Ji-Xin Cheng - US grants

The objective of this project is to develop coherent anti-Stokes Raman scattering (CARS) microscopy to study membrane rafts. The PI will develop three-color CARS microscopy in which two images, one tuned to the peak and the other tuned to the dip of a CARS band, are acquired simultaneously. The difference of the two images will permit high-sensitivity vibration imaging of single membranes. The resonant CARS signal from the aliphatic C-D vibration will be used to determine the partition ratio of a specific lipid between the liquid ordered (rafts) and disordered domains. The PI will investigate whether rafts are formed by an interaction between cholesterol and lipid hydrocarbon chains or by hydrogen bonding between cholesterol and sphingomyelin. He will use the polarization sensitivity of CARS to determine the orientation of the CH2 groups of lipid hydrocarbon chains in a bilayer containing cholesterol. This method will be able to measure the ordering degree of lipids in rafts. The PI will also develop coherent Raman microspectroscopy for spectral characterization of lipid-lipid characterization in single membrane domains. The PI will directly test the hypothesis of lipid rafts in the plasma membrane of live cells. Aggregation of fluorophore labeled rafts has been observed in different cellular processes. He will examine if these macroscopic domains are rich in sphingomyelin and cholesterol using CARS microscopy. The non-resonant background that arises from water and other cellular organelles will be removed by using the three-color excitation scheme.

This project provides an environment for interdisciplinary scientific training. Students involved in this research will need to combine the knowledge in chemistry, physics, and biology. The new imaging methods developed in this project will be incorporated in the PI's graduate level course of Biomedical Optics and undergraduate level course of Senior Engineering Design.

[unreadable] DESCRIPTION (provided by applicant): Chemically selective imaging provides a direct approach to unraveling cellular machinery and disease mechanisms. Our long term goal is to develop highly sensitive optical microscopy that allows selective imaging of single macromolecules or single molecular assemblies in live cells without fluorophore labeling, and to apply such tools for mapping intermediate metabolites in disease processes and drug pathways. The recently developed nonlinear optical microscopy based on coherent anti-Stokes Raman scattering (CARS) allows vibrational imaging with a high signal level. However, the nonresonant background limits the sensitivity and spectral selectivity. This proposal aims to push the detection sensitivity and molecular selectivity of CARS microscopy to a new level. We propose to develop three-color CARS microscopy in which three synchronized picosecond pulse trains are used to acquire on-resonance and off-resonance CARS image simultaneously. The vibrationally resonant CARS signal is extracted from the difference between the two images. We plan to apply three-color CARS microscopy to test the hypothesis of rafts in cellular membranes. We have been able to image specific lipids in model membranes using the CARS signal from the aliphatic CD stretch vibration of a lipid molecule with fully deuterated acyl chains. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding the molecular and cellular behavior in the live tissue (ex vivo) and live animal (in vivo) environment represents a frontier of current biomedical science. Our long term goal is building novel nonlinear optical (NLO) microscopy tools to explore the working mechanisms of biological systems at the live tissue and live animal level and to investigate the underlying molecular pathways in diseases. This proposal focuses on a significant and challenging topic - the central nervous system (CNS) which includes brain and spinal cord. The specific aims are: 1) Development of a compact multimodal multiphoton microscope and noninvasive imaging of live CNS tissues. Using a multiphoton imaging system that consists of three Ti:sapphire lasers and two laser scanning microscopes, we have demonstrated coherent anti-Stokes Raman scattering (CARS) imaging of myelin sheath, sum frequency generation imaging of astroglial processes, and two-photon excitation fluorescence imaging of calcium ion distribution in live spinal tissues. We will construct a compact multimodal NLO microscope using spectral detection and optical parametric generation. 2) Development of in vivo multimodal multiphoton microscopy. We have demonstrated 3D in vivo imaging of myelin sheath and collagen fibrils in a mouse sciatic nerve. We will build a video rate scanner for high- resolution CARS imaging of spinal cord and brain in live animals. 3) Multimodal multiphoton imaging to explore the molecular mechanisms of demyelination. We have revealed a putative role of cytosolic phospholipase A2 in lysolecithin induced myelin vesiculation via real time CARS imaging of myelin degradation. We will pursue a systematic study of the molecular pathway in inflammation induced myelin damage. The proposed instrumentation activities are expected to push the capability of NLO microscopy to an unprecedented level. The methods being developed for in vivo imaging of CNS can be generally applied to other tissues such as tumor. Moreover, our development of advanced imaging technologies is strongly coupled to a compelling biomedical problem - demyelination. The proposed biomedical applications are expected to provide new knowledge about molecular mechanisms of demyelination. The new knowledge will help us to identify the key steps to be inhibited for alleviating myelin degradation in spinal cord injury and demyelinating diseases.

CBET-0828832
Cheng

This proposal aims to develop a method that employs functionalized gold nanorods as an imaging and photothermal agent to target and eradicate macrophages in an atherosclerotic plaque by near infrared laser irradiation. preliminary studies have demonstrated two-photon luminescence imaging and optical hyperthermia of activated primary macrophages in vitro by gold nanorods conjugated with octaarginine (R8) peptides. By controlling the laser power and irradiation time, they have been able to induce apoptosis of macrophages confirmed by annexin V staining. The hypothesis is that laser irradiation of nanorods accumulated in a plaque could induce apoptosis of macrophages with minimal toxicity to the rest of the body. This hypothesis will be tested at the in vivo level using ApoE deficient mice with the following objectives. (1) Functionalize gold nanorods for prolonged blood circulation time and selective targeting to macrophages in an atherosclerotic plaque. (2) Determine the blood residence time, biodistribution, and organ toxicity of functionalized gold nanorods. (3) Optical hyperthermia of macrophages in atherosclerotic plaques of ApoE deficient mice.

DESCRIPTION (provided by applicant): A long term goal of our research is pursuing new understanding of molecular functions in health and disease via development of label-free microscopy. In this R21 application, we develop a new method termed vibrational photoacoustic (VPA) microscopy for 3-D vibrational imaging of tissues with a field of view and a penetration depth both in the mm scale. Our method is based on excitation of molecular overtone vibration and acoustic detection of the resultant pressure waves in the tissue. Our approach is significant in the following aspects: (i) Overtone excitation provides chemical bond selectivity and spectroscopic information in a label-free manner. (ii) Acoustic detection eliminates the tissue scattering problem encountered in near-infrared spectroscopy and enables depth-resolved signal collection in one scan. (iii) Our method provides a tissue penetration depth of a few mm, which is not accessible with existing vibrational microscopies. By excitation of the second overtone of the C-H bond stretch around 8300 cm-1, where blood interference is minimal, we have demonstrated preliminary VPA imaging of tissue phantoms and atherosclerotic plaques in arteries with a penetration depth in mm scale. These results show the great potential of developing VPA microscopy and endoscopy for label-free molecular imaging and spectroscopic analysis of lipid-related disorders in live animals and eventually in patients. The two specific aims are (1) developing high-speed VPA microscopy for in vivo molecular imaging and quantitative analysis and (2) developing VPA spectroscopy and endoscopy for in situ, depth resolved characterization of atherosclerotic plaques. Successful development of VPA microscopy should provide a new platform enabling label-free molecular imaging and spectroscopic analysis of biological specimen ex vivo and in vivo. Towards diagnosis of diseases such as atherosclerosis, VPA spectroscopy aided with principal component analysis should allow determination of lesion stages owing to the capability of identifying different pathophysiological compositions throughout deep tissues. Furthermore, our endoscopy development is expected to push the VPA method towards intravital imaging of plaques.

DESCRIPTION (provided by applicant): Flow cytometry is a widely used tool for high-throughput quantitative analysis of cell populations and intracellular content. Signals in flow cytometry arise from electrical impedance, forward or side light scattering, and fluorescence. Scattering and electrical impedance provide granularity and size/volume information, but with no chemical specificity. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, fluorescent tags are not applicable to all cases, especially small molecules (e.g. drugs) for which labeling may significantly perturb their properties. The current application aims to fill this gap through the development of a multichannel spectral flow cytometer using stimulate Raman scattering (SRS) signal from inherent molecular vibration. The stimulated Raman scattering overcomes the low signal level in spontaneous Raman scattering. It measures the light-matter energy transfer and is therefore free of the nonresonant background encountered in coherent anti-Stokes Raman scattering. Moreover, as a nonlinear optical process it is inherently phase matched, permitting a weakly focused collinear beam geometry that is compatible with high-speed detection of flowing objects. The planned instrumentation contains two specific aims. The first is to build a single-frequency SRS flow cytometer using a femtosecond laser source. The second is to build a multichannel SRS flow cytometer by multiplex detection of spectrally dispersed SRS signals. Based on the large signal level, we expect to reach the speed of 10,000 cells per second. Performance of the label-free spectral cytometer will be tested through quantitation of fat storage in adipocytes and of drug uptake by cancer cells.

DESCRIPTION (provided by applicant): While altered cell metabolism is an emerging hallmark of cancer, there is a crucial need of new tools for quantitation of metabolites. Though NMR spectroscopy, mass spectrometry, and Raman spectroscopy are widely used for molecular detection in tissue extracts or intact tissues, these tools do not tell the spatial locations of the analytes inside the cell. We address this unmet need via development of multiplex stimulated Raman scattering (SRS) microscopy to enable quantitative vibrational imaging of metabolites in live tumor cells and intact biopsies. The recently developed SRS microscopy allows high-speed, high-sensitivity imaging of single Raman bands in live cells. However, the single-frequency SRS imaging technique has limited capability because it cannot resolve molecular species that often have overlapped Raman bands. We propose to overcome this technical barrier via parallel detection of spectrally dispersed SRS signals enabled by a homebuilt tuned amplifier array. In a pilot study, we demonstrated multiplex SRS imaging of live pancreatic cancer cells with a pixel dwell time of 40 ¿s. In Aim 1, we will develop multiplex stimulated Raman loss (SRL) microscopy and multivariate analysis algorithm to enable quantitative vibrational imaging of lipid metabolites in live cells. In Aim 2, we will develop epi-detected multiplex SRL microscopy to enable high-speed, large-area spectroscopic imaging of tumor biopsies. By accomplishment of the two aims, we will generate a high- sensitivity, high-speed, spectral imaging platform for molecular analysis of live cells with sub-micron spatial resolution. This platform will permit label-free visualization of metabolic conversion in live cancr cells, which is not possible with proteomics tools. Such capability is critical for mechanistic understanding of cancer metabolism and precise evaluation of drugs targeting cancer metabolism. This platform will also permit large- area mapping of intact tumor biopsies and offer information about metabolic biomarkers (e.g. cholesteryl ester) that are indicative of cancer aggressiveness.

? DESCRIPTION: Quantitation of metabolic conversions in live cells and in real time is essential to determining how a cell responds to an intervention such as drug treatment or exposure to a risk factor. Nevertheless, most of our knowledge about cellular content is derived from in vitro analysis of isolated cells or measurement of tissue homogenates, either by biochemical assays, omics or sequencing technologies. This gap highlights a need of developing new techniques that are able to repetitively assess the same single cell in a vital organism. We propose to develop a new Raman imaging platform to enable repetitive assessment of single cell metabolism in a vital organism, using C.elegans as a test bed. Our innovation is spectral scanning of a femtosecond pulse at the Fourier plane of an angle-to wavelength pulse shaper, through which a SRS spectrum can be acquired on the scale of 20 ms per pixel. The long-term goal of our research is developing next generation technology to enable quantitative analysis of single live cell response to a stimuli or a treatment in 3D cultures or live animals. The specific objectives of this R21 application are constructing a ms time scale spectroscopic imaging system and longitudinally assessing the fat metabolism in vital C.elegans. An interdisciplinary team has been formed. Dr. Ji-Xin Cheng (PI) is an expert in label-free spectroscopic imaging. Dr. Heidi Tissenbaum (co-PI) is an expert in dissecting molecular mechanisms of the aging process using C.elegans as a model organism. The two investigators have an established collaboration in developing coherent Raman scattering microscopy to study lipid metabolism in live C.elegans. In feasibility studies, the team has demonstrated hyperspectral SRS imaging of lipid oxidation, lipid desaturation, and cholesterol storage in adult worms using fingerprint Raman bands. Moreover, the Cheng lab recently demonstrated spectral modulation SRS imaging with an angle-to- wavelength pulse shaper. Such development paves the foundation for acquisition of a SRS spectrum on the ms time scale. Our hypothesis that ms Raman spectroscopy is able to longitudinally assess lipid metabolism in vital C.elegans during aging, diet restriction or overfeeding. To test this hypothesis, we will design and construct, and test a microsecond Raman spectral imaging with an angle to wavelength pulse shaper. We will then use the system to longitudinally assess energy metabolism of single cells in wild-type and mutant C. elegans. Though C.elegans is used as a test bed, our platform heralds a broader impact on biomedical research via assessing single live cell response to an intervention, including monitoring live cell response to a risk factor or tissue regeneration in response to a stimulus.

? DESCRIPTION: In current medical treatment of atherosclerosis, a stent is placed for flow-limiting (i.e., >60% stenosis) plaques diagnosed by X-ray based angiography. Revascularization by stenting a flow-limiting coronary artery stenosis has profoundly improved survival. Plaque that is not flow-limiting and thus not stented, however, can be unstable and vulnerable to rupture, which is the major cause of acute myocardial infarction. Currently, no imaging tools exist to diagnose these non-flow-limiting yet potentially vulnerable plaques. The long-term goal of our project is to develop an intravascular vibrational photoacoustic (IVPA) catheter for clinica detection of vulnerable plaques. The objective of this R01 application is to demonstrate an IVPA system for real-time in vivo intravascular visualization of atherosclerotic plaque in a clinically relevant Ossabaw pig model. An interdisciplinary team has been assembled to approach this objective. Dr. Ji-Xin Cheng (Purdue University) is an expert in development and applications of vibrational spectral imaging. Dr. Michael Sturek (Indiana University School of Medicine) is an expert in cardiovascular research. Dr. Qifa Zhou (University of Southern California) is an expert in design and fabrication of ultrasound transducers. Consultant Art Coffey (M.D. in IU Health) is a cardiovascular surgeon who will provide clinical input on the IVPA catheter design. Our central hypothesis is that a high-speed IVPA system allows for in vivo assessment of lipid-rich, inflammatory plaques in an arterial wall. This hypothesis will be tested by 3 Specific Aims. (1) Develop a high-speed IVPA system and demonstrate real-time dual-modality PA/US sensing of an intact atherosclerotic artery in vitro. (2) Differentiate cholesterol crystal (CC) from cholesteol ester (CE)-rich lipid droplets inside an intact artery by spectral analysis of optically induced ultrasound signals. (3) Validate the IVPA system for in vivo assessment of atherosclerotic plaques in an Ossabaw swine model. Successful development of the IVPA system will be able to guide appropriate treatment, e.g. stent deployment, for the lipid-laden, unstable plaque that is not detectable by current intravascular catheters, thus potentially a life-saving technology.

Purdue University is planning to join the I/UCRC on Biophotonic Sensors and Systems (CBSS) as a new university site. CBSS has a goal to establish a national center of excellence in biophotonic sensor and system research, cultivate embryonic biosensor applications, develop methods for improved technology transfer and train a pipeline of skilled engineers and scientist to work in this field. Ultimately, CBSS intends to provide solutions for disease diagnosis, patient monitoring, drug efficacy testing and improved food and water safety. Purdue will expand the scope of corporate connections and brings exciting complementary areas of research to support the CBSS mission.

The Purdue team will offer research topics including: compressive optical imaging, flow cytometry, nonlinear optical microscopy, single cell detection, biodynamic imaging of 3D tissues, and point of care medical devices. These research topics fit perfectly with the current CBSS research thrusts and roadmap areas of Imaging, Bioanalytics and Diagnositcs.
The Purdue Site Director (Dr. Ji-Xin Cheng) has extensive connections in industry that include Olympus and Leica, which offer products in their portfolios based on his inventions. Companies that intend to participate in the planning meeting include companies such as Johnson & Johnson,Ocean Optics and Riverside Research, which are committing to full membership.

? DESCRIPTION (provided by applicant): Flow cytometry is one of the most important tools for high-throughput single cell analysis. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, fluorescent tags are not applicable to all cases especially small molecules (e.g. metabolites) for which labeling may significantly perturb their properties. Raman spectroscopic signals arising from inherent molecular vibrations provide a key approach to detect specific molecules inside cells and to differentiate cellular state. Raman-based microfluidic devices have been reported. However, the very small cross section of spontaneous Raman scattering results in low Raman signal level and consequently long data acquisition time, which is not compatible with the high- speed flow condition. The long-term goal of the proposed project is to establish a high-throughput high-content single cell analysis platform using molecular fingerprint vibrations as contrast. The specific objective of current application is to develop a vibrational spectroscopic cytometer based on the stimulated Raman scattering (SRS) process. Several recent advances in the Ji-Xin Cheng (PI) lab, including the highly sensitive femtosecond SRS imaging, lock-in free SRS signal detection and a tuned amplifier array for multiplex SRS imaging, pave the foundation for the planned instrumentation. The PI has assembled an interdisciplinary team for the proposed study. Dr. J. Paul Robinson (co-PI) is a leader in development and applications of fluorescence-based flow cytometer and he will bring expertise to the design of fluidics and multichannel detection systems. Dr. Bartek Rajwa (co-PI) will provide expertise for spectroscopic cytometry data analysis and machine learning. The team will design and construct a SRS flow cytometer by multichannel detection of dispersed SRS signal (Aim 1), construct a tandem system able to collect SRS and fluorescence data (Aim 2), develop spectral un-mixing and machine-learning analysis tools able to combine the information obtained from SRS spectra and labeled biomarkers for functional classification of cells (Aim 3), and validate the capability of SRS flow cytometer for label-free detection of single-cell metabolism (Aim 4). With a speed of analyzing thousands of cells per second, SRS flow cytometer will enable high-throughput analysis of single-cell chemical content which is beyond the reach by fluorescence-based flow cytometer.

Project summary: Current bioanalysis largely relies on tissue homogenization, separation, followed by various assays. These in vitro approaches tell the presence of molecules and their concentrations. Nevertheless, without spatial and temporal dynamics information, how molecules execute their functions in a living system remains unknown. Moreover, important target molecules with small quantities are often buried in the large background of dominant species in an in vitro assay. Chemical microscopy allows for label-free analysis of biomolecules in their natural environment. Based on molecular fingerprint vibration, recently developed coherent Raman scattering microscopy has enabled a broad spectrum of biomedical applications. Yet, broader use of coherent Raman scattering microscopy for single cell analysis is blocked by the extremely small cross section of Raman scattering (~10-30 cm2. In response to PAR-17-045, we propose to develop a new chemical imaging platform that exceeds the detection sensitivity limit of coherent Raman microscopy. We employ mid-infrared absorption (with a much larger cross-section ~10-22 cm2) as a contrast mechanism for imaging living cells. To bypass the water absorption obstacle, we indirectly detect the thermal effect induced by vibrational absorption. Our scientific premise is that after the mid-infrared photons induce the molecule to vibrate, the subsequent vibrational relaxation into heat causes a local change of the refractive index. Such change creates a phase delay and a thermal lens, both of which can be detected at sub-micron spatial resolution by a visible probe beam. In a proof of concept study in Science Advances (2016, 2: e1600521), we have reached an imaging speed of 8 seconds per frame, a lateral resolution of 0.6 micrometer, and a detection sensitivity level of 10 micro-molar for the endogenous C=O bond. In the proposed study, we will further improve the detection sensitivity by using better laser source and lab-built electronics (Aim 1). Moreover, we will improve the speed to 100 frames per second through wide-field illumination and quantitative phase detection (Aim 2). Finally, we will demonstrate volumetric chemical imaging through a light-sheet scheme (Aim 3). By accomplishing these Aims, we will have produced a highly sensitive, high-speed chemical imaging platform able to map drug distribution in a pharmaceutical formulation, metabolic activities inside a living cell, and membrane potential in a living neuron. These capacities will generate a profound impact on our understanding of life at the molecular level and on biomarker-based precise staging of diseases.

The focus of this multi-PI R01 application is to characterize and target a new metabolic vulnerability of ovarian cancer stem cells (CSCs) discovered by our collaborative team. By using hyperspectral-stimulated Raman scattering (SRS) imaging of single living cells and mass spectrometry analysis of extracted lipids we identified increased levels of unsaturated fatty acids (UFAs) in ovarian CSCs compared to non-CSCs. We demonstrated that UFAs are critical to the survival, proliferation, and tumorigenicity of ovarian CSCs. Here we propose to analyze the mechanisms by which increased lipid unsaturation mediated by ?9 desaturase (stearoyl-coA desaturase, SCD1) regulates retinoic acid signaling in ovarian CSCs to determine cellular fate and promote tumorigenicity. We will analyze whether the balance between saturated and unsaturated lipids enhance the survival of drug-tolerant cells after chemotherapy. We will use SCD1 knock down and chemical inhibitors to eradicate drug-tolerant cells persisting after treatment with platinum in ovarian xenografts and patient derived xenografts (PDX). Lipid unsaturation will be visualized in CSCs in situ by using a multimodal high-speed SRS microscope. Label-free molecular imaging will quantify CSCs and unsaturated lipids in human tumors and xenografts before and after treatment with platinum or desaturase inhibitors. Ultimately, in depth characterization of fatty acid metabolism in CSCs will reveal key pathways linked to stemness and persistence of chemotherapy-tolerant cells. In the long run, our studies will develop new strategies to attack deadly ovarian cancer.

Project Summary Ultrasound has been explored as a modality to modulate nerves and muscles back in the 1920s. A number of recent studies have demonstrated the feasibility of using ultrasound to stimulate peripheral nerves, spinal cord, and brain. Yet, it has been difficult to determine whether ultrasound stimulation is via direct modulation of the membrane voltage or via indirect synaptic or network pathways. In order to unveil the mechanisms of ultrasound modulation, we formed a team of complementary expertise (Xue Han: neuroscience and technology; Ji-Xin Cheng: imaging and opto-acoustic technology; Edward Boyden: neurotechnology). Specifically, we will deploy and integrate three novel technologies that have been established in the co-PI's labs recently. First, we will use a miniature fiber optoacoustic converter (FOC) (0.4 mm in dia.) that can be positioned inside the brain to deliver localized ultrasound with an unprecedented sub-millimeter spatial resolution. Second, we will use cutting-edge genetically encoded voltage sensors to quantify the effects of ultrasound stimulation on individual cells in the brain at a temporal resolution of 1 millisecond that is beyond commonly used Ca2+ imaging. Third, we will deploy submicron spatial resolution stimulated Raman scattering microscopy to map membrane voltage at threshold and sub-threshold level to monitor membrane response to ultrasound at different regions of a single neuron. Integrating these novel technologies with a large-scale imaging platform that allows simultaneous intracranial local drug delivery, recently developed in the Han lab, we will perform a systematic analysis of the cellular and the biophysical mechanisms of ultrasound stimulation at sub-cellular level in cultured primary neurons, and in different brain regions of awake mice. Specifically, we will (1) examine the spatial response profile of individual neurons in awake brains by FOC-based neurostimulation and large-scale Ca2+ imaging in vivo; (2) examine the temporal response profile of individual neurons in awake brains by FOC-based neurostimulation and in vivo voltage imaging with genetically encoded voltage sensors; and (3) examine the involvement of membrane deformation and mechanosensitive channel activation in ultrasound neuromodulation. Our proposed studies will deliver a systematic understanding of the spatiotemporal profiles of ultrasound neuromodulation in the brain, and identify the causal role of membrane deformation and mechanosensitive channels. These new knowledge will build a new foundation for rational design of ultrasound neuro-stimulators and for basic neuroscience research as well as treatment of neurological disorders.

Project summary: Although altered cell metabolism is becoming an established hallmark of cancer, there remains a crucial need of new tools for quantitation of metabolites at single living cell level. In particular, due to lack of specific labels for metabolites, there is an unmet need for high-resolution imaging tools capable of mapping metabolites and small molecules (fatty acids, carbohydrates, amino acids) that play essential roles in pathogenesis of cancer. Supported by a R21 grant through the IMAT program, our team partially addressed this need via developing a multiplex stimulated Raman scattering (SRS) microscope, which enabled vibrational imaging of metabolites in live tumor cells and intact biopsies at the speed of 5 microseconds per spectrum. This R33 application aims to push the hyperspectral stimulated Raman imaging technology to the next level through (i) technical simplification and validation, (ii) developing a robust hyperspectral image segmentation framework, and (iii) integrating the SRS modality with a commercial spontaneous Raman microscope towards broad use by non-experts. We have assembled an inter-disciplinary team for the proposed development. The three specific aims are: (1) Developing an easy-to-operate, highly sensitive line-by-line hyperspectral SRS microscope and validate its capacity for cancer metabolic imaging at single cell level. (2) Establishing a feature analysis framework for segmentation of hyperspectral SRS images using the non-parametric Bayesian model. (3) Integrating and validating the stimulated Raman imaging modality on a spontaneous Raman microscope. By completing the proposed development and validation activities, we will have generated a highly novel spectroscopic imaging system broadly applicable to analysis of chemical contents of cells and tissues for discovery-driven research and marker- based precision diagnosis.

Project Summary The increasing emergence of antimicrobial-resistant bacteria/fungi has become a growing global threat due to the misuse and overuse of antimicrobial drugs. In order to combat infections and reduce anti-microbial resistance, it is essential to detect and characterize bacterial/fungal susceptibility to antimicrobial in the early stages of infections to reduce the inappropriate use of antimicrobial drugs and the death rate. To address this urgent medical condition, it is critical to rapidly and accurately determine the antimicrobial susceptibility of bacteria/fungi so that optimal therapy drugs can be prescribed early in the disease process. Conventional methods for antimicrobial susceptibility testing, such as agar plates and broth dilution assays, detect phenotypic resistance based on bacterial/fungal growth in the presence of antimicrobial drugs being tested. A major limitation of these methods is that they are based on culture and require at least 16 to 24 h to conduct. To address this unmet need, a microsecond-scale stimulated Raman spectroscopic imaging platform is proposed to enable in situ detection of a single bacterium in complex environment at sub-micron resolution and early determination of its response to an antimicrobial drug. An interdisciplinary team will conduct the proposed study. Dr. Ji-Xin Cheng (PI) is an inventor and leading expert in coherent Raman scattering microscopy. Dr. Mohamed Seleem (co-PI) is a DVM-scientist with broad expertise in infectious diseases and microbiology. Dr. Ryan F. Relich (consultant), Medical Director of the Indiana University Health Clinical Virology and Serology Laboratories, has extensive experience in clinical diagnosis of infectious diseases. The team?s central hypothesis that microsecond-scale coherent Raman spectroscopic imaging will enable in situ analysis of single microbial cells enriched directly from a clinical sample (whole blood). To test this hypothesis, the team will demonstrate fast determination of antimicrobial response through microsecond-scale stimulated Raman imaging of metabolic activity in a single living bacterium (aim 1), develop a microsecond-scale broadband stimulated Raman spectroscopic microscope for label-free discrimination of bacteria and determination of anti-microbial susceptibility (aim 2), and demonstrate early detection and fast antimicrobial susceptibility profiling of fungal infections (aim 3). The proposed rapid AST method works for bacteria/fungi in complex environment and at the single cell level. Therefore, long-time specimen culture and subculture to get bacterial/fungal isolate can be avoided. The characteristics of this approach offer a significant advancement over current approaches for treatment of bacterial/fungal infections.

Project Summary: Vulnerable plaques are the main contributor to acute coronary syndrome. In early stages, vulnerable plaques are not blood flow limiting, thus not visible in X-ray angiography. Thin cap fibroatheroma, which is found to be the precursor lesion associated with plaque rupture, is featured by a thin fibrous cap, a large necrotic lipid pool, and inflammation. Under the support of R01HL125385, our team developed an intravascular photoacoustic (IVPA) imaging modality for in vivo detection of lipid-laden plaques, which simultaneously measures lipid-specific components and their depth in an artery wall, summarized in 9 journal articles and 2 book chapters. Towards the long-term goal of identification and quantification of lipid-laden vulnerable plaque in human patients, we recognize that current piezoelectric-transducer-based IVPA technology has intrinsic limitations. First, the sensitivity for lipid detection is limited by the insufficient coverage of the low-frequency PA signal; Second, the bandwidth of piezoelectric transducer is not large enough to provide sufficient axial resolution to identify the thin fibrous cap (typically < 65 ?m); Third, the piezoelectric transducer makes it difficult for the eventual size of an IVPA catheter to meet the clinical requirement (< 1 mm in diameter). To address the limitations, this competitive renewal R01 proposal aims to develop a novel all-optical IVUS/PA catheter and validate the system by in vivo imaging of arteries in a clinically relevant Ossabaw swine model. An interdisciplinary team is assembled to achieve this objective. Dr. Ji-Xin Cheng (PI, Boston University) is an expert in development and applications of novel label-free optical imaging methods. Dr. Michael Sturek (co-investigator, Indiana University School of Medicine) is an expert in vascular research and atherosclerotic animal model development. Dr. Islam A. Bolad (consultant, Indiana University School of Medicine) is an interventional cardiologist who has over 20 years of experience on clinical research with multimodal intravascular imaging tools; Dr. Qifa Zhou (consultant, University of Southern California) is an expert of ultrasound transducers. Dr. Yingchun Cao (Research Scientist, Boston University) is an expert on fiber optics and catheter development. We will take three steps to build this all-optical IVUS/PA catheter. In Aim 1, we will develop a dual-frequency IVPA/US catheter with optical-resolution PA imaging capacity. In Aim 2, we will develop and validate an all-optical IVPA/US catheter with high sensitivity and high axial resolution. In Aim 3, we will validate the all-optical IVUS/PA system by in vivo intracoronary imaging on an Ossabaw swine model. Our intravascular fiber-optic ultrasound generation and detection approach will provide significantly extended bandwidth, which not only allows sensitive detection of the low frequency PA signal, but also overcomes the long-standing insufficient spatial resolution of both IVPA and IVUS imaging.

Project Summary A central theme of research in the Cheng group is focused on basic understanding of how biomolecules and/or molecular assemblies function in space and time to drive life. For example, how does the membrane respond to the action potential in neurons? How is the metabolism remodelled during cell development or cancer progression? What happens to the chemistry inside a microorganism when exposed to a drug treatment? Answering these questions has broad implications for diagnosing and treating conditions ranging from infectious diseases to metastatic cancers. Towards this mission, Cheng and his team invent and apply highly sensitive chemical imaging technologies that are able to unveil hidden signatures in various living systems. The eventual goal is to enable molecule-based precision diagnosis and/or treatment of human diseases. The Cheng team further harnesses and manipulates the unique properties of photons to modulate the behaviour of cells. Two focused projects are photolysis of chromophores to eradicate drug-resistant bacteria and optoacoustic modulation of neural tissues at ultrahigh spatial precision. Overall, with integrated expertise in engineering, physics, chemistry, biology, medicine and entrepreneurship, the research team is devoted to three integrated thrusts: (1) Inventing label-free optical modulation and spectroscopic imaging technologies and pushing their physical limits; (2) Discovering molecular signatures that define cellular state and functions; (3) Converting label- free technologies and biological discoveries into molecule-based precision diagnosis and treatments. During the past 5 years (2013 to 2018), research by Cheng and co-workers has pushed the boundary of vibrational spectroscopic imaging in terms of speed, spectral bandwidth, imaging depth, and detection sensitivity (for a review, Science, 2015, 350: aaa8870). In parallel, via collaborations, Cheng and co-workers discovered significant metabolic signatures defining cancer aggressiveness (Cell Metabolism 2014), cancer cell stemness (Cell Stem Cell 2017), and antimicrobial resistance (Anal Chem 2017), as well as a spectroscopic indicator of membrane voltage in neurons (JPC Lett 2017). The overarching goal of this MIRA proposal is to further push the boundary of nonlinear vibrational spectroscopic imaging platforms in order to unveil the signatures that underlie initiation or progression of human diseases by ?watching the orchestra of molecules? in real space and time inside a living system. Cheng and co-workers will pursue this goal by advancing the capability of two complimentary vibrational imaging platforms, namely multiplex stimulated Raman scattering microscopy and infrared photothermal microscopy both invented in the Cheng lab, to reach single-molecule detection sensitivity, 100-nm spatial resolution, volumetric mapping at high speed, and deep-tissue penetration. As a focused application, the team will deploy the developed technologies to advance the basic understanding of how a biological cell reprograms its metabolism during development in vivo or in response to a stress.