Two-Photon Autofluorescence Imaging of Fixed Tissues: Feasibility and Potential Values for Biomedical Applications.

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50 µm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750 nm, blue emission filter; FAD channel: excitation 860 nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.

Optical Redox Imaging of Fixed Unstained Muscle Slides Reveals Useful Biological Information.

PURPOSE: Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information. PROCEDURES: Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences. RESULTS: We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups. CONCLUSION: Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications.

Two-channel autofluorescence analysis for oral cancer.

We created a two-channel autofluorescence test to detect oral cancer. The wavelengths 375 and 460 nm, with filters of 479 and 525 nm, were designed to excite and detect reduced-form nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Patients with oral cancer or with precancerous lesions, and a control group with healthy oral mucosae, were enrolled. The lesion in the autofluorescent image was the region of interest. The average intensity and heterogeneity of the NADH and FAD were calculated. The redox ratio [(NADH)/(NADH + FAD)] was also computed. A quadratic discriminant analysis (QDA) was used to compute boundaries based on sensitivity and specificity. We analyzed 49 oral cancer lesions, 34 precancerous lesions, and 77 healthy oral mucosae. A boundary (sensitivity: 0.974 and specificity: 0.898) between the oral cancer lesions and healthy oral mucosae was validated. Oral cancer and precancerous lesions were also differentiated from healthy oral mucosae (sensitivity: 0.919 and specificity: 0.755). The two-channel autofluorescence detection device and analyses of the intensity and heterogeneity of NADH, and of FAD, and the redox ratio combined with a QDA classifier can differentiate oral cancer and precancerous lesions from healthy oral mucosae.

Novel quantitative analysis of autofluorescence images for oral cancer screening.

OBJECTIVES: VELscope® was developed to inspect oral mucosa autofluorescence. However, its accuracy is heavily dependent on the examining physician's experience. This study was aimed toward the development of a novel quantitative analysis of autofluorescence images for oral cancer screening. MATERIALS AND METHODS: Patients with either oral cancer or precancerous lesions and a control group with normal oral mucosa were enrolled in this study. White light images and VELscope® autofluorescence images of the lesions were taken with a digital camera. The lesion in the image was chosen as the region of interest (ROI). The average intensity and heterogeneity of the ROI were calculated. A quadratic discriminant analysis (QDA) was utilized to compute boundaries based on sensitivity and specificity. RESULTS: 47 oral cancer lesions, 54 precancerous lesions, and 39 normal oral mucosae controls were analyzed. A boundary of specificity of 0.923 and a sensitivity of 0.979 between the oral cancer lesions and normal oral mucosae were validated. The oral cancer and precancerous lesions could also be differentiated from normal oral mucosae with a specificity of 0.923 and a sensitivity of 0.970. CONCLUSION: The novel quantitative analysis of the intensity and heterogeneity of VELscope® autofluorescence images used in this study in combination with a QDA classifier can be used to differentiate oral cancer and precancerous lesions from normal oral mucosae.

Continuous Wave Spectroscopy with Diffusion Theory for Quantification of Optical Properties: Comparison Between Multi-distance and Multi-wavelength Data Fitting Methods.

Typically, continuous wave spectroscopy (CWS) can be used to accurately quantify biological tissue optical properties (µ a and µ s ') by employing the diffuse reflectance information acquired at multiple source-detector separations (multi-distance). On the other hand, sample optical properties can also be obtained by fitting multi-wavelength light reflectance acquired at a single source detector separation to the diffusion theory equation. To date, multi-wavelength and multi-distance methods have not yet been rigorously compared for their accuracy in quantification of the sample optical properties. In this investigation, we compared the accuracy of the two above-mentioned quantifying methods in the optical properties recovery. The liquid phantoms had µ a between 0.004 and 0.011 mm(-1) and µ s ' between 0.55 and 1.07 mm(-1) whose optical properties mimic the human breast. Multi-distance data and multi-wavelength data were fitted to the same diffusion equation for consistency. The difference between benchmark µ a and µ s ' and the fitted results, ΔError (ΔE) was used to evaluate the accuracy of the two methods. The results showed that either method yielded ΔE within 15-30 % when values were within certain limits to standard values applicable to µ s ' and µ a for human adipose tissue. Both methods showed no significant differences in ΔE values. Our results suggest that both multi-distance and multi-wavelength methods can yield similar reasonable optical properties in biological tissue with a proper calibration.

Imaging heterogeneity in the mitochondrial redox state of premalignant pancreas in the pancreas-specific PTEN-null transgenic mouse model.

BACKGROUND: Metabolic alteration is one of the hallmarks of carcinogenesis. We aimed to identify certain metabolic biomarkers for the early detection of pancreatic cancer (PC) using the transgenic PTEN-null mouse model. Pancreas-specific deletion of PTEN in mouse caused progressive premalignant lesions such as highly proliferative ductal metaplasia. We imaged the mitochondrial redox state of the pancreases of the transgenic mice approximately eight months old using the redox scanner, i.e., the nicotinamide adenine dinucleotide/oxidized flavoproteins (NADH/Fp) fluorescence imager at low temperature. Two different approaches, the global averaging of the redox indices without considering tissue heterogeneity along tissue depth and the univariate analysis of multi-section data using tissue depth as a covariate were adopted for the statistical analysis of the multi-section imaging data. The standard deviations of the redox indices and the histogram analysis with Gaussian fit were used to determine the tissue heterogeneity. RESULTS: All methods show consistently that the PTEN deficient pancreases (Pdx1-Cre;PTENlox/lox) were significantly more heterogeneous in their mitochondrial redox state compared to the controls (PTENlox/lox). Statistical analysis taking into account the variations of the redox state with tissue depth further shows that PTEN deletion significantly shifted the pancreatic tissue to an overall more oxidized state. Oxidization of the PTEN-null group was not seen when the imaging data were analyzed by global averaging without considering the variation of the redox indices along tissue depth, indicating the importance of taking tissue heterogeneity into account for the statistical analysis of the multi-section imaging data. CONCLUSIONS: This study reveals a possible link between the mitochondrial redox state alteration of the pancreas and its malignant transformation and may be further developed for establishing potential metabolic biomarkers for the early diagnosis of pancreatic cancer.

Breast cancer detection of large size to DCIS by hypoxia and angiogenesis using NIRS.

This investigation aimed to test all tumor-bearing patients who undergo biopsy to see if angiogenesis and hypoxia can detect cancer. We used continuous-wave near-infrared spectroscopy (NIRS) to measure blood hemoglobin concentration to obtain blood volume or total hemoglobin [Hbtot] and oxygen saturation for the angiogenesis and hypoxic biomarkers. The contralateral breast was used as a reference to derive the difference from breast tumor as a difference in total hemoglobin Δ[HBtot] and a difference in deoxygenation Δ([Hb]-[HbO2]). A total of 91 invasive cancers, 26 DCIS, 45 fibroblastomas, 96 benign tumors excluding cysts, and 67 normal breasts were examined from four hospitals. In larger-size tumors, there is significantly higher deoxygenation in invasive and ductal carcinoma in situ (DCIS) than in that of benign tumors, but no significant difference was seen in smaller tumors of ≤ 1 cm. With the two parameters of high total hemoglobin and hypoxia score, the sensitivity and specificity of cancer detection were 60.3 % and 85.3 %, respectively. In summary, smaller-size tumors are difficult to detect with NIRS, whereas DCIS can be detected by the same total hemoglobin and hypoxic score in our study.

Characterizing the metabolic heterogeneity in human breast cancer xenografts by 3D high resolution fluorescence imaging.

We previously reported that tumor mitochondrial redox state and its heterogeneity distinguished between the aggressive and the indolent breast cancer xenografts, suggesting novel metabolic indices as biomarkers for predicting tumor metastatic potential. Additionally, we reported that the identified redox biomarkers successfully differentiated between the normal breast tissue and the cancerous breast tissue from breast cancer patients. The aim of the present study was to further characterize intratumor heterogeneity by its distribution of mitochondrial redox state and glucose uptake pattern in tumor xenografts and to further investigate the metabolic heterogeneity of the clinical biopsy samples. We employed the Chance redox scanner, a multi-section cryogenic fluorescence imager to simultaneously image the intratumor heterogeneity in the mitochondrial redox state and glucose uptake at a high spatial resolution (down to 50 × 50 × 20 µm(3)). The mitochondrial redox state was determined by the ratio of the intrinsic fluorescence signals from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp including FAD, i.e., flavin adenine dinucleotide), and the glucose uptake was measured using a near-infrared fluorescent glucose-analogue, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG). Significant inter- and intratumor metabolic heterogeneity were observed from our imaging data on various types of breast cancer xenografts. The patterns and degrees of heterogeneity of mitochondrial redox state appeared to relate to tumor size and metastatic potential. The glucose uptake was also heterogeneous and generally higher in tumor peripheries. The oxidized and reduced regions mostly corresponded with the lower and the higher pyro-2DG uptake, respectively. However, there were some regions where the glucose uptake did not correlate with the redox indices. Pronounced glucose uptake and high NADH were observed in certain localized areas within the tumor necrotic regions, indicative of the existence of viable cells which was also supported by the H&E staining. Significant heterogeneity of the redox state indices was also observed in clinical specimens of breast cancer patients. As abnormal metabolism including the Warburg effect (high glycolysis) plays important roles in cancer transformation and progression, our observations that reveal the 3D intratumor metabolic heterogeneity as a characteristic feature of breast tumors are of great importance for understanding cancer biology and developing diagnostic and therapeutic methods.

Heterogeneity of mitochondrial redox state in premalignant pancreas in a PTEN null transgenic mouse model.

Pancreas-specific deletion of PTEN in mice revealed progressive premalignant lesions such as ductal metaplasia with infrequent malignant transformation. In this study, we aimed at evaluating the mitochondrial redox state of the metaplastic pancreas in a pancreas-specific PTEN null transgenic mouse model. The two intrinsic fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) such as flavin adenine dinucleotide (FAD), in the respiratory chain in mitochondria are sensitive indicators of mitochondrial redox states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox ratio, Fp/(Fp+NADH) provides a sensitive index of mitochondrial redox state. We have obtained optical images of the in vivo mitochondrial redox states of the snap frozen pancreases from pancreas-specific PTEN null mice (Pdx1-Cre;PTEN(lox/lox), N=3) and the controls (PTEN(lox/lox), N=3) using the redox scanner at low temperature. The results showed high spatial heterogeneity of mitochondrial redox state in the mutated pancreases with hot spots of much higher Fp redox ratios whereas the normal ones, were relatively homogenous. The cystic dilation regions in the metaplastic pancreases showed little to no NADH or Fp signal. Histological analysis confirmed no cells existed in these regions. It is the first time that the in vivo mitochondrial redox states of the metaplastic mouse pancreas were optically imaged. Our previous results on human melanoma and breast cancer mouse xenografts have shown that mitochondrial redox state quantitatively correlates with cancer metastatic potential. The more oxidative mitochondrial redox state (higher Fp redox ratio) corresponded to the higher metastatic potential of the tumors. As mitochondrial redox state imbalance is associated with abnormal mitochondrial function, and redox state mediates the generation of reactive oxygen species and many signal transduction pathways, this research may provide insights for studying basic biology and developing early diagnostic imaging biomarkers for pancreatic cancer.

Optical tecnology developments in biomedicine: history, current and future.

Biomedical optics is a rapidly emerging field for medical imaging and diagnostics. This paper reviews several biomedical optical technologies that have been developed and translated for either clinical or pre-clinical applications. Specifically, we focus on the following technologies: 1) near-infrared spectroscopy and tomography, 2) optical coherence tomography, 3) fluorescence spectroscopy and imaging, and 4) optical molecular imaging. There representative biomedical applications are also discussed here.

IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES.

Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ∼200-440 µm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

Quantitative mitochondrial redox imaging of breast cancer metastatic potential.

Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

Comparisons of muscle oxygenation changes between arm and leg muscles during incremental rowing exercise with near-infrared spectroscopy.

Our purpose is to compare the changes in muscle oxygenation in the vastus lateralis (VL) and biceps brachii (BB) muscles simultaneously using near-infrared spectroscopy (NIRS) during incremental rowing exercise in eight rowers. Based on the BB and VL muscle oxygenation patterns, two points are used to characterize the muscle oxygenation kinetics in both the arm and the leg muscles. The first point is the breaking point (Bp), which refers to an accelerated fall in muscle oxygenation that correlates with the gas exchange threshold (GET). The second point is the leveling-off point (Lo), which suggests the upper limit of O(2) extraction. The GET occurred at 63.3+/-2.4% of maximal oxygen uptake (VO(2 max)). The Bp appeared at 45.0+/-3.8% and 55.6+/-2.4% VO(2 max) in the BB and VL, respectively. The Lo appeared at 63.6+/-4.1% and 86.6+/-1.0% VO(2 max) in these two muscles, respectively. Both the Bp and the Lo occurred earlier in BB compared with VL. These results suggest that arm muscles have lower oxidative capacity than leg muscles during rowing exercise. The rowers with higher exercise performances showed heavier workloads, as evaluated by Bp and Lo. The monitoring of muscle oxygenation by NIRS in arm and leg muscles during rowing could be a useful guide for evaluation and training.

Noninvasive determination of exercise-induced vasodilation during bicycle exercise using near infrared spectroscopy.

BACKGROUND: The purpose of this study was to examine the changes in total hemoglobin (Delta[tHb]) response during bicycle exercise at various constant workloads using near infrared continuous wave spectroscopy (NIRcws) in humans. We hypothesized that the Delta[tHb] during exercise may progressively increase as a result of a dilation of the vascular bed and/or capillary recruitment at lower constant work rates. MATERIAL/METHODS: Seven healthy subjects performed bicycle exercise at 20, 40, 60, 80, and 100% of maximal work rates (Wmax) for 5 min. Muscle oxygenation change (Delta[Oxy]) and Delta[tHb] at the right vastus lateralis were monitored using a NIRcws device. Exercise-induced Delta[tHb] and Delta[Oxy] responses at each constant workload were evaluated as functional Delta[tHb] change (f-Delta[tHb]) and functional oxygenation change (f-Delta[Oxy]), respectively. Blood lactate concentration [La] was also evaluated after each exercise stage. RESULTS: At work rates 60%Wmax and below, after an initial decrease at the start of exercise, both Delta[tHb] and Delta[Oxy] showed progressive increases until the end of exercise. A significant positive correlation was found between f-Delta[tHb] and f-Delta[Oxy] (p<0.01). In addition, there was a significant negative relationship of [La] to f-Delta[tHb] during exercise (p<0.05). CONCLUSIONS: These results provide evidence that increased muscle oxygenation during bicycle exercise up to 60%Wmax may be caused by increased O2 supply due to exercise-induced blood volume expansion. Subsequently, the cessation of increase in f-Delta[tHb] at higher intensity exercise may lead to lower muscle tissue oxygenation and higher lactate accumulation.

Histological basis of MR/optical imaging of human melanoma mouse xenografts spanning a range of metastatic potentials.

Predicting tumor aggressiveness will greatly facilitate cancer treatment. We have previously reported investigations utilizing various MR/optical imaging methods to differentiate human melanoma mouse xenografts spanning a range of metastatic potentials. The purpose of this study was to explore the histological basis of the previously reported imaging findings. We obtained the cryogenic tumor sections of three types of human melanoma mouse xenografts with their metastatic potentials falling in the rank order A375P

MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES.

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D high-resolution (~ 50 × 50 × 10 µm3) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 µm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.

QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE.

The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) including flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a three-dimensional (3D) low temperature imager previously developed by Chance et al., measures the in vivo metabolic properties of tissue samples by acquiring fluorescence images of NADH and Fp. The redox ratios, i.e. Fp/(Fp+NADH) and NADH/(Fp+NADH), provided a sensitive index of the mitochondrial redox state and were determined based on relative signal intensity ratios. Here we report the further development of the redox scanning technique by using a calibration method to quantify the nominal concentration of the fluorophores in tissues. The redox scanner exhibited very good linear response in the range of NADH concentration between 165-1318µM and Fp between 90-720 µM using snap-frozen solution standards. Tissue samples such as human tumor mouse xenografts and various mouse organs were redox-scanned together with adjacent NADH and Fp standards of known concentration at liquid nitrogen temperature. The nominal NADH and Fp concentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards. This calibration procedure allows comparing redox images obtained at different time, independent of instrument settings. The quantitative multi-slice redox images revealed heterogeneity in mitochondrial redox state in the tissues.

Pharmacokinetic-rate images of indocyanine green for breast tumors using near-infrared optical methods.

In this paper, we develop a method of forming pharmacokinetic-rate images of indocyanine green (ICG) and apply our method to in vivo data obtained from three patients with breast tumors. To form pharmacokinetic-rate images, we first obtain a sequence of ICG concentration images using the differential diffuse optical tomography technique. We next employ a two-compartment model composed of plasma, and extracellular-extravascular space (EES), and estimate the pharmacokinetic rates and concentrations in each compartment using the extended Kalman filtering framework. The pharmacokinetic-rate images of the three patient show that the rates from the tumor region and outside the tumor region are statistically different. Additionally, the ICG concentrations in plasma, and the EES compartments are higher around the tumor region agreeing with the hypothesis that around the tumor region ICG may act as a diffusible extravascular flow in compromised capillary of cancer vessels. Our study indicates that the pharmacokinetic-rate images may provide superior information than single set of pharmacokinetic rates estimated from the entire breast tissue for breast cancer diagnosis.

Characterization of breast tumors with NIR methods using optical indices.

This work describes the characterization efficiency of optical properties of breast tumors based on the features obtained using in vivo near-infrared (NIR) spectroscopy measurements. Three features, relative blood concentration, oxygen saturation and the size of the tumor, are used to diagnose benign and malignant tumors. The performance of the proposed set of features are evaluated by various classifiers using data acquired from 44 patients with malignant tumors, and 72 patients with benign tumors. The area under the receiver operating characteristics (ROC) curve of the scaled nearest mean classifier (NMSC) using the three features yields a value of 0.91 with a significance level of 0.05. Our results suggest that the features, relative blood concentration, and oxygen saturation can differentiate breast tumors with a relatively high precision.