Chem-SIM: Super-resolution Chemical Imaging via Photothermal Modulation of Structured-Illumination Fluorescence

Abstract: Structured illumination microscopy (SIM) has attained high spatiotemporal delineation of subcellular architecture, yet offers limited insight into chemical composition. Here, we present Chem-SIM, a structured-illumination fluorescence detected mid-infrared photothermal microscopy, for super-resolution chemical imaging of microorganisms and mammalian cells. A computational pipeline combining Poisson maximum-likelihood demodulation and spectral normalization across wavenumber is implemented to robustly recover the weak IR-induced fluorescence intensity change under low photon budgets and convert the fluorescence intensity modulation to chemical fingerprints. A photothermal gating scheme further rejects water backgrounds in aqueous samples, while the IR pump maintains cellular activity at near-physiological temperature. Chem-SIM preserves full vibrational fingerprints, achieves SIM-grade lateral resolution, and operates in a high-throughput, camera-based format with minimal modifications and low photothermal load. At the single-bacterium level, Chem-SIM distinguishes stationary phase from log phase cells through chemical content mapping. In ovarian cancer cells, Chem-SIM delivers readouts of lipid chemistry under deuterium fatty acid treatment and resolves lipid droplets dynamics in live cells. Together, Chem-SIM provides an accessible route to super-resolved mapping of organelle chemistry, metabolism, and dynamics.