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With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake (“Warburg effect”), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease.Implications:This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Supplementary Figure 1: 3D Rendering of mammosphere stained with 2-NBDG and TMRE. Supplementary Figure 2: Western blot confirms loss of Her2 expression following withdrawal of Dox in media conditions. Supplementary Figure 3: Quantification of immunohistochemistry results confirm changes in proliferation and apoptosis. Supplementary Figure 4: Average 2-NBDG and TMRE per mammosphere and group across each time point. Supplementary Figure 5: Annexin V and TMRE flow cytometry show increased TMRE is independent of increased apoptosis.

BACKGROUND: Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. AIM OF REVIEW: In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.

Significance: Optical fluorescence spectroscopy technique has been explored extensively to quantify both glucose uptake and mitochondrial metabolism with proper fluorescent probes in small tumor models in vivo. However, it remains a great challenge to rapidly quantify the intrinsic metabolic fluorophores from the optically measured fluorescence spectra that contain significant distortions due to tissue absorption and scattering. Aim: To enable rapid spectral data processing and quantify the in vivo metabolic parameters in real-time, we present an empirical ratio-metric method for rapid fluorescence spectra attenuation correction with high accuracy. Approach: A first-order approximation of intrinsic fluorescence spectra can be obtained by dividing the fluorescence spectra by diffuse reflectance spectra with some variable powers. We further developed this approximation for rapid extraction of intrinsic key metabolic probes (2-NBDG for glucose uptake and TMRE for mitochondrial function) by dividing the distorted fluorescence spectra by diffuse reflectance intensities recorded at excitation and emission peak with a pair of system-dependent powers. Tissue-mimicking phantom studies were conducted to evaluate the method. Results: The tissue-mimicking phantom studies demonstrated that our empirical method could quantify the key intrinsic metabolic probes in near real-time with an average percent error of ∼5%. Conclusions: An empirical method was demonstrated for rapid quantification of key metabolic probes from fluorescence spectra measured on a tissue-mimicking turbid medium. The proposed method will potentially facilitate real-time monitoring of key metabolic parameters of tumor models in vivo using optical spectroscopy, which will significantly advance translational cancer research.

We report a point-of-care optical spectroscopy platform for simultaneous quantification of key metabolic and vascular parameters of tumors in vivo. Both phantom and preclinical animal studies were conducted to demonstrate the capability of the system.

Overexpression of heat shock protein 90 (Hsp90) on the surface of breast cancer cells makes it an attractive molecular biomarker for breast cancer diagnosis. Before a ubiquitous diagnostic method can be established, an understanding of the systematic errors in Hsp90-based imaging is essential. In this study, we investigated three factors that may influence the sensitivity of ex vivo Hsp90 molecular imaging: time-dependent tissue viability, nonspecific diffusion of an Hsp90 specific probe (HS-27), and contact-based imaging. These three factors will be important considerations when designing any diagnostic imaging strategy based on fluorescence imaging of a molecular target on tissue samples.

Targeting a tumor&rsquo, s metabolic dependencies is a clinically actionable therapeutic approach, however, identifying subtypes of tumors likely to respond remains difficult. The use of lipids as a nutrient source is of particular importance, especially in breast cancer. Imaging techniques offer the opportunity to quantify nutrient use in preclinical tumor models to guide development of new drugs that restrict uptake or utilization of these nutrients. We describe a fast and dynamic approach to image fatty acid uptake in vivo and demonstrate its relevance to study both tumor metabolic reprogramming directly, as well as the effectiveness of drugs targeting lipid metabolism. Specifically, we developed a quantitative optical approach to spatially and longitudinally map the kinetics of long-chain fatty acid uptake in in vivo murine models of breast cancer using a fluorescently labeled palmitate molecule, Bodipy FL c16. We chose intra-vital microscopy of mammary tumor windows to validate our approach in two orthotopic breast cancer models: a MYC-overexpressing, transgenic, triple-negative breast cancer (TNBC) model and a murine model of the 4T1 family. Following injection, Bodipy FL c16 fluorescence increased and reached its maximum after approximately 30 min, with the signal remaining stable during the 30&ndash, 80 min post-injection period. We used the fluorescence at 60 min (Bodipy60), the mid-point in the plateau region, as a summary parameter to quantify Bodipy FL c16 fluorescence in subsequent experiments. Using our imaging platform, we observed a two- to four-fold decrease in fatty acid uptake in response to the downregulation of the MYC oncogene, consistent with findings from in vitro metabolic assays. In contrast, our imaging studies report an increase in fatty acid uptake with tumor aggressiveness (6NR, 4T07, and 4T1), and uptake was significantly decreased after treatment with a fatty acid transport inhibitor, perphenazine, in both normal mammary pads and in the most aggressive 4T1 tumor model. Our approach fills an important gap between in vitro assays providing rich metabolic information at static time points and imaging approaches visualizing metabolism in whole organs at a reduced resolution.

In most biomedical optical spectroscopy platforms, a fiber-probe consisting of single or multiple illumination and collection fibers was commonly used for the delivery of illuminating light and the collection of emitted light. Typically, the signals from all collection fibers were combined and then sampled to characterize tissue samples. Such simple averaged optical measurements may induce significant errors for in vivo tumor characterization, especially in longitudinal studies where the tumor size and location vary with tumor stages. In this study, we utilized the Monte Carlo technique to optimize the fiber-probe geometries of a spectroscopy platform to enable tumor-sensitive diffuse reflectance and fluorescence measurements on murine subcutaneous tissues with growing solid tumors that have different sizes and depths. Our data showed that depth-sensitive techniques offer improved sensitivity in tumor detection compared to the simple averaged approach in both reflectance and fluorescence measurements. Through the numerical studies, we optimized the source-detector distances, fiber diameters, and numerical apertures for sensitive measurement of small solid tumors with varying size and depth buried in murine subcutaneous tissues. Our study will advance the design of a fiber-probe in an optical spectroscopy system that can be used for longitudinal tumor metabolism and vasculature monitoring.

Redox metabolism plays essential functions in the pathology of cancer. As tumor redox profiles uniquely reflect cancer stage and in select cases, therapeutic sensitivity, the capability to image redox molecular features is essential to improve diagnosis, treatment, and overall quality-of-life (QOL) of cancer patients. While a number of radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate feasibility of diagnosing radiation resistant tumors, which display lower [18F]DCP signal. These findings are consistent with our previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We anticipate further development and implementation of this concept in clinical practice to improve the diagnosis of patients with radiation resistant tumors and the accuracy of prognosis for patients undergoing radiation treatment.Single Sentence SummaryThe study introduces a new PET radiotracer for profiling tumor protein oxidation as a prognostic indicator of resistance to radiation therapy.

Flap transfer has become a common technique in reconstructive surgery. However, a significant number of compromised skin flaps are not successfully salvaged because the current clinical method for flap assessment relies heavily on the clinician's experience. Vascular occlusion is the major reason for flap failure, thus the accurate and objective early prediction of blood vessel occlusion is vitally important. Our parallel point measurement study has demonstrated the great potential of joint diffuse reflectance and autofluorescence spectroscopy in the early detection and differentiation of venous and arterial occlusion in skin flaps. Unfortunately, the technique of point measurements is not suitable to examine a large skin flap when a high spatial resolution is required. In this study, we attempted to overcome this problem by performing spectral diffuse reflectance and autofluorescence imaging on a rat skin flap model. Both imaging data and reconstructed spectra were used to statistically differentiate control flaps, arterially occluded flaps and venously occluded flaps. Our preliminary results suggest that the technique of joint diffuse reflectance and autofluorescence spectroscopic imaging can achieve high classification accuracy thus could be used to detect and differentiate flaps with venous and arterial occlusion accurately at an early time point in a large skin flap. Typical reconstructed spectra of (a) diffuse reflectance and (b) autofluorescence after normalization.

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake (“Warburg effect”), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. Implications: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Overexpression of heat shock protein 90 (Hsp90) on the surface of breast cancer cells makes it an attractive molecular biomarker for breast cancer diagnosis. Before a ubiquitous diagnostic method can be established, an understanding of the systematic errors in Hsp90-based imaging is essential. In this study, we investigated three factors that may influence the sensitivity of ex vivo Hsp90 molecular imaging: time-dependent tissue viability, nonspecific diffusion of an Hsp90 specific probe (HS-27), and contact-based imaging. These three factors will be important considerations when designing any diagnostic imaging strategy based on fluorescence imaging of a molecular target on tissue samples.

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 (“Warburg effect”), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor’s adaptive behavior.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.

Drug-carriers, capable of releasing the drug at the target sites upon external stimuli, are attractive for theranostic applications. In recent years, photo-responsive nanoparticles (NPs) have received considerable attention because of their potentials in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the process of development and are unprocurable by the clinics. Here, we demonstrated facile fabrication of these photo-responsive NPs by loading hydrophilic gold NPs within thermo-responsive liposomes. Calcein was used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Furthermore, we characterized their size, morphology, phase transition temperature and stability. Finally, we demonstrated that this photo-triggered release might be due to the membrane disruption caused by microbubble cavitation.

For the first time, we performed simultaneous in vivo microscopic imaging of 2-NBDG and TMRE in a preclinical model, to successfully capture glucose and mitochondrial membrane potential on the same tissue site at one time.

Our previous preclinical study demonstrated that both visible diffuse reflectance and autofluorescence spectroscopy, each of which yields a different set of physiological information, can predict skin flap viability with high accuracy in a MacFarlane rat dorsal skin flap model. In this report, we further evaluated our technique for the early detection and differentiation of venous occlusion and arterial occlusion in a rat groin flap model. We performed both diffuse reflectance and autofluorescence measurements on the skin flap model and statistically differentiated between flaps with and without occlusions as well as between flaps with venous occlusion and those with arterial occlusion based on these non-invasive optical measurements. Our preliminary results suggested that visible diffuse reflectance and autofluorescence spectroscopy can be potentially used clinically to detect both venous and arterial occlusion and differentiate one from the other accurately at an early time point.

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.

We investigate the potentials of digitally sampling scintillation pulses techniques for positron emission tomography (PET) in this paper, focusing on the determination of the event time. We have built, and continue building, a digital library of PET event waveforms generated with various combinations of photo-detectors and scintillator materials, with various crystal sizes. Events in this digital library are obtained at a high sampling of 20 GSps (Giga-samples per second) so that their waveforms are recorded with high accuracy. To explore the potential advantages of digitally sampling scintillation pulses, we employ a dataset in the above-mentioned library to evaluate two methods for digitizing the event pulses and linear interpolation techniques to analyze the resulting digital samples. Our results show that the two digitization methods that we studied can yield a coincidence timing resolution of about 300 ps FWHM when applied to events generated by a pair of LSO + PMT detector units. This timing resolution is comparable with that is achieved by the same detector pair with a constant fraction discriminator (CFD). As a benchmark, regular-time sampling (RTS) method, usually implemented with very fast traditional analog-to-digital converters (ADCs) for digitizing scintillation pulses, is not feasible for a multi-channel system like a PET system. Digitizing scintillation pulses with multi-voltage threshold (MVT) method could be implemented at a reasonable cost for a PET system. With digitized PET event samples, various digital signal processing (DSP) techniques can be implemented to determine event arrival time. Our results have therefore demonstrated the promising potentials of digitally sampling scintillation pulses techniques in PET imaging.

This dissertation presents a series of studies in the development of contact and non-contact optical measurement techniques, and Monte Carlo (MC) based data analysis methods in visible diffuse reflectance and auto-fluorescence spectroscopy/imaging for tissue characterization. Their applications in early epithelial cancer diagnosis and tissue viability prediction are demonstrated. Firstly, the background in epithelial cancer and flap tissue viability as well as the principles/state of art of optical spectroscopy and imaging in tissue optics was presented in Chapter 1. Then a general survey on the capability of MC modeling of light transport in tissues was provided in Chapter 2 due to the importance of MC modeling in the field of tissue optics. The recent progress in the development of methods for speeding up MC simulations and the potential directions of future development were discussed. Based on the literature review on MC methods given in the previous Chapter, we developed a MC method to simulate diffuse reflectance and fluorescence from a layered tissue with embedded objects to mimic an early epithelial caner model in Chapter 3. With the help of this MC method, a series of numerical studies were performed to provide the guidelines for the selection of a proper epithelial cancer model in MC simulations. However, it is very time consuming to use the standard MC method for the simulation of light transport in a layered tissue with embedded object. To overcome this problem, a hybrid method, in which the scaling method and the perturbation MC method were integrated coherently, was proposed and validated to accelerate the simulations of diffuse reflectance from a layered tissue with embedded objects in Chapter 4. This method is suitable for simulating diffuse reflectance spectra or creating a MC database to extract optical properties of an early epithelial cancer model. All the above studies were designed for contact optical measurements using fiber-optics. However, inconsistent probe-sample contact could induce significant errors in diagnosis of early epithelial cancer. To address this problem, lens based setup was investigated for non-contact optical measurements in Chapter 5 and Chapter 6. We firstly developed a MC method to simulate diffuse reflectance and fluorescence measurements by convex lenses based non-contact setup. Then a series of numerical studies were performed to achieve depth sensitive diffuse reflectance and fluorescence measurements on the early epithelial cancer model. After that experimental studies were performed by a lens based optical system to validate the MC results and confirm the findings obtained in simulation study. We further extended the lens based non-contact spectroscopy system to an imaging setup with a larger field of view to perform depth sensitive color imaging on an early epithelial cancer phantom in Chapter 7. In the proposed setup, a micro-lens array was used to induce multi-focal illumination and a tunable lens was utilized to map multiple light foci into the tissue phantom at a range of depths. Another imaging lens was used to image light into a 3-CCD camera. The study performed on the epithelial cancer phantoms demonstrated that our method could be potentially used as a clinical tool for the diagnosis of early epithelial cancer. In addition to the numerical and phantom studies in epithelial cancer diagnosis, an animal study was also performed in Chapter 8 to predict tissue viability in flap surgery using a dual-modal system capable of performing both visible diffuse reflectance and auto-fluorescence spectroscopy. The results showed that either visible diffuse reflectance spectroscopy or auto-fluorescence spectroscopy alone can predict the skin viability accurately; however, auto-fluorescence spectroscopy was more sensitive to tissue changes in the first two hours after the induction of ischemia. It was feasible to predict flap failures in the first two hours when using auto-fluorescence spectroscopy alone. Moreover, it is possible to predict flap failures even in the first 15 minutes with high accuracy when using diffuse reflectance and auto-fluorescence spectroscopy simultaneously. In Chapter 9, we summarized that UV-visible diffuse reflectance and auto-fluorescence spectroscopy or imaging are promising tools for early epithelial cancer diagnosis and tissue viability prediction. Further refinement of these techniques would help advance the use of optical spectroscopy and imaging in clinical settings for tissue characterization in an even larger range of clinical applications. DOCTOR OF PHILOSOPHY (SCBE)

Key tissue parameters, e.g., total hemoglobin concentration and tissue oxygenation, are important biomarkers in clinical diagnosis for various diseases. Although point measurement techniques based on diffuse reflectance spectroscopy can accurately recover these tissue parameters, they are not suitable for the examination of a large tissue region due to slow data acquisition. The previous imaging studies have shown that hemoglobin concentration and oxygenation can be estimated from color measurements with the assumption of known scattering properties, which is impractical in clinical applications. To overcome this limitation and speed-up image processing, we propose a method of sequential weighted Wiener estimation (WE) to quickly extract key tissue parameters, including total hemoglobin concentration (CtHb), hemoglobin oxygenation (StO2), scatterer density (α), and scattering power (β), from wide-band color measurements. This method takes advantage of the fact that each parameter is sensitive to the color measurements in a different way and attempts to maximize the contribution of those color measurements likely to generate correct results in WE. The method was evaluated on skin phantoms with varying CtHb, StO2, and scattering properties. The results demonstrate excellent agreement between the estimated tissue parameters and the corresponding reference values. Compared with traditional WE, the sequential weighted WE shows significant improvement in the estimation accuracy. This method could be used to monitor tissue parameters in an imaging setup in real time. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Lens based setups have been explored for non-contact diffuse reflectance measurements to reduce the uncertainty due to inconsistent probe-sample pressure in the past years. However, there have been no reports describing the details of Monte Carlo modeling of lens based non-contact setup for depth sensitive diffuse reflectance measurements to the best of our knowledge. In this study, we first presented a flexible Monte Carlo method to model non-contact diffuse reflectance measurements in a lens based setup. Then this method was used to simulate diffuse reflectance measurements from a squamous cell carcinoma (SCC) tissue model in the cone shell, cone and hybrid configurations, in which the cone shell configuration has not been previously proposed in optical spectroscopy. Depth sensitive measurements were achieved by adjusting the following two parameters: (1) the depth of focal point of the imaging lens in the SCC model; and (2) the cone radius in the cone configuration or the ring radius in the cone shell configuration. It was demonstrated that the cone shell and the hybrid configurations in general have better depth sensitivity to the tumor and the stroma than the more commonly used cone configuration for diffuse reflectance measurements in the SCC model. Published version

Background: Accurate and early prediction of skin flap viability is vitally important in reconstructive surgery. To the best of the authors’ knowledge, this is the first pilot study to evaluate the simultaneous use of both visible diffuse reflectance and autofluorescence spectroscopy on a reverse MacFarlane rat dorsal skin flap model in the early prediction of skin viability. Methods: A total of 62 flap measurement sites from 11 Sprague-Dawley rats were monitored for 72 hours. Both statistical analysis using measured spectra and quantification of physiologically relevant tissue parameters using empirical methods were performed. Results: The statistical analysis results suggest that either visible diffuse reflectance spectroscopy or autofluorescence spectroscopy alone can predict the skin viability accurately; however, autofluorescence spectroscopy is more sensitive to tissue changes in the first 2 hours after induction of ischemia. The pilot study shows that it is feasible to predict flap failures in the first 2 hours when using autofluorescence spectroscopy alone; moreover, it is possible to predict flap failures even in the first 15 minutes with high accuracy when using diffuse reflectance and autofluorescence spectroscopy simultaneously. Meanwhile, several physiologically relevant parameters including hemoglobin oxygenation, total hemoglobin concentration, and redox ratio indicators estimated from diffuse reflectance and autofluorescence spectra show distinctively different trends over time for nonviable and viable skin. Conclusions: These findings will be helpful to clinicians for making a precise judgment on flap viability. Furthermore, the authors’ results highlight the advantage of using autofluorescence spectroscopy in the early prediction of skin flap viability relative to diffuse reflectance spectroscopy. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Accepted version

The accurate assessment of skin flap viability is vitally important in reconstructive surgery. Early identification of vascular compromise increases the change of successful flap salvage. The ability to determine tissue viability intraoperatively is also extremely useful when the reconstructive surgeon must decide how to inset the flap and whether any tissue must be discarded. Visible diffuse reflectance and auto-fluorescence spectroscopy, which yield different sets of biochemical information, have not been used in the characterization of skin flap viability simultaneously to our best knowledge. We performed both diffuse reflectance and fluorescence measurements on a reverse MacFarlane rat dorsal skin flap model to identify the additional value of auto-fluorescence spectroscopy to the assessment of flap viability. Our result suggests that auto-fluorescence spectroscopy appears to be more sensitive to early biochemical changes in a failed flap than diffuse reflectance spectroscopy, which could be a valuable complement to diffuse reflectance spectroscopy for the assessment of flap viability.

In this study, we propose a low-cost cross-polarized dark field microscopy system for in vivo vascular imaging to detect head and neck cancer. A simple-to-use Gabor-filter-based image processing technique was developed to objectively and automatically quantify several important vascular features, including tortuosity, length, diameter and area fraction, from vascular images. Simulations were performed to evaluate the accuracies of vessel segmentation and feature extraction for our algorithm. Sensitivity and specificity for vessel segmentation of the Gabor masks both remained above 80% at all contrast levels when compared to gold-standard masks. Errors for vascular feature extraction were under 5%. Moreover, vascular contrast and vessel diameter were identified to be the two primary factors which affected the segmentation accuracies. After our algorithm was validated, we monitored the blood vessels in an inducible hamster cheek pouch carcinogen model over 17 weeks and quantified vascular features during carcinogenesis. A significant increase in vascular tortuosity and a significant decrease in vessel length were observed during carcinogenesis.

We present a hybrid method that combines a multilayered scaling method and a perturbation method to speed up the Monte Carlo simulation of diffuse reflectance from a multilayered tissue model with finite-size tumor-like heterogeneities. The proposed method consists of two steps. In the first step, a set of photon trajectory information generated from a baseline Monte Carlo simulation is utilized to scale the exit weight and exit distance of survival photons for the multilayered tissue model. In the second step, another set of photon trajectory information, including the locations of all collision events from the baseline simulation and the scaling result obtained from the first step, is employed by the perturbation Monte Carlo method to estimate diffuse reflectance from the multilayered tissue model with tumor-like heterogeneities. Our method is demonstrated to shorten simulation time by several orders of magnitude. Moreover, this hybrid method works for a larger range of probe configurations and tumor models than the scaling method or the perturbation method alone. Published version

We present a hybrid method to speed up the Monte Carlo simulation of diffuse reflectance from a multi-layered tissue model with finite-size tumor-like heterogeneities. The proposed method consists of two steps. In the first step, a set of photon trajectory information generated from a baseline Monte Carlo simulation is utilized to scale the exit weight and exit distance of survival photons for the multi-layered tissue model by using a multiple scaling method. In the second step, another set of photon trajectory information including the locations of all collision events from the baseline simulation and the scaling result obtained from the first step are employed by the perturbation Monte Carlo method to estimate diffuse reflectance from the multi-layered tissue model with tumor-like heterogeneities. Our method is demonstrated to be able to shorten simulation time by several orders of magnitude. Moreover, this hybrid method works for a larger range of probe configurations and tumor models compared to the scaling method or the perturbation method alone. Published Version

A Monte Carlo code based on a public domain code, MCML, was developed for the simulation of light transport in layered tissue models with buried tumor-like targets. We used the code to investigate the validity of the semi-infinite tumor assumption in an epithelial tissue model. The epithelial tissue model consists of three layers, which includes the top epithelium, the middle tumor and the bottom stroma. Diffuse reflectance is simulated for a fiber-optic probe in commonly used configurations. It is demonstrated that the validity of the semifinite tumor layer model depends on the fiber-optic probe configuration and the tumor dimensions. The results of this study provide a guide in the selection of appropriate light transport models and fiber-optic probe configurations in the optical diagnosis of early epithelial cancers.

We present our work toward implementing all-digital signal processing for Positron Emission Tomography (PET) event detection. In the conventional PET system, proper calibration and extending event processing are challenging tasks due to the huge number of channels and multiplexing of input signals in the mixed-signal front-end. To alleviate such limitations, we have proposed a simple all-digital PET system utilizing digital signal processing (DSP) technologies for analyzing event pulses generated in PET. In this work, we implement a Gaussian shaper circuit for scintillation pulses, which followed by a moderate sampling rate Analog-to-Digital Converter (ADC). We also evaluate two DSP algorithms for extracting time information from the digitized pulse samples, and the two algorithms examined could generate a coincidence timing resolution of ~ 2.4ns FWHM, by using a 125MSps sampling rate ADC.

We investigate the use of digital signal processing (DSP) techniques for event processing in positron emission tomography (PET). In this work, we focus on the determination of the event time. We are building a digital library of pulse waveforms generated by using different photo-detectors, scintillator materials, crystal sizes, and read-out electronics of interest in PET. We employ a dataset from our library and investigate two methods for obtaining digital samples of an event pulse and several DSP algorithms for estimating the event time. Our first results, by using non-optimized DSP methods, are encouraging.

Experimental investigation and optimization of various optical parameters in the design of depth sensitive optical measurements in layered tissues would require a huge amount of time and resources. A computational method to model light transport in layered tissues using Monte Carlo simulations has been developed for decades to reduce the cost incurred during this process. In this work, we employed the Monte Carlo method to investigate the depth sensitivity achieved by various illumination and detection configurations including both the traditional cone configurations and new cone shell configurations, which are implemented by convex or axicon lenses. Phantom experiments have been carried out to validate the Monte Carlo modeling of fluorescence in a two-layered turbid, epithelial tissue model. The measured fluorescence and depth sensitivity of different illumination–detection configurations were compared with each other. The results indicate excellent agreement between the experimental and simulation results in the trends of fluorescence intensity and depth sensitivity. The findings of this study and the development of the Monte Carlo method for noncontact setups provide useful insight and assistance in the planning and optimization of optical designs for depth sensitive fluorescence measurements. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

We propose a multifocal noncontact setup to perform depth-sensitive fluorescence imaging on a two-layered epithelial tissue model. The combination of a microlens array and a tunable lens enables the depth of the multifocal plane to be conveniently adjusted without any mechanical movement of the imaging lens or the sample. This advantage is particularly desirable in the clinical setting. Results from the phantom study demonstrate that the setup can achieve depth-sensitive color imaging for fluorescence measurements, which is further confirmed by spectral measurements. Published version

A general survey is provided on the capability of Monte Carlo (MC) modeling in tissue optics while paying special attention to the recent progress in the development of methods for speeding up MC simulations. The principles of MC modeling for the simulation of light transport in tissues, which includes the general procedure of tracking an individual photon packet, common light-tissue interactions that can be simulated, frequently used tissue models, common contact/noncontact illumination and detection setups, and the treatment of time-resolved and fre- quency-domain optical measurements, are briefly described to help interested readers achieve a quick start. Following that, a variety of methods for speeding up MC simulations, which includes scaling methods, perturbation methods, hybrid methods, variance reduction techniques, parallel computation, and special methods for fluorescence simulations, as well as their respective advantages and disadvantages are discussed. Then the applications of MC methods in tissue optics, laser Doppler flowmetry, photodynamic therapy, optical coherence tomography, and diffuse opticaltomography are briefly surveyed. Finally, the potential directionsfor the future development of the MC method in tissue optics are discussed. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. (DOI: 10.1117/1.JBO.18.5.050902)

The accurate understanding of optical properties of human tissues plays an important role in the optical diagnosis of early epithelial cancer. Many inverse models used to determine the optical properties of a tumor have assumed that the tumor was semi-infinite, which infers infinite width and length but finite thickness. However, this simplified assumption could lead to large errors for small tumor, especially at the early stages. We used a modified Monte Carlo code, which is able to simulate light transport in a layered tissue model with buried tumor-like targets, to investigate the validity of the semi-infinite tumor assumption in two common epithelial tissue models: a squamous cell carcinoma (SCC) tissue model and a basal cell carcinoma (BCC) tissue model. The SCC tissue model consisted of three layers, i.e. the top epithelium, the middle tumor and the bottom stroma. The BCC tissue model also consisted of three layers, i.e. the top epidermis, the middle tumor and the bottom dermis. Diffuse reflectance was simulated for two common fiber-optic probes. In one probe, both source and detector fibers were perpendicular to the tissue surface; while in the other, both fibers were tilted at 45 degrees relative to the normal axis of the tissue surface. It was demonstrated that the validity of the semi-infinite tumor model depends on both the fiber-optic probe configuration and the tumor dimensions. Two look-up tables, which relate the validity of the semi-infinite tumor model to the tumor width in terms of the source-detector separation, were derived to guide the selection of appropriate tumor models and fiber optic probe configuration for the optical diagnosis of early epithelial cancers.