Multiscale and multicontrast mid-infrared photothermal microscopy

As biological organization extends from molecules to tissues, understanding how chemical events lead to function requires imaging tools that can directly connect molecular composition to biological processes across scales. Conventional optical methods often rely on labeling or lack molecular specificity, leaving gaps between biochemical composition and biological function. Mid-infrared photothermal (MIP) microscopy overcomes these barriers by detecting molecular vibrations through the photothermal effect, providing chemical contrast with submicrometer resolution. We demonstrate that MIP microscopy can operate across diverse biological scales and contrast mechanisms. At cell scale, MIP reveals vibrational signatures that distinguish molecular components within individual bacteria and mammalian cells. At larger scales, metabolic probes enable quantitative mapping of metabolic activity in complex organoids. Furthermore, integration with fluorescence in situ hybridization introduces an additional contrast that correlates cell identity with the chemical information. Together, these studies highlight the versatility of MIP as a powerful multiscale and multicontrast imaging tool.