Background-Suppressed Infrared Photothermal Microscopy at Low Temperature

The short-wave infrared region provides reduced scattering and improved penetration depth compared to visible and near-infrared wavelengths. However, strong water absorption across this spectral window generates background signals that limit the contrast and obscure weak biomolecular features. To address this challenge, we implement a low-temperature approach that exploits the temperature dependence of the water photothermal response to suppress background while preserving lipid-derived signals in photothermal microscopy. Sample cooling during imaging enhanced the signal-to-background ratio (S/B) by up to 5.5-fold in cellular imaging and improved the contrast-to-noise ratio (CNR) of weak lipid features by up to 1.6-fold at greater tissue depths, enabling reliable volumetric imaging in complex biological systems. We further show that a low-temperature approach is also applicable to mid-infrared photothermal microscopy, thus providing an effective means to enhance the photothermal contrast of aqueous specimens.