Stephen A. Boppart - US grants

0086696
Boppart
The applicants propose to develop an integrated microscope capable of optical coherence tomography (OCT) and multi-photon microscopy (MPM) for simultaneous acquisition of microstructural and functional image data in microfluidic systems. This microscope will be used to investigate the design and performance of microfluidic mixing systems and to non-invasively monitor molecular beacon hybridization in real-time for the detection of specific nucleic acids from microbial pathogens and microorganisms in the environment.

The microscope will enable the complementary use of OCT to provide microstructural and flow information and MPM to visualize the spatial distribution of fluorescent molecular beacons with respect to the microstructure. Optical coherence tomography is an emerging high-speed high-resolution imaging technology which has largely been applied to medical and biological applications. OCT performs optical ranging with micron-scale resolution in a manner similar to ultrasound, except reflections of near-infrared light are detected rather than sound. Optical Doppler OCT, a technique analogous to laser Doppler velocimetry, can acquire image-based data of fluid flow profiles and make quantitative measurements of flow velocities. The capabilities are ideally suited for diagnostic monitoring of three-dimensional microfluidic systems, which typically have microstructural dimensions of 10-100 mm. Molecular beacons, oligonucleotides that fluoresce only upon hybridization to a nucleic acid target site, are promising optical probes for the detection of microbial pathogens and other microbial populations. The optical sectioning capability of MPM will be used to simultaneously monitor the three-dimensional spatial distribution of fluorescing molecular beacons within microfluidic systems.

Description (provided by applicant): The goal of tissue engineering is to augment, replace, or restore complex human tissue function by combining synthetic and living components in appropriate configurations and environmental conditions. There are three key aspects to consider in any tissue-engineered construct - the cells, the matrix or biomaterial construct, and cell-material interactions. Although increasing numbers of research groups are developing techniques to control cell growth in artificial matrices, few have investigated cell-matrix interactions and the evolving mechanical properties of three-dimensional (3-D) mechanically-stimulated engineered tissues. The primary limitation has been inadequate imaging technology for high-resolution, real-time, non-invasive imaging deep within scattering tissue. The 3-0 arrangement of cell populations strongly influences the way in which cells dynamically respond within the engineered tissue. Optical imaging techniques that permit deep-tissue 3-D imaging offer the opportunity to non-invasively track the formation of engineered tissues. This project will integrate and apply two complementary state-of-the-art optical imaging techniques, optical coherence tomography and multi-photon microscopy, which are capable of performing these imaging tasks. Both optical techniques utilize the same laser source and will be integrated in a single microscope to investigate dynamic cell-matrix interactions and the evolving mechanical properties of two model engineered tissues. These optical imaging techniques will permit high-resolution, real-time, deep-tissue imaging in 3-D to non-invasively and non-destructively track changes in the tissue formation of 3-D blocks of cardiac myocytes and vascular structures composed of fibroblasts and endothelial cells. We will use these imaging capabilities and biomolecular focal adhesion assays to determine the influence that the 3-D microenvironment and dynamic mechanical forces have on the growth, organization, and mechanical properties of these model engineered tissues. The development and application of this unique investigative microscope will improve our understanding of cell function and tissue dynamics in 3-D mechanically-stimulated culture environments, enabling generation of engineered tissues with increasingly complex functionality.

0347747
Boppart
Optical coherence tomography (OCT) is an emerging high-resolution biomedical imaging modality with potential applications in a wide range of medical and biological specialties. Studies to date have used OCT primarily to non-invasively image morphology. Doppler and polarization-sensitive OCT have been used to assess functional properties of tissue, but most commonly in blood flow and tissue injury, respectively. The capabilities of OCT, however, extend far beyond microanatomical imaging. Just as functional magnetic resonance imaging has had a significant impact on our understanding of cognitive processes in the brain, functional OCT (fOCT) techniques have the potential for investigating neural activity and communication patterns at the cellular and molecular level.

For this CAREER Development Award, fOCT methods will be developed to visualize not only anatomical microstructure but also physiological function in neurons and in neural tissue. Novel optical methods will be developed to non-invasively characterize electrical and molecular function. To investigate and demonstrate the capabilities of these imaging methods, fOCT will be used in three representative cell culture and small animal models. Functional OCT detecting scattering and birefringence changes will be used to non-invasively detect electrical neural activity in cultured neuron populations without the addition of exogenous fluorophores and without the need for multiple electrode arrays and electronics. In the abdominal ganglion of Pleurobranchaea (sea slug), neural pathways will be mapped out using real-time fOCT imaging and compared to known networks. Differences in functional molecules (oxygen, acetylcholine, Fragile X mental retardation protein [FMRP]) in the brains of normal and Fragile X mouse models will be characterized using spectroscopic and nonlinear OCT methods. This project will develop novel fOCT methods that comprise an integrated imaging system. This system will establish fOCT and these methods as powerful neural imaging tools for a wide-range of investigations.

The research will be integrated with educational plans to engage K-12, undergraduate and graduate students and post-doctoral fellows of highly diverse backgrounds. Two exhibits will be designed and constructed for the local Science Museum targeted toward the K-12 audience, one on "Biophotonics" and the other on "Nature's Optics." Research results will be integrated into existing undergraduate courses in Optical Imaging and Biomedical Instrumentation and efforts will intensify to develop an optics track and initiate a new course in Biophotonics. An international graduate student exchange with colleagues' laboratory will be arranged and a graduate-level seminar series on Biophotonics will be initiated.

0519920
Boppart
The goal is to develop optical coherence elastography and use it to understand the role of micromechanical forces on development of biological tissues. The mechanical properties will be used in mechanical hydrogel models to determine rules governing organization and patterning of biological tissue. Measurements will validate the models. The project will create a new tool for biomechanical measurement and imaging, improved or refined biomaterial design, insights into functional properties of tissues, potential impact on future engineered and functionally-inspired tissue Using an experimental approach similar to what has been used for ultrasound sonoelasticity imaging and ultrasound elastography, promising preliminary results are provided, demonstrating the ability of OCT imaging to visualize changes that can be induced in engineered tissue following controlled loading of the sample. Low frequency vibrational energy will be used to slowly load the tissue.

[unreadable] DESCRIPTION (provided by applicant): This project will develop a novel class of optical molecular imaging probes that exhibit a magnetomotive response to alter optical contrast when excited by a controllable external magnetic field. Investigational methods for in vivo molecular imaging of targeted magnetomotive probes are demonstrated using optical coherence tomography (OCT). This method will provide a unique resolution scale for the in vivo study and characterization of disease, for high detection sensitivity and dynamic range of induced optical contrast, for the evaluation of the efficacy of targeted drugs, and for basic biomedical research on the pharmacokinetic properties of these magnetomotive molecular probes. This proposal addresses the current need for highly sensitive and specific in vivo molecular imaging that has potential clinical utility. Magnetomotive-OCT (MMOCT), with micron-scale resolution combined with millimeters of penetration, will serve in investigating the dimensional- and surfactant-dependence of probe extravasation and diffusion. Externally-applied magnetomotive forces can be optimized in situ for highly sensitive detection, and can also be used to elucidate the binding strengths and ratios of the probes to their targeted sites. The potential of these probes for contrast in MRI and ultrasonic imaging, and their use in hyperthermic therapy and ultrasound-mediated drug delivery suggests a wide array of future translational investigations with unique capabilities for improving currently available cancer therapies. Specifically, we aim to: 1) Fundamentally understand the magnetomotive contrast of these molecular probes and produce probes optimized for the tissue type and desired information, 2) Develop novel, biocompatible, molecularly-targeted magnetomotive probes with high circulation time, low non-specific uptake, and magnetically-inducible optical contrast, 3) Determine efficacy of probes in targeting tumor cells using in vitro cell assays, and 4) Investigate the delivery mechanisms, extravasation, and specific uptake of targeted agents in vivo in a carcinogen-induced rat mammary tumor model using MMOCT with particular emphasis on tumor uptake at various stages of growth. These aims are synergistic, with the goal of producing optimized magnetomotive molecular probes for a given application (early-stage diagnosis or therapy). Conversely, we expect that the capability afforded by MMOCT to screen for efficacy and specificity will drive further development of more effective probes. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Image-guided surgical interventions have the potential to improve surgical outcomes by providing improved visualization and differentiation of normal and pathological tissue intraoperatively and in real-time. Pathology, however, exists across a large range of size scales within the human body, ranging from large tumor masses to genetic mutations in single cells. Currently, the majority of the surgical treatments for cancer are performed at the macro tissue scale. In virtually all surgical oncology interventions, the goal is to remove the entire tumor mass and every tumor cell. Therefore, a critical need exists for an image-guided interventional technology that is capable of real-time intraoperative imaging for the complete resection of a tumor mass, for the detection and removal of individual tumor cells that have migrated across tumor margins, and for the detection of cells that have metastasized to regional lymph nodes. Optical coherence tomography (OCT) is an emerging high-resolution real-time biomedical imaging technology capable of intraoperative imaging for tumor detection and intervention. We propose to investigate the use of OCT for performing high-resolution image-guided surgical interventions for the identification and resection of solid tumors, tissue regions suspicious for occult tumor cells, and loco-regional lymph nodes that show evidence of metastatic involvement. We will develop a high-speed surgical microscope-based OCT system with fast digital-signal-processing hardware. This system will allow for the real-time acquisition, processing, and display of OCT image data. A real-time image-tissue registration system will illuminate regions in the open surgical field that correspond to regions highlighted by the operator on the real-time OCT images. Breast cancer remains a formidable diagnostic and surgical challenge. To date, no OCT imaging studies have investigated the use of this diagnostic technique for this disease. We will utilize a well-characterized rat mammary tumor model to demonstrate the capabilities of our techniques by performing OCT image-guided surgical interventions of mammary tumors in an open surgical field and with an integrated tissue-sampling OCT needle biopsy probe. Studies will also be performed to determine if the use of novel OCT contrast agents can improve the identification and complete resection of the tumors. The use of OCT for image- guided surgical interventions has the long-term goal of improving surgical outcomes by enabling complete resection of tumor by visualizing the surgical field with resolutions that approach histopathology. [unreadable] [unreadable] [unreadable]

0619257
Boppart

This Instrument Development proposal details the construction, integration, and application of a novel multimodality microscope technology that will be enabling for a wide range of biological and medical investigations. A state-of-the-art multimodality microscope is proposed that combines the emerging technology of optical coherence tomography (microscopy) (OCT/OCM) with advanced multiphoton microscopy (MPM). This instrument is greater than the sum of its parts by providing complementary data on both the structure (OCM) and function (MPM) of biological systems.

[unreadable] DESCRIPTION (provided by applicant): This project will develop a novel class of optical molecular imaging probes that exhibit a magnetomotive response to alter optical contrast when excited by a controllable external magnetic field. Investigational methods for in vivo molecular imaging of targeted magnetomotive probes are demonstrated using optical coherence tomography (OCT). This method will provide a unique resolution scale for the in vivo study and characterization of disease, for high detection sensitivity and dynamic range of induced optical contrast, for the evaluation of the efficacy of targeted drugs, and for basic biomedical research on the pharmacokinetic properties of these magnetomotive molecular probes. This proposal addresses the current need for highly sensitive and specific in vivo molecular imaging that has potential clinical utility. Magnetomotive-OCT (MMOCT), with micron-scale resolution combined with millimeters of penetration, will serve in investigating the dimensional- and surfactant-dependence of probe extravasation and diffusion. Externally-applied magnetomotive forces can be optimized in situ for highly sensitive detection, and can also be used to elucidate the binding strengths and ratios of the probes to their targeted sites. The potential of these probes for contrast in MRI and ultrasonic imaging, and their use in hyperthermic therapy and ultrasound-mediated drug delivery suggests a wide array of future translational investigations with unique capabilities for improving currently available cancer therapies. Specifically, we aim to: 1) Fundamentally understand the magnetomotive contrast of these molecular probes and produce probes optimized for the tissue type and desired information, 2) Develop novel, biocompatible, molecularly-targeted magnetomotive probes with high circulation time, low non-specific uptake, and magnetically-inducible optical contrast, 3) Determine efficacy of probes in targeting tumor cells using in vitro cell assays, and 4) Investigate the delivery mechanisms, extravasation, and specific uptake of targeted agents in vivo in a carcinogen-induced rat mammary tumor model using MMOCT with particular emphasis on tumor uptake at various stages of growth. These aims are synergistic, with the goal of producing optimized magnetomotive molecular probes for a given application (early-stage diagnosis or therapy). Conversely, we expect that the capability afforded by MMOCT to screen for efficacy and specificity will drive further development of more effective probes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The trend in biomedical imaging is to detect and visualize diseases such as cancer at their earliest time-points, when the disease is most likely to be cured. Molecular imaging techniques are being developed across virtually all imaging modalities for this purpose. Optical molecular imaging offers the potential for not only high imaging resolution, but also multiple approaches for obtaining molecular sensitivity. We have developed an optical molecular imaging technique called Nonlinear Interferometric Vibrational Imaging (NIVI). This technique images the three-dimensional spatial distribution of molecules based on their vibrational resonance frequencies. Molecular vibrational resonances are detected by the nonlinear optical signals from Coherent Anti-Stokes Raman Scattering (CARS). Taking advantage of the coherent nature of CARS, we perform depth-resolved coherence-gating using heterodyne interferometric detection, leveraging many of the principles found in optical coherence tomography (OCT). For the R21 phase of this project, we will extend the imaging capabilities of NIVI to biological molecules associated with cancer. We will improve the sensitivity of detection by rejecting nonresonant signals that are commonly generated from water in biological environments and significantly contribute to background noise, and will implement spectral-domain detection to detect multiple Raman frequencies simultaneously. Before advancing to the R33 phase, we will demonstrate the biological application of NIVI by establishing sensitivity detection limits for biological macromolecules, and by differentiating neoplastic from normal tissue from a rat mammary tumor model based on molecular composition and classification. By demonstrating the potential use of NIVI for biomolecular diagnostics, we will advance this technology toward in vivo imaging in the R33 phase. To accomplish this, we will construct an ultra-broadband NIVI instrument that utilizes femtosecond pulse-shaping techniques to rapidly stimulate multiple vibrational resonances in specific molecules. We will establish the lower sensitivity limits of this technique as well as quantify the resolution at which similar molecular vibrations can be differentiated. Finally to demonstrate the real-time molecular imaging capabilities of our system, we will perform cell, tissue, and in vivo animal studies using a well-characterized carcinogen-induced rat mammary tumor model. Throughout carcinogenesis, tumors will be imaged in vivo to characterize their changing molecular composition and quantify the concentration and spatial distribution of DMA. This research will establish NIVI as a unique in vivo nonlinear optical molecular imaging technique for distinguishing and spatially mapping the distribution of molecules associated with cancer.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)

0852658
Boppart

Skin is our largest organ, serving critical roles in fluid homeostasis, thermoregulation, immune surveillance, and self-healing. Disease and/or the loss of major portions of a human?s skin can be disabling and potentially life threatening, and is a major health problem in the U.S. and throughout the world. Stem cells play a critical role in repairing and regenerating many tissues, including the skin. Elucidating the roles that stem cells play in the skin will therefore have a significant impact on not only our fundamental understanding of stem cell dynamics, but also on our treatment of skin diseases, for replacing skin in medical applications, or in rejuvenating skin in our aging population.

Recent advances in optical imaging techniques offer an unprecedented combination of high spatiotemporal imaging resolution that can now be applied to visualizing the complex three-dimensional (3-D) dynamics of skin stem cells within normal skin, and in response to skin injury and skin replacement, such as after grafting engineered skin replacements. The intellectual merit of this proposal is represented for the first time by an advanced optical biomedical imaging approach for elucidating the complex dynamics of skin stem cells in vivo and within engineered skin grafts. The hypothesis of this research is that optical coherence and multi-photon microscopy, in an integrated platform, can uniquely track and quantify the different dynamic 3-D in vivo stem cell behaviors in and around autologous and allogeneic engineered skin grafts.

With the recent discovery of the skin stem cell niche located within the bulge region of hair follicles, many questions arise as to the dynamic behavior of these stem cells as they migrate from the bone marrow and into the skin niche, as well as in and out of the niche in response to skin injury and disease. This project therefore has intellectual merit in four areas. First, an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy will be utilized to uniquely visualize the structural and functional relationships of stem cells within in vivo skin. Second, this project investigates and longitudinally images in 3-D the migration patterns and dynamics of skin stem cells. This will provide fundamental insight into the role they play in maintaining the function and health of skin. Third, the effects of skin injury, induced by the placement of an autologous skin graft (skin punch biopsy), will be investigated, providing insight into the stem cell dynamics in the healing response. Fourth, this project will longitudinally image the stem cell and tissue responses in vivo following the grafting of allogeneic engineered skin constructs, contributing significantly to the understanding of how skin stem cells interact with engineered tissue grafts within biological hosts. The development and application of more quantitative imaging techniques to analyze the dynamics at the single-cell or cell-population levels will provide further insight into the ability to understand the role of skin stem cells, and ultimately provide a better approach for the treatment of human pathologies that require skin grafting. Taken together, this project is novel in each of these four areas, and the use of these advanced imaging techniques to carry out these investigations is transformative for the fields of stem cell biology and tissue engineering.

Considering the broader scope, the outcome of this project is likely to have a significant and broad impact on both the fundamental and clinical understanding on how stem cells behave dynamically in vivo. This project addresses major challenges in stem cell biology and tissue engineering: how to visualize and track cells and small populations of cells in vivo, in 3-D, and longitudinally over time in highly-scattering engineered and natural tissues.

This project will integrate state-of-the-art research with educational elements to advance discovery and promote teaching, training, and learning. Undergraduate students, in addition to graduate students, will complete theses related to this work. These students, as well as post-doctoral fellows and research scientists, will develop lifelong career skills in optics, image processing, cell and tissue culture and biology, and the use of pre-clinical models. Under-represented groups including women and minorities will be targeted for research opportunities, and annual laboratory and campus-wide open-house events will be held for outreach to K-12 and community groups. The results and image databases from this project will be disseminated widely through our educational website, local and national conferences, and leading scientific, engineering, and medical publications.

DESCRIPTION (provided by applicant): Molecular imaging is frequently performed with contrast agents that have been specifically designed to target to cells or molecules associated with the disease. These agents also produce a distinct signal that can be detected by an imaging method. In this project, we develop a novel type of molecular contrast agent based on engineered protein microspheres functionalized to target the vascular endothelium in atherosclerotic cardiovascular disease. These agents are designed specifically for enhancing contrast in optical coherence tomography (OCT), an emerging high-resolution biomedical imaging modality that can be used for intravascular detection and assessment of atherosclerotic lesions. Objective/Hypothesis: Multifunctional microsphere contrast agents will be developed to provide molecularly-specific contrast enhancement in OCT. Our hypothesis is that these engineered microspheres, targeted to the 1v23 integrin, can provide molecular contrast in OCT for in vivo catheter-based localization of atherosclerotic lesions. Specific Aims: Aim 1: Optimize the optical scattering properties of multifunctional microspheres. Aim 2: Determine targeting efficiency of microspheres to the 1v23 integrin in in vitro cell culture. Aim 3: Characterize and quantify OCT imaging and molecular contrast enhancement in an ex vivo hyperlipidemic pre-clinical model of atherosclerosis using targeted microspheres. Aim 4: Characterize and quantify OCT imaging and molecular contrast enhancement in vivo in a hyperlipidemic pre-clinical model of atherosclerosis using targeted microspheres. Study Design: This project will involve interdisciplinary efforts including the chemistry of fabricating microsphere contrast agents, the engineering and optimization of their optical properties, the use of novel optical imaging technology and data acquisition systems, and immunological targeting concepts for applications in in vitro cell culture and ex vivo and in vivo pre-clinical models. Clinical Relevance: This research is highly significant in the areas of detection, imaging, and assessment of atherosclerotic cardiovascular disease. The novel use of high-resolution OCT for tracking the spatial localization of molecularly-targeted optical contrast agents in pre-clinical ex vivo experiments and in in vivo catheter-based imaging of atherosclerosis enables a new imaging methodology and paradigm in the detection, treatment, and monitoring of atherosclerotic cardiovascular disease. PUBLIC HEALTH RELEVANCE: This proposal describes the development and application of targeted multifunctional microspheres for enhancing contrast in optical coherence tomography (OCT), an emerging high-resolution biomedical imaging modality. Our hypothesis is that these engineered microspheres can provide molecularly-sensitive contrast for OCT. The use of these agents therefore offers the potential for improving the diagnostic utility of OCT for investigations in the detection, imaging, assessment, and monitoring of cardiovascular disease.

0922539
Boppart

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."

The acquisition and use of this molecular imaging instrument will enable new research directions and educational opportunities and programs that will significantly contribute to the intellectual base of knowledge in these disparate but unified areas, and do so on national and international levels. The five research areas include (1) monitoring dynamic chemical reactions within advanced self-healing materials, (2) tracking nanoparticle biodistribution and visualizing their incorporation into the physiological processes of living organisms over time, (3) following the homing pathways of stem cells and their functional significance, (4) investigating metabolomics to understand the biological and physiological basis of nutrition, and (5) monitoring the dynamic three-dimensional distributions of materials, chemicals, and microbes in environmental samples. The acquisition of this molecular imaging instrument will not only enable the rapid expansion of existing research programs, but also provide the opportunity for new directions of investigation.

1033906
Boppart

Despite important advances within the past decade, current tissue engineered skin equivalents represent little more than a fragile, short-term dressing for patients that need viable skin replacements. The major weakness in current skin equivalents is that the constituent cells are cultured and applied under conditions that are very different from that of natural skin. It is believed that this is principally due to our limited understanding of the roles that 3-D scaffold topography and mechanical stimuli have on the intercellular organization, connectivity, and communication of engineered tissues. In this project an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy, and optical coherence elastography is utilized to uniquely visualize the structural and functional relationships of cells within 3-D engineered skin constructs, and measure the evolving biomechanical properties. Second, this project investigates and longitudinally images in 3-D the growth of engineered skin constructs on varying microtopographic substrates. This will provide fundamental insight into the mechanical influences at the dermal-epidermal junction on the keratinocytes and fibroblasts. Third, the effects of mechanical stimuli on these constructs will be investigated, defining how varying stimuli affect the 3-D cell dynamics and tissue organization over time. such stimulation effects will provide a more physiologically-relevant culture condition. Finally, this project will longitudinally image the cell and tissue responses in vivo following the grafting of the skin constructs to host pre-clinical models, contributing significantly to our understanding of how engineered tissue grafts interface with biological hosts.

1040462
Popescu

This proposal is for instrument development of a spatial light interference microscopy facility that will measure samples in both transmission and reflection modes. This quantitative phase imaging instrument will benefit diverse research efforts in the materials and life sciences. In particular, it will enable: (1) non-destructive inspection of nanostructures, semiconductor devices, and new materials such as graphene and carbon/semiconductor nanotubes, (2) observation of the dynamics of live cells and transport in neurons, and (3) exploration of new cancer detection techniques. Current topographic imaging technology severely constrains the size and sheer number of samples that can be measured at high resolution. Thus, the information gathered and new understanding obtained is thereby limited. Numerous new lines of research and opportunities for discovery in fields ranging from medicine and life sciences to semiconductors and material sciences will be enabled once this new form of fast microscopy is made accessible. Further, development of this transformative scientific instrument will provide rich opportunities to broadly integrate research and education.

DESCRIPTION (provided by applicant): The surgical treatment of solid tumors frequently involves the resection and post-operative histopathological assessment of sentinel and loco-regional lymph nodes to determine the extent to which the cancer may have spread. This information is also used to stage the disease. The stage of the disease determines the prognosis and dictates treatment strategies. Assessing lymph node status (whether each is normal, reactive, or contains metastatic disease) and staging the disease intraoperatively has significant opportunity to improve the treatment of cancer, and the use of high- resolution real-time intraoperative imaging has the potential to guide interventions, rather than relying solely on post-operative histopathology. This project involves the clinical translation and investigation of intraoperative three-dimensional optical coherence tomography (3-D OCT) for assessing the micro- architecture of lymph nodes. In contrast to all other imaging techniques that either require resection, bisection, and disruption of lymph nodes, or offer insufficient resolution to visualize morphology in situ, 3- D OCT imaging can be performed through the intact capsule of surgically-exposed lymph nodes that can remain in situ. Through preliminary results, we have demonstrated that 3-D OCT can differentiate between normal, reactive, and metastatic lymph nodes based on image biomarkers. The specific aims of this project outline a systematic approach for characterizing image biomarkers in both ex vivo and in situ lymph nodes, and with an intraoperative 3-D OCT system that incorporates an advanced handheld MEMS-scanner-based imaging probe. A novel computational reconstruction algorithm called Interferometric Synthetic Aperture Microscopy (ISAM) is applied to acquired image data to determine if higher spatially-invariant resolution increases the sensitivity and specificity of lymph node status determination. The successful completion of these aims and project will result in a new statistically- validated imaging technology capable of performing image-guided surgical interventions. The intraoperative assessment of lymph node status and the staging of cancer has the potential to update and direct the surgical intervention in real-time, to reduce or eliminate the need for the surgical removal of lymph nodes, to reduce costs, and most importantly, to reduce or eliminate the risks of lymphedema, a highly morbid and lifelong complication from the surgical treatment of many types of cancer. PUBLIC HEALTH RELEVANCE: The intraoperative assessment of lymph nodes during cancer surgery has the potential to reduce patient complications, reduce costs, and provide point-of-care feedback to update the surgical treatment plan. Three-dimensional optical coherence tomography (3-D OCT) can image and assess lymph node status, providing real-time feedback and diagnostic information during surgical interventions.

DESCRIPTION (provided by applicant): Primary Care Medicine, including Family Practice and Pediatrics, has traditionally relied on physical exam skills and simplistic instruments for critical diagnostic decision making, monitoring, and referral to medical specialists. The otoscope and ophthalmoscope are two historical and ubiquitous instruments that largely only illuminate and magnify tissue surfaces in the ear and eye, respectively. This Partnership will develop a new Primary Care Imaging system integrating optical coherence tomography (OCT) imaging with these instruments in a handheld scanner and portable system to advance the technological diagnostic and monitoring capabilities in primary care, and more effectively manage and refer patients based on quantitative data. A partnership composed of collaborating academic, clinical, and industrial institutions and investigators will develop and clinically evaluate this new point-of-care diagnostic technology. OCT is the optical analogue to ultrasound imaging, but generates 3-D images based on the backscatter of near-infrared light rather than sound. This Primary Care Imaging system with a MEMS-based handheld scanner and interchangeable tips enables high-resolution real-time 3-D imaging of the multiple tissue sites commonly examined during primary care outpatient exams including the eyes, ears, oral and nasal mucosa, skin, and cervix. For this project, system demonstration will focus on two increasingly prevalent diseases encountered in the primary care office, namely otitis media (middle ear infections) and diabetic retinopathy. Recent evidence has strongly associated chronic, recurrent episodes of otitis media with the presence of middle ear bacterial biofilms, and currently no non-invasive means exists to detect and quantify, let alone longitudinally monitor, these structures, which act as reservoirs to antibiotic-resistant bacterial for seeding recurrent infections. OCT enables quantitative assessment of middle ear biofilms and effusions, with the potential to significantly improve the antibiotic regimens and clinical management of this common disease. The rapidly rising prevalence of obesity has already been followed by increases in diabetes among increasingly younger patients, along with the associated complications of this disease, such as diabetic retinopathy. The successful use of OCT in ophthalmology can subsequently be advanced to the front-line of primary care to monitor patients for early evidence of diabetic retinopathy, and to quantify longitudinal changes during treatment. With an increasing reliance on effective primary care patient management for the expected increase in numbers of patients, new advanced diagnostic and quantitative technologies and instruments are needed in the outpatient primary care clinic for the early detection of disease, for quantitative monitoring of disease progression or regression, and for more efficient and evidence-based referrals to medical specialists. This Primary Care Imaging system addresses this critical need, and for the first time, brings advanced diagnostic imaging technology to the primary care office.

DESCRIPTION (provided by applicant): Diagnostic Photonics, Inc. (DxP) is a medical device company developing an intraoperative imaging system for surgical guidance and real-time assessment of cancer tumor margins based on interferometric synthetic aperture microscopy (ISAM), a novel modality that uses light defraction to assess tissue. Because of the fatty nature of the breast, current intraoperative histopathologic assessments are time consuming and unreliable. This project tests ISAM while the long term goal is to reduce the number of re-excisions for the management of breast cancer by providing rapid, reliable intraoperative margin assessment without tissue destruction. This will result in significant cost savings by reducing repeat operations. It may eventually result in a reduced volume of resection due to the ability to more accurately identify the extent of resection needed. In Phase I, three specific aims will be accomplished. Phase I aims include the development of a handheld probe and instrumentation support, user interface, and sterile disposable needed for execution of in vivo clinical studies as well as a clinical evaluation of ISAM margin detection versus pathology. The clinical goal in Phase I will be an initial evaluation of ISAM by comparing ISAM images against corresponding histology slides. This trial is necessary before proceeding to larger trials and will also serve asa control group for Phase II. The ex vivo assessment of tumor margins using the ISAM device will provide sensitivity and specificity data for the device vs. the gold standard of post-operative histology. The trial results and other data collected such as patient or pathology factors that increase or decrease the rate of margins will allow us to more accurately determine sample size requirements for Phase II. In Phase II, the ISAM technology will be applied in vivo. Correct labeling and assessment of tumor margins has long been considered a weakness of the current margin assessment methods and has been well documented to be unreliable. It is crucial to develop a technique that can assess margins both ex vivo and in vivo in the specimen cavity. Successfully identifying positive margins at the time of the operation allows the surgeon to act on those positive margins immediately, reducing the number of re-excisions. Aims of Phase II include assessment of in vivo margin analysis, evaluation of the utility of the ISAM system and time required during surgery, and the ultimate reduction in repeat surgeries from positive margins. PUBLIC HEALTH RELEVANCE: Breast cancer is one of the leading causes of cancer death among women of all races in the United States. Over 182,000 cases of breast cancer are diagnosed annually An estimated 128,000 of these patients with early stage breast cancer undergo breast conservation surgery (BCS), i.e., lumpectomy/partial mastectomy. The literature reports post-surgical positive margins in 20-70% of these patients which then requires a repeat surgery. New methods of reliable cost-effective intra-operative margin assessment are needed to move diagnostic capabilities to the operating room for point-of-care decision-making, and thus reducing the number of repeat surgeries that add cost and risk to the patient. DxP aims to fulfill this unmet need starting with the goals set for this grant application.

DESCRIPTION (provided by applicant): Margin status during the surgical treatment of solid tumors is the most critical factor in determining local recurrence rates. For breast cancer, breast conserving surgeries or lumpectomies are routinely performed. Currently, the surgeon is unable to visualize the microscopic structure at the margin, and conventionally relies on post-operative histological assessment of surgical margins to verify complete resection of the tumor. However, a critical intraoperative decision must be made to determine how much tissue around the primary tumor must be removed, or, where one must define the surgical margin. Therefore, a critical need exists to assess the surgical margin microscopically in real-time in the operating room, with spatial resolutions and pathological accuracies commensurate with post-operative histology, so that intraoperative feedback can be obtained during the surgical intervention. A partnership composed of collaborating academic, clinical, and industrial institutions and investigators will address this critical need by nonlinear interferometric vibrational imaging (NIVI). NIVI can images a wide variety of intrinsically vibrating biomolecules at a depth up to 1 mm with no labeling. The sensitive interferometric detection of quantitative Raman spectra allows NIVI to perform as a high-speed imaging analogue to Raman micro-spectroscopy. Through preliminary results obtained in our optical laboratory, we have demonstrated NIVI to have combined advantages of coherent anti-Stokes Raman scattering (CARS) spectroscopy and optical coherence tomography (OCT), and have successfully employed NIVI to detect tumor margins of breast cancer through specific abnormalities of endogenous biomarker molecules. This partnership will thus pursue the clinical translation of NIVI into an intraoperative imaging tool. The goal of this partnership will be achieved through a systematic approach. First, the extensive use of fiber-optic components will improve the robustness and the portability of the system, so that NIVI will largely retain the simplicity of a standard fiber-based OCT system. Also, an advanced handheld MEMS-scanner- based imaging probe will be incorporated into the system to flexibly access the surgical margin intraoperatively on resected tissue. Finally, the sensitivity and specificity of detecting positive margins will be determined to screen and flag margin locations suspicious for residual breast tumor. The successful completion of this project will result in a statistically-validated high-resolution molecular imaging technology capable of performing image-guided surgical interventions during breast cancer surgery. The intraoperative assessment of surgical tumor margin status has the potential to update and direct the surgical intervention in real-time, to reduce or eliminate reoperations, to minimize costs, and most importantly, to reduce the risk of local recurrence. Although the intraoperative imaging by NIVI in breast cancer surgery is the main focus of this project, we envision that this technique will benefit many other areas of surgical oncology.

PI: Boppart, Stephen A.
Proposal: 1450829
Title: BRAIN EAGER: Spatially-Resolved In Vivo Optogenetic Stimulation and Imaging Platform

Significance
The successful outcome of this research project will have a broad impact in neuroscience in addition to optical science and engineering. The PI will use implanted imaging fiber bundles that will enable
in vivo imaging as well as spatially-controlled optical stimulation and optical feedback of
large-area neural circuits. Current fibers only indiscriminately illuminate large-areas. Optogenetics is expected to make a broad impact in neuroscience, as well as medical science and clinical medicine in the future. This proposed research offers the potential to have an even greater impact by controlling the light stimulus and enhancing specificity in the control of neural circuits. The results of this project will be shared widely amongst the scientific and engineering communities, and also across wide segments of society in outreach activities. The new imaging and visualization capabilities will inspire K-12 students to think about how technology can be used to see things one cannot normally see, and how we can invent new ways of seeing the world around us and discovering new knowledge. Outreach activities will include demos of these imaging fiber bundles and novel light sources to K-12 and community groups through
annual Engineering Open House events, as well as integration of these technological methods in Prof. Boppart?s undergraduate ECE/BioE 467 Biophotonics and ECE/BioE 380 Biomedical Imaging
courses.

Technical Description
Optogenetics is a rapidly developing field with an ever-expanding toolkit of molecular biology
techniques to enable light-activated switching and control of cells, most commonly neurons.
Equally significant advances have occurred in optical science and engineering. By understanding
and exploiting physics-based principles of how light interacts in photonic crystal fibers (PCFs) and within imaging fiber bundles, it is possible to generate, control, and optimize a wide range of new optical parameters for in vivo optogenetic stimulation. Traditionally in in vivo optogenetic applications, light has been sent down single multi-mode optical fibers to diffusely illuminate the brain, relying on the molecular biology of optogenetically-modified neurons for cell and circuit specificity. This EAGER project will uniquely develop and demonstrate the use of imaging fiber bundles, and the generation of specific light pulse parameters to enable spatially-resolved optogenetic stimulation and imaging of neural circuits in vivo. These novel neurotechnologies will enable new investigations underlying behavior and cognition.

Proposal Number: 1403660
P.I.: Boppart, Stephen A.
Title: Enhanced Optogenetic Control of Neuronal Activity with Tailored Light Stimuli

Non-Technical Explanation

Significance:

This project will investigate how new forms of light may enhance the electrical output and control of neurons that have been genetically modified to be light-sensitive. Optogenetics is a rapidly developing field that uses molecular biology techniques to enable cellular functions to be controlled with light. When neurons (nerve cells) are genetically modified to express a light-activated membrane channel, they can be made to trigger electrical activity when exposed to light. This new level of light-activated control over neuronal activity has yet to be fully exploited, but is offering new opportunities for understanding how neurons and their electrical circuits function within the brain to form thoughts, memories, behaviors, and emotions. In optical science and engineering, advances now make it possible to generate a wide range of new forms of light with customized properties, or what is called tailored light. This research is highly significant and important for the fields of neuroscience and biophotonics. Biophotonics is the science of how light interacts with biological cells and tissues, and neuroscientists seek to understand how the brain and mind work. The new optical sources for generating tailored light in this project will change the way in which we use optogenetics to investigate and understand the function of neurons, neural circuits, and the brain. This project is also highly interdisciplinary, and will provide a unique educational and training opportunity for undergraduate and graduate students to help them solve the complex interdisciplinary problems in engineering and biology in the future. Results from this research will also be integrated into undergraduate and graduate courses in biophotonics, neuroscience, and advanced microscopy. The long-term societal benefits of this research will include raising the public's scientific literacy of how neurons and neural circuits in our brain function, and how new types of light and lasers can be used to probe the complex functions of our brain.

Technical Description

Optogenetics is a rapidly expanding field, and one that originated out of the field of neuroscience, where genetic modifications to mammalian neurons enabled photo-activated control of membrane channels to elicit action potentials. While this concept has provided a unique toolkit for exploring neuroscience questions and envisioning new medical science applications, there have been relatively few advances or contributions to optogenetics from the fields of optical science and engineering. This proposal addresses this gap by using advanced optical sources and precise control over the optical properties of the light stimuli to enhance the neural control in optogenetics.

The innovation of this research project is the ability to generate new forms of tailored light, and apply this light as new forms of stimuli to excite, modulate, and control the output of optogenetically-modified neurons in new ways. Conceivably, it is much more practical to modify and control the light stimulus than to genetically modify the biological properties of cells and tissues. As optogenetics advances to in vivo applications, this practical advantage will be even more significant. Therefore, our hypothesis is that by precisely controlling the spectral, temporal, and spatial parameters of novel tailored light stimuli, it is possible to provide enhanced modulation and control of the electrical output activity of optogenetically-modified neurons. To prove our hypothesis, our research plan will be guided by three objectives. First, we will construct an optical stimulus and microscope system to generate these new forms of tailored light. Second, we will optically stimulate and electrically/optically record from cultured hippocampal neurons that have been genetically modified to express Channelrhodopsin-2, a light-gated membrane ion channel, to investigate how tailored light stimuli alters the electrical output and activity from these cells. Third, we will implement an optical feedback system that will measure the optical response of the neurons and adjust the light stimuli parameters to optimize, modulate, and control the electrical output.

The successful outcome of this research project will have far-reaching impact in not only the field of biophotonics, but also in neuroscience and optical science and engineering. Just as optogenetics is expected to make a broad impact in neuroscience, as well as medical science, this research will potentially have an even greater and more rapid impact because it will conceivably be more practical to tailor the light stimulus than to modify the biology to enhance the optogenetic control in the future.

This award is being made jointly by two Programs- (1) Biophotonics, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate), and (2) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate).

PI: Boppart, Stephen
Instituation: University of Illinois at Urbana-Champaign
Proposal: 1445111
EAGER: Smart Phone Platform for Personal High-Resolution 3D Optical Imaging

The successful outcome of this research project will have a far-reaching and broad impact on many segments of our society. Most significantly, the technology and innovation in this project will enable novel mobile health applications and capabilities that will enable more sophisticated personalized health monitoring for the general public as well as for those in rural or developing regions of our world. This technology, for a number of very common diseases in the ear and skin, will serve as a means for early detection of disease. In addition, after treatment has been prescribed, it will serve for monitoring of the regression or progression of the disease.

This project will design and develop a smart phone platform that enables personal high-resolution 3D optical imaging. In addition to the traditional CCD cameras currently found in every smart phone and used for photography, this platform will implement a low-cost, compact, and portable optical imaging system based on optical coherence tomography (OCT). This self-contained handheld device with a unique set of optical elements will utilize the smart phone light source and camera, as well as its on-board processing and wireless communication hardware.

This group will develop a novel high-resolution 3D optical imaging platform designed for integration into the next-generation of personal smart phones. This platform which is based on low-coherence interferometry is regularly utilized in OCT, and therefore will have widespread applications not only along the medical and biological development paths established by OCT, but also a larger number of other applications because of the compact, portable, low-cost, and virtually ubiquitous nature of the technology.

Ear Nose Throat (ENTs) specialists and other healthcare professionals that diagnose and treat middle ear disease need to know if the patient has an infection, whether that infection is bacterial or viral, and determine some measure of the severity of the infection. They need to be able to make this assessment quickly because their interpretation of the middle ear health is the basis for significant interventional treatment, such as the prescription of antibiotics or decision to perform interventional surgery to minimize pain, illness, hearing loss, and delays in speech and language development. Despite the obvious importance and prevalence of this disease, the clinical gold standard for diagnosing ear infections is essentially a magnifying glass. The otoscope is a tool healthcare professionals use to evaluate middle ear health. The problem is that the otoscope only allows visualization of the surface of the eardrum, when the disease actually takes place in the middle ear. This team has developed a handheld medical device that can not only view the surface of the eardrum, but can also see through the eardrum and into the middle ear. The proposed improved diagnostics will lead to better clinical outcomes due to better management of antibiotics and interventional surgery.

The proposed technology uses harmless near-infrared light to produce three-dimensional images of the eardrum and middle ear contents. This provides physicians access to a wealth of new information, such as eardrum thickness, which has shown to vary with infection severity, effusion purulence, which is a strong indicator of the degree of infection, and most importantly, the presence or absence of a bacterial biofilm, which has been linked to chronic recurrent cases of ear infection that are generally resistant to antibiotic therapy. With 3-D information, quantitative measures and metrics can be visualized and recorded for objective diagnoses and longitudinal monitoring of therapeutic interventions. The device is used in the same way that a traditional otoscope is used, and since it can provide objective measurements, it allows a technician or nurse to obtain the same quality of diagnostic information as that of an experienced physician.

BROADER SIGNIFICANCE OF THE PROJECT:

The REU site "Discoveries in Bioimaging" at the University of Illinois at Urbana-Champaign (UIUC) will provide a state-of-the-art, interdisciplinary research environment in bioimaging to inspire and train undergraduates from under-represented demographics in engineering to encourage their transition to graduate degree programs. Throughout the educational process, visuals are a powerful means to clearly convey complex ideas and to stimulate curiosity and interest. Likewise, investigations in the biological and medical sciences invariably require clear pictures of cells, tissues, and organs to discover mechanisms of life and to detect and understand the complex pathological processes of disease. The unifying link between bioscience, discovery, and bioimaging will be the inspirational centerpiece for the new REU site. Students who take part in this REU program will gain an intimate familiarity with how biological discoveries directly arise from imaging, and gain an advanced skillset in state-of-the-art imaging technologies and interdisciplinary research that they can carry with them to graduate school. They will be exposed to the vast breadth of bioimaging techniques and applications while simultaneously focusing on a specific project at the forefront of biology and medical science. Furthermore by implementing literature-supported mentorship and training strategies, this program will directly address pipeline leaks for the transition of under-represented groups to engineering graduate degree programs. Those who choose to not pursue graduate studies will still be enriched with technical skills, professionalism, and career-readiness. The attributes of our program, both those that are successful, or perhaps ineffective, will be constantly assessed and refined, and findings will be disseminated to the public through publications in educational journals.

PROJECT DESCRIPTION:

With the support from the NSF Division of Engineering Education and Centers, the REU Site will be dedicated to inspiring and training undergraduates in STEM fields through a summer experience in bioimaging research. The goal is to create a coherent interdisciplinary program centered on the visualization of biology and medical science at all scales in which students develop a network of role models, mentors, and peers to support and encourage their transition to graduate school. Undergraduates finishing their freshman, sophomore, and junior years in a variety of STEM disciplines will be recruited with special efforts directed toward females and under-represented minorities, especially those at minority-serving institutions and small colleges with limited research opportunities. In addition, highly motivated community college students who are en route to 4-year colleges and universities, will be included. Students will begin this 10-week summer program with a 5-day "Bioimaging Bootcamp" to provide foundational knowledge in biology, imaging, and microscopy through lectures, demonstrations, lab activities, and tours, in conjunction with social introductions. Over the next nine weeks, students will engage in intensive research projects in imaging and biological visualization, closely mentored by a faculty member and graduate student, with assistance from an experienced UIUC undergraduate. All research projects, ranging from imaging single molecules using fluorescence microscopy to tracking cell dynamics in the skin of live animals, will have three essential elements: (1) imaging and/or microscopy, (2) an application directed toward biological or medical sciences, and (3) an analytical or computational component. Students will share experiences with their cohort through biweekly social activities, attend research seminars and professional development sessions, and interact with participants in other REUs on campus to expand their network. Summer achievements will culminate in the submission of an abstract to the Biomedical Engineering Society (BMES) Annual Fall Meeting, where they will reconvene in the fall for continued education and career-enriching opportunities.

In this proposed departmental revolution, the Bioengineering Department at the University of Illinois is aligning its undergraduate curriculum with medical practice and education by designing the curriculum around a simple message, "no solution without a need." The rising cost of healthcare, the increased role of technology in medicine, and emerging ethical dilemmas created by an increasing and aging population require that all bioengineers and healthcare providers understand the social and ethical context of their work to derive solutions that can meet these complex needs. However, students are typically isolated from these social contexts during their technical training, limiting their ability to identify and understand the needs of society and healthcare providers. Consequently, these students are limited in their ability to derive optimal engineering or technological solutions. The Bioengineering curriculum is being changed to achieve four objectives: 1) redesign the curriculum so that societal needs for healthcare and medicine drive the technical content, 2) integrate out-of-class experiences so that students receive hands-on practice with identifying and understanding societal and healthcare provider needs, 3) translate medical assessment practices to align clinical experiences with the curriculum, and 4) develop faculty teaching skills to meet these new challenges by engaging their intrinsic motivations to revolutionize the department. This revolution is driven locally by the creation and launch of the first Engineering-Based College of Medicine in the nation in fall 2018 that will integrate instruction in engineering with clinical and biological sciences.

Catalyzed by the Grinter Report, engineering education was previously revolutionized by aligning its practice and education with science. This alignment created a social-technical duality in engineering where the technical skills were elevated and social skills were relegated. In response, calls have risen for holistic training of engineering students who understand the societal needs and the societal implications of their practice. This change can be accomplished by aligning sub-disciplines of engineering with other holistic disciplines. The next revolution in bioengineering education can be brokered by realigning with healthcare and medicine - areas of impact and practice that holistically integrate social and technical aspects. This revised curriculum integrates clinical experiences that provide a context for students to learn ethnographic methods for user-oriented needs identification and problem scoping. While traditional curricula organize courses by their technical content, the new curriculum organizes courses by the needs that the curriculum will empower students to solve. Needs such as age-related disease, global health, and cancer provide the starting point for students who are navigating their curriculum. The use of new assessment tools such as competency-based models and e-portfolios integrate these new curricular tracks with the clinical experiences. Finally, to help faculty learn how to execute this new curriculum, the Bioengineering faculty are organized into Communities of Practice to execute course and assessment revisions. This process is driven by departmental administration who are guided and supported by organizational change researchers. This revolution is being spread beyond the Illinois campus through accreditation agencies and technical societies.

SUMMARY Biofilms in the human middle-ear are known to be directly associated with chronic otitis media (OM), and the bacteria that reside within these protective biofilms are likely responsible for contributing to the persistence and re-seeding of the infection despite repeated doses of antibiotics. While prior sampling of human middle-ear biofilms was performed invasively during a surgical procedure, developments and advances in the use of innovative optical imaging technologies and systems have made it possible to non-invasively detect, characterize, and monitor any biofilm that may be affixed to the tympanic membrane and/or any effusion that may be present within the middle ear. Optical coherence tomography (OCT) has been developed for imaging applications in otolaryngology and primary care, and over the last four years, feasibility studies have demonstrated its use for imaging various tissue sites examined during a primary care exam. In this renewal project, we propose to fundamentally investigate both the clinical and biological significance of the middle-ear biofilms and effusions that are detectable with OCT systems, as well as develop low-cost solutions that will enable this technology to be economical for primary care physicians and front-line healthcare providers. Guided by a central hypothesis and five specific aims, we intend to successfully demonstrate how non-invasive OCT of the middle ear can be used to improve the clinical management of OM by identifying the presence and dynamics of biofilms and effusions in this highly prevalent disease. To accomplish our goals, we have evolved and expanded our successful bioengineering research partnership to include new world-renowned expertise in the biological and clinical investigation of middle ear biofilms, as well as a start-up company that emerged during our initial project period to fully translate and commercialize this novel technology. We expect that this new technology and its capabilities will provide new metrics for improved detection, diagnosis, and monitoring of OM. The long-term outcome and impact will be a new standard-of-care for the management of ear infections, with improved patient care.

SUMMARY Optical imaging, which offers sufficient spatial resolution, specificity, sensitivity, and temporal resolution, has provided substantial insights into important biological processes at the cellular and molecular levels. Although high quality optical images can be obtained from in vitro cell cultures and/or thin tissue sections, intravital cell imaging in a complex, three-dimensional living tissue environment remains quite challenging. Because biological tissue naturally does not favor the propagation of light, and because of the inevitable presence of strong ambient light in the environment, special challenges arise in virtually every in vivo biomedical optical imaging, namely weak light signals and strong light backgrounds (including the multiply-scattered light in the tissue and the environment light). These challenges have significantly limited the full application of optical imaging in in vivo pre-clinical and clinical investigations. Currently, the detection of weak light signals for optical imaging relies heavily on the use of highly sensitive electronic detectors and electronic amplifiers. Although high-end electronic photo receivers are often very sensitive, the high sensitivity leads to the imaging systems being extremely prone to random photon noise, such environmental background (room) light, which is problematic for practical applications (e.g. in vivo studies and clinical practice). Furthermore, electronic detectors are incapable of distinguishing image-bearing ballistic photons from the multiply-scattered light background, which as a predominant source of noise in optical imaging of biological samples, can be overwhelming and significantly degrade resolution when imaging microstructure deep in tissue. In this proposed project, we will develop a multimodal microscope that utilizes a novel high speed optical parametric amplifier (OPA) to optically amplify weak back-scattered light signals, and demonstrate its capabilities by investigating in vivo cellular apoptosis events in murine skin. As shown by our preliminary data, the OPA will not only provide a high level of signal gain to improve detection sensitivity, but also provide an inherent nonlinear optical gate to both extract imaging-bearing signals and reject the noise sources from environmental photons and multiply-scattered background light. We will systematically explore the benefits afforded by this OPA for multimodal imaging that will include label-free reflectance confocal microscopy and optical coherence tomography. Improvement in resolution, contrast, imaging depth, and reduced photo-damage, will be investigated. The successful completion of this project will demonstrate a high-speed, robust, optical intravital microscope that combines multiple modalities with enhanced performance and new fascinating imaging functions uniquely enabled by the OPA. This intravital microscope will not only enable new biological and clinical studies, but also promote the development of new optical imaging technologies based on optical amplifiers.

SUMMARY Breast cancer is a global healthcare burden, not only for the patients diagnosed with this disease, but also their families and friends. The surgical treatment of breast cancer, while successful, has significant limitations that increase patient anxiety, increase costs, and can increase the risk for local recurrence and lifelong post-operative complications. A primary limitation stems from the lack of an intraoperative microscopic assessment of surgical tumor margins. Our cohesive and productive team with academic, clinical, and industrial representation has successfully developed and demonstrated for the first time the use of intraoperative optical coherence tomography (OCT) for in vivo human imaging of tumor margins during breast cancer surgery using a novel handheld surgical imaging probe. Additionally, the development and use of interferometric synthetic aperture microscopy (ISAM) for in vivo imaging has shown an important improvement in resolution and depth-of-field. Despite these advances, challenges remain for identifying tissue microstructure, particularly between normal fibrous stroma and dense tumor tissue, which are both highly scattering structures. To address these challenges, we propose the novel and innovative application of polarization-sensitive OCT (PS-OCT) and PS-ISAM for intraoperative in vivo imaging in human breast cancer surgery, and hypothesize that these will improve the detection sensitivity and specificity of positive breast tumor margins over standard OCT/ISAM. Realizing that the presence and progression of cancer significantly alters the collagen-based tissue microenvironment, the use of PS-OCT to sensitively detect and quantify birefringence of tissue collagen offers the potential for earlier detection of cancer and the altered microenvironment. By leveraging ISAM and other computational optical image segmentation algorithms, we can more fully characterize the tissue/tumor microenvironment. Through four specific aims, we will implement hardware and innovative software contributions to construct an intraoperative multi-mode system capable of real-time OCT/ISAM and PS-OCT/PS-ISAM, then use this system to investigate the performance of these imaging modes in clinical human studies to determine the sensitivity and specificity of ex vivo and in vivo PS-OCT/PS-ISAM over standard OCT/ISAM, and against the standard-of-care assessments which include post-operative histopathology and intraoperative visual/tactile cues. The successful completion of this project is expected to establish the clinical intraoperative use of these new optical imaging techniques, with the goal of reducing the current unacceptably high reoperation rates in the surgical treatment of breast cancer.

This project will develop and demonstrate a unique live-tissue imaging platform that can detect the presence of opioids in live brain slices from mice, and image their effects on the cellular metabolism and coupled neural brain activity. This platform will allow the first visualization of how the presence of opioids affects the metabolism of neurons and astrocytes, and the subsequent neural spontaneous depolarization activity. The establishment and demonstration of this imaging platform will enable future comparative studies using morphine (the prototypical opioid), caffeine, and dopamine to elucidate how opioids differ from non-addictive compounds (e.g. caffeine and anesthetics) and prevalent neurotransmitters (e.g. dopamine) to modulate the cellular metabolism of the brain.

This project will impact opioid addiction and related neuroscience and impact the broader research of drug development involving different diseases, organs, and preclinical models. The proposed imaging platform will be generally applicable to drug screening and discovery through preclinical imaging when the opioid is replaced by the drug of interest.
The results of this project will be shared amongst the scientific/engineering and pharmaceutical
communities, and across wide segments of society in outreach activities. The new imaging and
visualization capabilities will inspire K-12 students to think about how technology can be used to benefit scientific investigations. Outreach activities will include demonstrations of this imaging platform to community groups through annual Engineering Open House events, as well as integration of these technological methods in Prof. Boppart's undergraduate Biophotonics and Biomedical Imaging courses.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary: An imaging modality that allows for fast, simultaneous, noninvasive probing of both 3D cellular resolution retinal morphology by optical coherence tomography (OCT) and molecular-specific functions by autofluorescence (AF) could substantially improve both basic understanding and the early diagnosis of age- related macular degeneration (AMD), the leading cause of blindness in the developed world. The evaluation and management of AMD utilize several investigation modalities, but advancements in OCT technology have significantly contributed to better understanding of the disease, and have helped with monitoring progression and therapeutic efficacy. However, due to optical aberrations of the eye, the transverse resolution of conventional OCT is generally limited to 10-15 µm, inadequate for visualizing individual retinal cells in vivo. The integration of adaptive optics (AO) into OCT has demonstrated an immense success in mitigating these aberrations. Among various AO-OCT techniques, computation-based AO (CAO) becomes the spotlight of research because it shows unique advantages in data postprocessing flexibility and a reduced system cost. However, CAO is extremely sensitive to phase stability. The rapid motion of the eye can easily scramble the phase of reflected photons, restricting imaging to a single en-face layer. To overcome this problem, we will integrate a snapshot hyperspectral imaging method, Image Mapping Spectrometry (IMS), with full-field spectral-domain OCT. The integrated system will enable 3D imaging of retina within a single camera exposure. Next, to improve resolution in 3D, we will adapt an established CAO algorithm to correct for wavefront aberrations and improve transverse resolution to 2 µm. The resultant method, which we term snapshot ultra-high-resolution OCT, will allow an acquisition of a quarter million A-scans simultaneously. Given a typical flash illumination duration (4 µs), the equivalent A-scan rate is 62.5 GHz, which is approximately three orders of magnitude faster than the state-of-the-art methods. Furthermore, to expand the system?s functionality to molecular imaging, we will add a second IMS imaging channel for simultaneous hyperspectral imaging of retinal pigment epithelium (RPE) autofluorescence, enabling spectral biopsy of the RPE and subRPE lesions such as drusen, the hallmark lesion of early AMD. The resultant dual-channel OCT/AF system will be the first imaging modality that can provide both structural and molecular information about the retina in vivo and in 3D. We envision such a system would shift the landscape of AMD evaluation and management. The insights so obtained will be of high value in clinical diagnosis and treatment. In addition, such a system will accelerate translational research with sensitive and early outcome testing of prospective therapeutic agents, saving sight and thereby providing enormous benefit to society.

PROJECT SUMMARY Otitis media (middle ear infection) is a highly prevalent disease, especially in young children. It has been documented that more than 75% of children will have at least one episode of otitis media (OM) by age 3, and many children will have recurrent or chronic OM. In most cases, diagnosis of an ear infection is performed based on the appearance of the tympanic membrane and presence of fluid via an otoscope. In the absence of a method to identify bacterial infections, broad-spectrum antibiotics are prescribed without definitive knowledge of whether i) an active infection present, and ii) whether it is a bacterial infection (versus a viral infection). Previous clinical data also suggests that antibiotic therapy is only effective for one-third of OM patients, with two thirds of cases likely caused by antibiotic resistant bacteria, or more likely, viral pathogens, resulting in a misdirected expense of more than $700 million, annually. Therefore, there is a clear and urgent need for a point-of-care diagnostic tool to determine the presence of an active infection and whether an infection is bacterial in origin. Further, if we can distinguish the bacterial species present, unnecessary administration of broad-spectrum antibiotics can be eliminated, thus improving the efficacy of treatment and management of this highly prevalent disease. This proposal seeks to fulfill this unmet need by developing integrated Raman Spectroscopy-Optical Coherence Tomography (RSOCT) for the real-time detection and bacterial differentiation of pathological microorganisms in the middle ear. The scientific premise of the proposed research is that OCT can be used for image-guided placement of the RS probing beam, as well as visualization of any biofilm affixed to the tympanic membrane which would be indicative of a bacterial infection. This imaging system will be coupled with Raman spectroscopy for a more direct assessment of any biofilm and/or effusion by determining the presence of OM-causing bacteria and speciation via its biochemical fingerprint. The overall objective of the project will be accomplished by technological innovation and integration, followed by a series of systematic in vitro, ex vivo, and in vivo human studies with rigorous data analysis methods outlined in the specific aims. This research will have a profound impact on how we diagnose and care for these common ear infections. Most importantly, this project will provide critical data (identifying specific bacterial species) to monitor the development of antibiotic resistance, to reduce the overuse of antibiotics, and ultimately, to efficiently improve the health of patients.

Summary A label-free imaging technology is proposed for general cancer research, termed as Optical Fiber-Tethered Simultaneous Lifetime-resolved Autofluorescence and Multiharmonic (OFT-SLAM) microscopy, to overcome the lack of a versatile tool to simultaneously visualize tumor and non-tumor cells in authentic tumor microenvironment. The non-tumor cells broadly include the fibroblastic cells, angiogenic vascular cells, and infiltrating immune cells that engage normal biological functions such as embryonic/adult development and inflammatory/immune response (e.g. wound healing). However, in the tumor microenvironment where the overall metabolism is known to switch from energy consumption to proliferative biosynthesis (the Warburg effect), these normal (neutral) cells have all been recently recognized as the accessories to the crime (cancer). Thus, the proposed development of this imaging technology will interrogate the interrelations between metastatic tumor cells (principal) and various non-tumor cells (accessories) that conspire to kill a cancer patient (crime). This interrogation will be more comprehensive than imaging-based cancer research that has typically focused on one specific cell type of interest (the principal or one accessory of the crime). Without a label-free imaging technology like OFT-SLAM to avoid cell-specific labeling, simultaneous visualization of various cells would perturb the tumor microenvironment by exogenous staining, cell/tissue transplantation, and genetic modification. We will build the ?SLAM? of OFT-SLAM based on multimodal multiphoton microscopy and fluorescence-lifetime imaging, and invoke general intrinsic contrasts of cellular optical heterogeneity and metabolic activity to reveal and differentiate tumor and non-tumor cells. We will then empower the ?SLAM? with the ?OFT? to flexibly access different anatomical sites in intravital animal/preclinical microscopy and ex vivo human/clinical histopathology. We will subsequently employ the resulting OFT-SLAM to image the formalin-fixed human specimens of breast cancer from Cooperative Human Tissue Network (CHTN), including the primary breast tumors, breast cancer- induced lung and brain metastases, and surrounding peri-tumoral fields at different stages from different patients (n > 200). In parallel, we will apply OFT-SLAM to long-term (imaging window-assisted) intravital microscopy of three prototypical breast cancer rat/mouse models, covering all known steps throughout the invasion-metastasis cascade. With the unique capability of OFT-SLAM to bridge otherwise isolated ex vivo human histopathology (snapshots taken by pathologists in a clinical setting) and intravital animal microscopy (movies acquired by biologists in a laboratory), we will strive to identify various cancer-associated cells and their interrelations in an evolving tumor microenvironment and their dependence on spatial heterogeneity and individual variability. The successful outcome of this project will demonstrate a versatile visualization tool to interrogate tumor microenvironment with built-in translational ability, and thus transform cancer diagnosis and therapy.

The broader impact/commercial potential of this I-Corps project encompasses laser science, microscope technology, and the entire spectrum of biological imaging of live tissues, cells, and microorganisms. This includes basic life science and environmental research where advanced microscopes and cell measurement instruments are used routinely to collect data from cell cultures, animals, and human specimens. Most of these measurements currently require the use of expensive chemical or biological "labels" to identify the targets of interest. This labeling process is time consuming, expensive, and often results in irreversible damage to the specimen. This technology largely eliminates the need for these labels, substantially reducing both the cost and time required to perform experiments. This means that precious public and private research funds will accomplish more. In addition, the potential commercialization plan for this technology includes offering it to existing microscope and instrument manufacturers so that it can readily be integrated into existing systems.

This I-Corps project and its intellectual merit involves an advanced technology for rapidly capturing images of live tissue at microscopic resolution, and immediately extracting a wealth of information about the viability and function of the cells and tissue from those images, all without the need for chemical staining or tissue preparation. This technology is the result of over a decade of research conducted at a major university where the research team created a new programmable laser light source that tailors and launches short pulses of light to interact with specific molecular components within the live tissue, essentially making blood vessels, different cells, and cancerous tissue "light-up" with unique light signatures. By combining this laser source with an advanced microscope and software also developed by the research team, a next-generation imaging device has been developed that could dramatically improve the accuracy and speed, and lower the cost of histology, which is currently used to microscopically assess dead, fixed, and stained tissue sections.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY Racial and ethnic minorities and women have been historically under-represented in quantitative sciences. Even within biology, diversity in quantitative sub-branches is much lower than that in experimental counterparts, with the historical data clearly showing that the more mathematical and computational skills a discipline requires, the fewer the enrollment of these under-represented students. The proposed training program seeks to ameliorate these especially pronounced disparities with the biomedical sciences by establishing a streamlined bridge between Master?s programs at Fisk University and doctoral programs at the University of Illinois, Urbana- Champaign (UIUC). Our bridge program designed to nurture a diverse future generation of active minds specifically in the areas of biomedical data science and quantitative biology is named FUTURE-MINDS-QB (Fisk- UIUC Training of Under-represented Minds in Data Science and Quantitative Biology), where quantitative biology encompasses bioinformatics, computational biology, genomic biology, and biophysics. This training program will significantly contribute to diversifying the pool of Ph.D. researchers to include those currently under- represented in biomedical discovery and leadership To achieve our goal, we will accomplish the following short-term and medium-term objectives: (a) establish pathways for transitioning 20 Fisk M.S. students to UIUC Ph.D. programs over five years by providing ample opportunities to strengthen their background in relevant fields and acquire core computational and mathematical skill sets; (b) ensure the trainees? timely Ph.D. attainment within 5 years after Master?s degree; (c) accelerate the admission to and completion of Ph.D. programs by creating a new 4+1 M.S. track at Fisk, rigorously preparing undergraduates for a shortened 1-year M.S degree at Fisk and successful completion of a Ph.D. degree at UIUC; (d) create an inclusive and diverse inter-institutional environment by training both students and faculty in equity- focused teaching, mentoring, peer interactions, rigor, reproducibility, and the responsible conduct of research; (e) devise effective career development plans and opportunities; (f) implement a longitudinal survey of the development of individual trainees, and disseminate an open network of current trainees, graduates, and faculty; and, (g) make FUTURE-MINDS-QB a dynamic entity that continually improves by integrating feedback from trainees, faculty, oversight committees, and independent evaluators. As outcomes of our training, we expect that our seamless infrastructure and appealing inclusive environment will significantly increase the recruitment of under-represented students to quantitative biomedical sciences and that the reinforced academic and psychological preparation will increase the completion of doctoral degrees by under-represented students and ultimately improve their long-term retention in biomedical sciences. We thus expect FUTURE-MINDS-QB to establish an exemplary foundation for training under-represented graduate students and have a long-lasting scientific and socioeconomic impact stemming from their persistence and leadership in their careers.