Project Summary In order to combat infections and reduce antimicrobial resistance, it is essential to detect and characterize bacterial/fungal susceptibility to antimicrobials in the early stages of infections to reduce the inappropriate use of antimicrobials and the rate of death. Under R01AI141439 (2018 to 2022), we have been addressing this urgent need by developing a rapid AST through high-speed stimulated Raman scattering (SRS) imaging of single-cell metabolism. Through SRS imaging of glucose-d7 metabolism, we determined the antimicrobial susceptibility profile of bacteria and fungi within 1 cell cycle. More recently, through SRS imaging of D2O incorporation, we obtained a single-cell metabolism inactivation concentration (SC-MIC) in less than 2.5 h from culturing a colony to obtaining results. Despite this initial success, there are remaining gaps in translating this technology into clinic. First, the single-color SRS signal depends on the bacterial size and thus is not quantitative in predicting single- cell metabolic activity. Second, the laser used in our past studies is too bulky to be used in clinic. Third, SRS imaging alone does not tell the identity of the pathogens. Fourth, the single-well imaging chamber does not allow high-throughput measurement of samples separately treated by a panel of antibiotics at various concentrations. In this competitive renewal of R01AI141439, we propose to overcome these barriers through three technical innovations. To address the first and second gaps, we will build a fast-tuning SRS microscope based on a compact fiber-OPO laser. We have demonstrated the feasibility of this approach in multi-window SRS imaging of single cell metabolism. By fast tuning between C-D and C-H vibration, we will be able to use CD/(CD+CH) SRS signal ratio to quantify the metabolic activity of a single cell. To address the third gap, we will build multi- color fluorescence in situ hybridization (FISH) into the compact SRS microscope to enable rapid identification and fast AST on a single microscope platform. We have demonstrated the feasibility of FISH-SRS for imaging metabolic activity of bacteria in a gut microbiome. To address the fourth gap, we will demonstrate high-throughput AST through robotic handling of liquid specimens and a multi-well cartridge design. A team with complementary expertise will carry out the proposed research. While we use urinary tract infection as a focused testbed in this proposal, our technology is broadly applicable to rapid determination of susceptibility for other important infections (e.g., bloodstream infections, meningitis, pneumonia), thus having a broad, positive impact on global public health.