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.