Time-resolved Mid-infrared photothermal Imaging

For biological applications, the molecular fingerprint region in the mid-infrared (mid-IR, 3 µm – 12 µm) has attracted interest since it contains characteristic vibrational resonances of many biophysically relevant compounds, including proteins, lipids, and nucleic acids. In mid-IR photothermal microscopy, targeting these resonances with a mid-infrared pump beam and a shorter wavelength probe beam leads to imaging with high specificity and sensitivity with sub-diffraction limited resolution smaller than 1 µm.

Time-resolved mid-IR photothermal imaging is discussed to study heat diffusion dynamics in various complex environments including water interfaces and nanoparticle clusters. Boxcar detection enables the extraction of hyper-temporal image stacks along with local temporal traces of the photothermal signal with high a signal-to-noise ratio and no complex post-processing. Environments with multiple absorbers in close proximity to each other or features in highly absorbing media can be analyzed with characteristic thermal decay profiles. In particular, liquid water (H2O) shares a peak at the same wavenumber (~ 6.1 µm) as the Amide I band. Even though this can be overcome by performing imaging in less absorbing aqueous environments (like D2O), heat transfer effects will vary significantly for different media, resulting in various imaging outputs (contrast and resolution). To have a complete picture of how biological samples behave in physiologically relevant environments, time-resolved imaging can offer an effective way to separate the water background based on its inherently different transient response.