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ULTRA-BROADBAND MULTIMODAL MICROSCOPY WITH A CATADIOPTRIC LENS - PROJECT SUMMARY CELL-BASED ASSAYS PLAY A CRUCIAL ROLE IN PRECLINICAL DRUG DEVELOPMENT PROGRAMS AS WELL AS IN BASIC BIOLOGICAL RESEARCH. THE GOAL OF OUR COMPANY IS TO PROVIDE SPATIAL BIOLOGY SOLUTIONS TO SUPPORT FOUNDATIONAL BIOLOGY STUDIES AND IMPROVE TARGET VALIDATION THROUGHOUT IN VITRO HIT-TO-LEAD (H2L) SCREENING AND LEAD OPTIMIZATION DURING PRECLINICAL RESEARCH. THIS IS ACHIEVED BY DELIVERING A SPATIAL BIOLOGY PLATFORM DESIGNED TO QUANTITATIVELY ASSES THE METABOLIC COMPOSITION OF CELLS AND THEIR SURROUNDING MICROENVIRONMENTS IN ORDER TO EVALUATE THE TRAITS AND CHARACTERISTICS THAT DEFINE PHENOTYPE. SPATIAL METABOLOMICS IS COMPLEMENTARY TO MULTIOMIC TECHNOLOGIES THAT LOCALIZE DNA, RNA, AND PROTEINS BIOMOLECULES, AND WHEN USED TOGETHER OFFER A MORE COMPREHENSIVE UNDERSTANDING OF COMPLEX BIOLOGICAL SYSTEMS. OUR APPROACH FOR RAPID, SUBCELLULAR LOCALIZATION OF METABOLITES IS BASED ON A WIDEFIELD VIBRATIONAL SPECTRO-MICROSCOPY, SPECIFICALLY PHOTOTHERMAL (PT) MICROSCOPY. THE TECHNIQUE USES A TUNABLE MID-INFRARED (MIR) LIGHT SOURCE TO EXCITE IR-ACTIVE MOLECULAR VIBRATIONS, WITH A VISIBLE LIGHT SOURCE TO MAP THE SPATIAL DISTRIBUTION OF THESE ABSORPTIONS. A SIGNIFICANT CHALLENGE IN DEPLOYING PT MICROSCOPY IS THE INCOMPATIBILITY OF STANDARD COMMERCIAL OBJECTIVE LENSES AND MIR LIGHT DUE TO THE LIMITED SPECTRAL TRANSMISSION OF THE REFRACTIVE ELEMENTS IN THE LENS. MOREOVER, THESE LENSES SUFFER FROM CHROMATIC ABERRATIONS AND INTRODUCE GROUP DELAY DISPERSION (GDD), WHICH CAUSES TEMPORAL BROADENING IN ULTRAFAST LIGHT SOURCES USED IN MULTIPHOTON MICROSCOPES. IN PHASE I, WE ADDRESSED THESE LIMITATIONS BY DEVELOPING A NOVEL OBJECTIVE LENS CONSTRUCTED ENTIRELY FROM CURVED MIRRORS IN A NON-CONCENTRIC GEOMETRY, DELIVERING ULTRA-BROAD TRANSMISSION FROM THE VISIBLE THROUGH THE MIR WITH PERFECT COLOR CORRECTION AND MINIMAL GDD, ALL WHILE REMAINING DIFFRACTION-LIMITED ACROSS THE ENTIRE FIELD-OF-VIEW (FOV). IN PHASE II, WE AIM TO INCREASE THE NUMERICAL APERTURE OF THE LENS BY INCLUDING A LIQUID IMMERSION. THIS WILL PROVIDE VERY HIGH-RESOLUTION IMAGING OVER A VIRTUALLY UNLIMITED SPECTRAL BANDWIDTH. WE WILL LEVERAGE OUR EXPERIENCES FROM PHASE I TO DESIGN, MANUFACTURE, AND VALIDATE THE NEXT GENERATION OBJECTIVE LENS. FOLLOWING THESE EFFORTS, WE WILL INTEGRATE THE LENS ONTO OUR SPATIAL METABOLITE IMAGING PLATFORM AND DEMONSTRATE ITS UTILITY TO CONDUCT IN VITRO STUDIES BY MONITORING THE SMALL MOLECULE PYRIDOXAL 5’-PHOSPHATE (VITAMIN B6) IN A PANEL OF HUMAN COLORECTAL CANCER (CRC) CELLS. THESE STUDIES MAY REVEAL KEY ROLES FOR VITAMIN B6 IN CELL GROWTH AND PROLIFERATION DURING TUMORIGENESIS, POTENTIALLY UNCOVERING NEW THERAPEUTIC VULNERABILITIES IN CRC CELLS. WE EXPECT OUR LENS TO REACH COMMERCIAL READINESS AND OUR PLATFORM WILL BE READY FOR ROUTINE BETA- TESTING BY EARLY ADOPTERS BY THE CONCLUSION OF THIS PROJECT.

IN VITRO METABOLITE PROFILING WITH VIBRATIONAL PHASE-CONTRAST IMAGING - PROJECT SUMMARY CELL-BASED ASSAYS ARE CRITICAL IN PRECLINICAL DRUG DEVELOPMENT PROGRAMS AS THEY PROVIDE RELIABLE PREDICTIONS OF A SMALL MOLECULE CHEMICAL COMPOUND’S METABOLISM, PHARMACOKINETIC PROPERTIES, AND POTENTIAL AS A THERAPEUTIC. IN VITRO METABOLITE PROFILING AND MAPPING STUDIES ARE RELATIVELY COST- EFFECTIVE WAYS TO OPTIMIZE LEAD CANDIDATES AND PROVIDE IMPORTANT INFORMATION FOR DESIGNING SECONDARY AND IN VIVO ASSAYS. METABOLITE CHARACTERIZATION IS ACHIEVED USING COMMERCIAL ANALYTICAL CHEMISTRY INSTRUMENTS, CURRENTLY LED BY MASS SPECTROMETRY, UV/VIS AND FLUORESCENCE SPECTROSCOPY, HOWEVER RECENT ADVANCEMENTS IN NOVEL VIBRATIONAL SPECTRO-MICROSCOPY METHODS WITH INCREASED SENSITIVITY, RESOLUTION, AND THROUGHPUT NOW OFFER A COMPELLING TECHNIQUE TO NON- DESTRUCTIVELY PROFILE METABOLITES WHILE RETAINING SPATIAL INFORMATION. THE METABOLITE PROFILING DATASETS OUR INSTRUMENT WILL GENERATE ARE COMPLIMENTARY TO OTHER SPATIAL BIOLOGY MULTIOMIC PLATFORMS AND WILL BE VERY USEFUL FOR EVALUATING PHENOTYPE. SUCH TECHNOLOGIES WILL LIKELY HAVE UTILITY STUDYING TISSUE SECTION SAMPLES AND ENGINEERED CELLS USED IN SYNTHETIC BIOLOGY AS WELL. OUR PLATFORM INSTRUMENT IS BASED ON WIDEFIELD PHOTOTHERMAL (PT) MICROSCOPY WHERE A TUNABLE MID-INFRARED LIGHT SOURCE IS USED TO EXCITE MOLECULAR VIBRATIONS. TO IMPROVE SENSITIVITY AT LOWER CHEMICAL CONCENTRATIONS, WE WILL INTEGRATE THE PT MODALITY WITH SPATIAL LIGHT INTERFERENCE MICROSCOPY (SLIM), A HIGHLY SUCCESSFUL QUANTITATIVE PHASE IMAGING (QPI) TECHNIQUE OPTIMIZED FOR SPECKLE-FREE IMAGING AND NANOMETER PHASE SENSITIVITY. WE WILL USE A POLARIZATION CAMERA TO CAPTURE QPI IMAGES IN A SINGLE-FRAME, ALLOWING TRUE VIDEO-RATE CHEMICAL IMAGING WITH SUB-MICRON RESOLUTION. WE WILL COMPARE KEY METRICS OF OUR INSTRUMENT TO THE PT MICROSCOPE IN THE LABORATORY OF PROF. JI-XIN CHENG AT BOSTON UNIVERSITY. WE CONCLUDE THIS PHASE I EFFORT WITH AN IN VITRO CELL SCREENING DEMONSTRATION AT THE UNIVERSITY OF CALIFORNIA, IRVINE WHERE WE WILL MONITOR PRESENCE OF THE SMALL MOLECULE PYRIDOXAL 5’-PHOSPHATE (VITAMIN B6) IN LIVE COLORECTAL CANCER (CRC) CELLS. WE ANTICIPATE LOWER B6 LEVELS IN CRCS WITH HYPERACTIVE LYSOSOMES AND DEGRADED PDXK ENZYMATIC ACTIVITY. THESE RESULTS IMPLICATE KEY ROLES FOR VITAMIN B6 IN THE PROCESSES OF CELL GROWTH/ PROLIFERATION AND HINT AT A THERAPEUTIC VULNERABILITY TO EXPLOIT IN CRCS.

RAPID AND HIGH-CONTRAST PHOTOTHERMAL MICROSCOPY WITH A NOVEL TUNABLE ZGP SOURCE - ABSTRACT. NATURAL PRODUCTS OFFER EXCELLENT SOURCES OF HEALTH-PROMOTING MEDICINES YET CHALLENGES IN EFFICIENTLY AND ECONOMICALLY SOURCING THESE VALUE-ADD BIO-COMPOUNDS HAVE PREVENTED THEIR GREATER AVAILABILITY AND PROMOTION TO PUBLIC HEALTH. WE ADDRESS THIS PROBLEM BY DEVELOPING A NOVEL IMAGE-BASED SCREENING TECHNOLOGY THAT OFFERS HIGHLY SENSITIVE, PHENOTYPIC BIOASSAYS OF METABOLITE CONCENTRATIONS ON LIVE SINGLE CELLS. THIS ALLOWS A GREATLY IMPROVED METHOD FOR IDENTIFICATION OF BETTER NATURAL PRODUCT PRODUCERS, AS WELL AS QUANTITATIVE INFORMATION ABOUT THE PRODUCTION PER CELL AND PRODUCTION DISTRIBUTION ACROSS THE POPULATION. THIS HIGH-RESOLUTION, NON- DESTRUCTIVE, LABEL-FREE CELL SCREENING AND CELL SORTING SYSTEM IS BASED ON VIBRATIONAL PHOTOTHERMAL MICROSCOPY WHICH USES MID-INFRARED LIGHT TO PROBE MOLECULAR VIBRATIONAL ABSORPTIONS AT RESOLUTIONS COMPARABLE TO VISIBLE LIGHT MICROSCOPES. TO DEMONSTRATE THE UTILITY OF THE APPROACH WE FOCUS ON THE PRODUCTION OF ESSENTIAL POLYUNSATURATED FATTY ACIDS (PUFAS) IN LIPID DROPLETS SOURCED FROM MICROALGAE (THRAUSTOCHYTRIDS) – A NATURAL PRODUCER OF PUFAS. THESE LONG-CHAIN FATTY ACIDS PLAY A VITAL ROLE IN THE PHYSIOLOGICAL HEALTH OF CELLS AND TISSUES THROUGHOUT THE BODY ARE COMMERCIALIZED AS A PROMINENT NUTRACEUTICAL. TO DEVELOP ENRICHED CELL LINES, WE WILL ENGINEER AND PROTOTYPE A MULTICHANNEL MICROFLUIDIC CHIP WITH AN ACTIVE PIEZOELECTRIC ACTUATOR TO SELECTIVELY SAVE CELLS IDENTIFIED THROUGH PHOTOTHERMAL AS BEING HIGHLY PRODUCTIVE. OUR GOAL IS TO PROVIDE AN ATTRACTIVE METHOD TO NON-DESTRUCTIVELY EVALUATE METABOLITE CONTENT IN ENGINEERED MICROBES INCLUDING BACTERIA AND YEAST, NOT JUST ALGAE, AND FOR A VARIETY OF HIGH-VALUE METABOLITES. TO ACCOMPLISH THESE GOALS, WE HAVE ESTABLISHED A TEAM OF QUALIFIED EXPERTS INCLUDING SPECIALISTS IN ADVANCED MICROSCOPY, MICROFLUIDICS DESIGN, AND ALGAE CULTIVATION FOR INDUSTRIAL APPLICATIONS. ULTIMATELY, THE METHODS DEVELOPED HERE WILL SHOWCASE A ROADMAP TOWARD RARE-CELL DISCOVERY AND ACTIVE CELL SORTING BASED ON CHEMICAL MAPPING ALLOWED THROUGH THE PHOTOTHERMAL IMAGING CONTRAST.

ULTRA-BROADBAND MULTIMODAL MICROSCOPY WITH A CATADIOPTRIC LENS - ABSTRACT. VIRTUALLY ALL OPTICAL MICROSCOPES FOR BIOLOGICAL IMAGING ARE BASED ON REFRACTIVE OBJECTIVE LENSES. THE PERFORMANCE OF THESE LENSES APPROACHES THE THEORETICAL LIMIT, HOWEVER, THEIR USE IS LIMITED TO THE VISIBLE TO NEAR-INFRARED SPECTRAL RANGE. EVEN WITHIN THIS RANGE, THEIR PERFORMANCE IS ONLY GUARANTEED OVER A RELATIVELY NARROW RANGE, AND BROADBAND USE IS INVARIABLY AFFECTED BY CHROMATIC ABERRATIONS. ANOTHER PROBLEM IS THE GROUP DELAY DISPERSION THAT THESE LENSES INTRODUCE TO SHORT OPTICAL PULSES, WHICH REDUCES THE EFFICIENCY OF NONLINEAR OPTICAL (NLO) SIGNAL GENERATION IN THE MICROSCOPE. TAKEN TOGETHER, THESE SHORTCOMINGS SERIOUSLY COMPROMISE THE IMAGING PROPERTIES OF SEVERAL NLO IMAGING MODALITIES SUCH AS THREE-PHOTON EXCITED FLUORESCENCE AND THIRD-HARMONIC GENERATION. IN ADDITION, REFRACTIVE OBJECTIVES SIMPLY CANNOT BE USED FOR NLO TECHNIQUES THAT INCORPORATE EXCITATION LIGHT IN THE MID-INFRARED (MIR) RANGE, SUCH AS PHOTOTHERMAL IMAGING AND SUM-FREQUENCY GENERATION, PROMISING TECHNOLOGIES BASED ON MIR MOLECULAR CONTRAST. THE ONLY VIABLE ALTERNATIVE IS THE ALL-REFLECTIVE SCHWARZSCHILD-CASSEGRAIN (SC) OBJECTIVE, WHICH IS INHERENTLY ACHROMATIC BUT SUFFERS FROM A NON-IDEAL POINT SPREAD FUNCTION AND A CENTER OBSCURATION THAT LIMITS THROUGHPUT. BECAUSE OF THESE LIMITATIONS, SC LENSES HAVE NOT FOUND WIDESPREAD USE IN BIOLOGICAL IMAGING APPLICATIONS. THIS LACK OF PERFORMANCE IS ALSO THE REASON WHY ADVANCES IN EXCITING NEW MIR-BASED NLO IMAGING TECHNOLOGIES HAVE BEEN STIFLED: THERE SIMPLY ARE NO HIGH-PERFORMANCE HIGH NUMERICAL FOCUSING OPTIONS AVAILABLE TO SUPPORT THESE EMERGING IMAGING TECHNOLOGIES. IN THIS PROJECT, WE DEVELOP A NOVEL HIGH NUMERICAL APERTURE LENS THAT OVERCOMES ALL LIMITATIONS OF THE SC FOCUSING LENS. LEVERAGING REFRACTIVE AND REFLECTIVE ELEMENTS BASED ON A NON- CONCENTRIC LAYOUT, THIS NEW CATADIOPTRIC DESIGN FEATURES TRANSMISSION FROM THE ULTRA-VIOLET TO THE MID-INFRARED, EXHIBITS A WIDE FIELD OF VIEW AND EXTENDED WORKING DISTANCE, DRAMATICALLY REDUCES GROUP DELAY DISPERSION AND SIGNIFICANTLY IMPROVES THROUGHPUT BY ELIMINATING THE CENTER OBSCURATION ALL TOGETHER. THIS LENS NOT ONLY ADVANCES EXISTING NLO MODALITIES THAT RELY ON BROADBAND RADIATION, BUT ALSO ENABLES NEW TECHNOLOGIES SUCH AS PHOTOTHERMAL IMAGING AND SFG MICROSCOPY THAT HAVE THUS FAR SUFFERED FROM LOW PERFORMANCE FOCUSING OPTICS. ULTIMATELY, THIS IMAGING TOOL WILL ENABLE RESEARCHERS TO PERFORM SINGLE-CELL AND TISSUE STUDIES FOR A VARIETY OF CROSS- CUTTING BIOMEDICAL APPLICATIONS REGARDLESS OF THE ILLUMINATION SOURCE USED.

SBIR PHASE I: BROADBAND FOCUSING FOR NON-INVASIVE CELL METABOLOMICS -THE BROADER IMPACT OF THIS SMALL BUSINESS INNOVATION RESEARCH (SBIR) PHASE I PROJECT IS AIMED TOWARD ADVANCING SUSTAINABLE PRODUCTION OF CHEMICALS USING SYNTHETIC BIOLOGY. HERE, SINGLE-CELL MICROBES ARE ENGINEERED TO PRODUCE VALUABLE METABOLITES USING ENZYMES RATHER THAN SOURCING THESE CHEMICALS FROM PETROLEUM. DEVELOPING GENETICALLY ENGINEERED CELL STRAINS WITH HIGH YIELD REMAINS AN ONGOING EFFORT DUE TO THE COMPLEXITIES IN HOW GENETIC CODE LEADS TO PHENOTYPE EXPRESSION. THIS PROBLEM IS ADDRESSED USING A BOTTOM-UP APPROACH TO SCREEN MICROBE POPULATIONS AT THE SINGLE-CELL LEVEL. THE METHOD DEPLOYED TO IDENTIFY METABOLITE CONTENT IN INDIVIDUAL CELLS IS BASED ON INFRARED (IR)-ABSORPTION SPECTROSCOPY WHICH IS LABEL-FREE, QUANTITATIVE, AND NON-DESTRUCTIVE. SYNTHETIC BIOLOGY IS POISED TO DISRUPT THE CHEMICAL VALUE-CHAIN BY PROVIDING AN ALTERNATIVE TO PETROLEUM-BASED CHEMICALS THAT IS SUSTAINABLE AND CARBON-NEUTRAL. ONCE A HIGHLY PRODUCTIVE CELL IS IDENTIFIED IT CAN BE SELECTIVELY PROPAGATED TO CREATE ENRICHED CELL LINES. INNOVATIONS IN OPTICAL MICROSCOPY ARE REQUIRED TO IMPROVE THE PERFORMANCE OF THE CELL SCREENING INSTRUMENTS, WHICH WILL ALLOW HIGH-RESOLUTION FOCUSING ACROSS A BROAD SPECTRAL RANGE. THE UPGRADED PLATFORM WILL OPTIMIZE YIELD MORE QUICKLY, PROVIDING VALUE BY REDUCING THE UPFRONT COST TO DEVELOP NEW INDUSTRIAL CELL STRAINS. THE PROPOSED PROJECT EMPHASIZES OPTICAL ENGINEERING TO DEVELOP A MICROSCOPE DESIGNED FOR HIGH-RESOLUTION CHEMICAL IMAGING BASED ON MOLECULAR VIBRATIONAL IR-ABSORPTIONS. THIS IS ACHIEVED BY DEPLOYING FOCUSING ELEMENTS THAT OPERATE OVER A BROAD SPECTRAL RANGE THAT EXTEND STANDARD OPTICAL MICROSCOPES TO INCLUDE MID-INFRARED LIGHT SOURCES. THE OPTICAL INSTRUMENT WILL BE USED TO EVALUATE CHEMICAL CONTENT IN INDUSTRIAL MICROBE STRAINS AND DEVELOP ENRICHED CELL LINES. THESE SINGLE-CELL MICROBE POPULATIONS ARE ENGINEERED TO PRODUCE ENZYMES USED TO CATALYZE THE SYNTHESIS OF VALUABLE METABOLITES. YIELDS FROM INDIVIDUAL CELLS, HOWEVER, ARE VARIABLE DUE TO GENETIC MUTATIONS IN THE POPULATION. THEREFORE, A QUANTITATIVE ANALYTICAL TOOL BASED ON IR-SPECTROSCOPY THAT CAN NON-DESTRUCTIVELY IDENTIFY HIGHLY PRODUCTIVE CELLS FOR SELECTIVE PROPAGATION IS EXTREMELY DESIRABLE. THIS BOTTOM-UP APPROACH FOR METABOLOMIC CELL SCREENING AND DIRECTED EVOLUTION IS AN INNOVATION AS IT IS LABEL-FREE, NON-INVASIVE, AND HAS STRONG CHEMICAL SPECIFICITY. IN THIS PROJECT, THE TEAM WILL STUDY AN INDUSTRIAL MICROALGAE STRAIN USED AS A LOW-COST FEEDSTOCK SUPPLEMENT AND IDENTIFY CELLS RICH IN PROTEIN CONTENT TO ENHANCE THE OVERALL PROTEIN YIELD. 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.

QUANTIFYING THE ALGAL METABOLOME THROUGH PHOTOTHERMAL MICROSCOPY