prognosis, and treatment of various cancers. Recent development of methodologies to convert measured reflectance and fluorescence spectra from tissue to cancer-relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation have significantly advanced the field of tissue optical spectroscopy. The number of publications reporting quantitative tissue spectroscopy results in the UV-visible wavelength range has increased sharply in the past three years, and includes new and emerging studies that correlate optically measured parameters with independent measures such as immunohistochemistry, which should aid in increased clinical acceptance of these technologies.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Significance Engineering of highly functional skeletal muscle tissues can provide accurate models of muscle physiology and disease and aid treatment of various muscle disorders. Previous tissue-engineering efforts have fallen short of recreating structural and contractile properties of native muscle in vitro. Here, we describe the creation of biomimetic skeletal muscle tissues with structural, functional, and myogenic properties characteristic of native muscle and contractile stress values that surpass those of neonatal rat muscle. When implanted and real-time imaged in live animals, engineered muscle grafts undergo robust vascularization and perfusion, exhibit continued myogenesis, and show further improvements in intracellular calcium handling and contractile function. This process is significantly enhanced by myogenic predifferentiation and formation of aligned muscle architecture in vitro. Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Autofluorescence spectroscopy of normal and malignant human breast cell lines

Abstract The fluorescence of tryptophan, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized in normal human breast cells as well as in malignant human breast cells of similar and dissimilar genetic origins. Fluorescence measurements of each cell line were made over a wide range of cell concentrations, and the fluorescence per cell was determined from the slope in the linear range of the fluorescence intensity vs cell concentration plot. All of the malignant cells showed a statistically significant decrease in the tryptophan fluorescence per cell relative to that of the normal cells. No statistically significant differences were observed in the NAD(P)H or FAD fluorescence per cell between the normal and any of the malignant cell types. NAD(P)H fluorescence was also imaged from monolayers of the normal and malignant cells (of similar genetic origin) using two-photon fluorescence microscopy. A statistically significant decrease in the NAD(P)H fluorescence with malignancy was observed, suggesting that fluorescence imaging of single cells or the cell monolayer preparation may provide more contrast than volume-averaged fluorescence measurements of cells in suspension. In conclusion, the differences in normal and malignant human breast tissue fluorescence spectra may be attributed in part to differences in the intrinsic cellular fluorescence of normal and malignant breast epithelial cells.

Autofluorescence and diffuse reflectance properties of malignant and benign breast tissues

BackgroundFluorescence spectroscopy is an evolving technology that can rapidly differentiate between benign and malignant tissues. These differences are thought to be due to endogenous fluorophores, including nicotinamide adenine dinucleotide, flavin adenine dinucleotide, and tryptophan, and absorbers such as β-carotene and hemoglobin. We hypothesized that a statistically significant difference would be demonstrated between benign and malignant breast tissues on the basis of their unique fluorescence and reflectance properties.MethodsOptical measurements were performed on 56 samples of tumor or benign breast tissue. Autofluorescence spectra were measured at excitation wavelengths ranging from 300 to 460 nm, and diffuse reflectance was measured between 300 and 600 nm. Principal component analysis to dimensionally reduce the spectral data and a Wilcoxon ranked sum test were used to determine which wavelengths showed statistically significant differences. A support vector machine algorithm compared classification results with the histological diagnosis (gold standard).ResultsSeveral excitation wavelengths and diffuse reflectance spectra showed significant differences between tumor and benign tissues. By using the support vector machine algorithm to incorporate relevant spectral differences, a sensitivity of 70.0% and specifcity of 91.7% were achieved.ConclusionsA statistically significant difference was demonstrated in the diffuse reflectance and fluorescence emission spectra of benign and malignant breast tissue. These differences could be exploited in the development of adjuncts to diagnostic and surgical procedures.

Molecular Imaging of Hypoxia

A wide variety of imaging approaches have been developed in the past few decades for monitoring tumor oxygenation and hypoxia in vivo. In particular, nuclear medicine has seen the development of several radiolabeled hypoxia markers and is the preferred method for imaging of tumor hypoxia. Hypoxia imaging is increasingly being used in the clinical setting and is progressing from a mere detection method to application in individualization of chemoradiotherapy.