Stimulated Raman scattering (SRS) microscopy has emerged as a powerful platform for chemically specific imaging in living systems, enabling minimally perturbative visualization of molecular composition and metabolic dynamics at subcellular resolution. However, its potential for metabolomic studies remains limited by intrinsic challenges in sensitivity, multiplexing applications, and quantitative interpretation of metabolized signals. This thesis addresses these limitations through advances in instrumentation, probe palette design, and computational analysis to expand the capabilities of SRS microscopy for metabolic imaging. In Chapter 2, visible enhanced stimulated Raman scattering microscopy via frequency-doubling of picosecond pulses, we develop a new form visible-SRS microscopy (VIS-SRS) microscopy through direct frequency-doubling of narrowband picosecond laser sources for a more practical route to multiplexed VIS-SRS imaging. By shifting excitation into the visible regime with picosecond pulses, this approach enhances Raman scattering cross-sections and improves spatial resolution while simplifying instrumental implementation compared to femtosecond-based VIS-SRS modalities. VIS-SRS holistically provides a new route toward increased sensitivity for imaging low-abundance molecular species and opening new opportunities for electronic pre-resonance imaging of new target analytes. In chapter 3, MetaboRamics: highly multiplexed metabolic imaging using stimulated Raman scattering microscopy for live-cell spatial metabolomics, we introduce a new vibrational palette for live-cell metabolic analysis. By leveraging minimally perturbative, metabolically active probes in combination with label-free contrast and advanced spectral unmixing, MetaboRamics enables super-multiplexed imaging across multiple metabolic pathways. This platform allows high-dimensional characterization of metabolic states and reveals interconnected metabolic rewiring in response to biological perturbations with single-cell and subcellular resolution. In chapter 4, Optical Metabolic Imaging of Tricarboxylic Acid Cycle Activity, we introduce MATRIX-SRS, a framework for quantitative imaging of tricarboxylic acid (TCA) cycle metabolism in living systems. By integrating hyperspectral SRS imaging with density functional theory (DFT)-informed spectral modeling, MATRIX-SRS enables new quantitative insights into metabolized probe signals. This approach allows, for the first time, spatially resolved and absolute quantification of metabolically incorporated species using optical imaging. Together, these advances position SRS microscopy as an increasingly powerful platform for metabolic investigations. By improving sensitivity, enabling super-multiplexed spatial metabolomics, and enhancing chemical insight into metabolic activity, this work opens new opportunities to study cellular heterogeneity, metabolic dynamics, and disease-associated metabolic rewiring.
Adrian Colazo (Tue,) studied this question.