This paper introduces overtone photothermal (OPT) microscopy, a novel technique for high-resolution and high-sensitivity vibrational imaging. OPT microscopy utilizes a visible probe beam to detect thermal lensing effects caused by overtone vibrational absorption in the C-H overtone region, excited by a tunable femtosecond laser in the SWIR window. The technique's capabilities are demonstrated through imaging of polymer nanostructures, depth-resolved intracellular chemical mapping of cancer cells, and imaging of multicellular C. elegans organisms and brain tissues. OPT microscopy bridges the gap between visible and mid-infrared photothermal microscopy, offering both high chemical specificity and deep tissue penetration, complementary to large-scale shortwave infrared imaging approaches.
