Cell-in-water: complementary optical photothermal infrared and Raman microscopy for submicron chemical imaging of...

Conventional Fourier transform infrared absorption spectroscopy is constrained by diffraction-limited spatial resolution and overwhelming absorption of water, typically requiring sample dehydration, which disrupts native cellular morphology. While Optical Photothermal Infrared microscopy overcomes the resolution barrier, reliable imaging of cells in their native, hydrated state remains a significant methodological challenge. In this work, an optimized Cell-in-Water protocol is presented for submicron, label-free chemical imaging of hydrated cells. The implementation of a silicone-sealed microchamber ensures a stable aqueous environment, preventing evaporation and enabling stable imaging for up to 4 h. Using a robust ratiometric data analysis framework, subcellular organelles, including nuclei and lipid droplets, are visualized with a lateral resolution of 300-500 nm. The protocol's reliability was validated through a comparative study of individual cells in both hydrated and dehydrated states, revealing critical spectral shifts and morphological distortions inherently associated with the drying process. These results provide a standardized, reproducible workflow for non-invasive, high-resolution biochemical mapping in aqueous environments, bridging the gap between conventional vibrational spectroscopy and the requirements of advanced biological imaging.