Project Grant R44GM149018

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.