Photothermal imaging has proven a powerful label-free chemical imaging technique. With time-resolved mid-infrared photothermal imaging heat transfer dynamics across aqueous interfaces can be studied. However, liquid water has been a limiting factor in mid-infrared imaging and spectroscopy due to its high absorption spanning across the molecular fingerprint region so that cellular imaging is often performed in less absorbing heavy water instead. Time-resolved measurements via boxcar detection enable the separation of water background and reveal how heat transfer dynamics across aqueous interfaces strongly depend on hydration and the surrounding environment. Mid-infrared photothermal imaging of extracted axon-bundles from crayfish is presented in a saline solution where the water background can be separated based on its inherently different transient response.
Time-resolved mid-infrared 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.
We present time resolved mid-infrared photothermal imaging that allows studying thermal dynamics of samples based on intrinsic vibrational absorption contrast. By using a gated detection in a boxcar configuration, temporal heat profiles at the sub-micron scales can be obtained. Further, the signal to noise is enhanced compared to standard lock-in detection without the need of averaging and post image processing. This imaging platform can be attractive to simultaneously obtain chemical identification and heat diffusion dynamics for a wide range of samples in a label-free manner.
Mid-infrared photothermal microscopy has demonstrated unique capabilities in the field of chemical imaging including sub-diffraction limited resolution and sub cellular imaging. Vibrational Infrared Photothermal and Phase Signal (VIPPS) introduces an additional contrast mechanism based on different thermal properties. This enables high contrast imaging of features with overlapping absorption profiles but different thermal diffusion characteristics. Our approach paves the way for high contrast sub-diffraction limited imaging of secondary protein conformations in fibroblast cells grown in a protein rich collagen matrix at the subcellular level.
Mid-infrared photothermal imaging is a novel chemical imaging modality that combines high sensitivity with enhanced spatial resolution. Subcellular features in fibroblast cells and tissues are imaged and analyzed with regards to their molecular structures without the need of exogenous fluorophores at a resolution that overcomes the diffraction limited spot size of the mid-infrared excitation beam. With a phase-sensitive lock-in detection scheme, changes in the thermal diffusion properties can be detected and can provide a complementary sample characterization.
Mid-infrared spectroscopy and imaging has attracted significant interest due to the presence of characteristic vibrational resonances of numerous molecular compounds in the fingerprint region. However, the imaging resolution in the midinfrared can be significantly weakened by the relatively large spot sizes of the beam used to target the absorption resonances of the sample. Photothermal spectroscopy and imaging as a label-free and nondestructive technique has shown great potential for pushing the imaging resolution to values below the mid-infrared diffraction limit while offering enhanced chemical sensitivity and specificity. Mid-infrared photothermal spectroscopy relies on a pump-probe setup, in which a shorter wavelength probe (removed from the targeted resonances) is co-aligned with the resonant pump beam. The modulated photothermal signal is then detected by a lock-in amplifier, whose output can provide information on the signal amplitude as well as phase. However, most commonly, amplitude images have been evaluated, without considering the recorded photothermal phase. We will provide a detailed analysis of the mid-infrared photothermal amplitude and phase information from a melamine bead sample embedded in a 2.5 μm-thick 4-Octyl-4’-Cyanobiphenyl (8CB) liquid crystal environment. Studies related to thermal transport phenomena as well as increases in imaging sensitivity will be presented for sub-diffraction limited resolution, label-free imaging. The concept of wavelength optimization to minimize thermal blurring effects at the zerocrossing for the out-of-phase contribution is introduced as a novel approach to enhance spatial resolution and sensitivity in mid-infrared label-free photothermal imaging.
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