Time-Resolved Mid-Infrared Photothermal Microscopy for Imaging Water-Embedded Axon Bundles

[Image: see text] Few experimental tools exist for performing label-free imaging of biological samples in a water-rich environment due to the high infrared absorption of water, overlapping with major protein and lipid bands. A novel imaging modality based on time-resolved mid-infrared photothermal microscopy is introduced and applied to imaging axon bundles in a saline bath environment. Photothermally induced spatial gradients at the axon bundle membrane interfaces with saline and surrounding biological tissue are observed and temporally characterized by a high-speed boxcar detection system. Localized time profiles with an enhanced signal-to-noise, hyper-temporal image stacks, and two-dimensional mapping of the time decay profiles are acquired without the need for complex post image processing. Axon bundles are found to have a larger distribution of time decay profiles compared to the water background, allowing background differentiation based on these transient dynamics. The quantitative analysis of the signal evolution over time allows characterizing the level of thermal confinement at different regions. When axon bundles are surrounded by complex heterogeneous tissue, which contains smaller features, a stronger thermal confinement is observed compared to a water environment, thus shedding light on the heat transfer dynamics across aqueous biological interfaces.