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The transient dynamics of photothermal signals provide interesting insights into material properties and heat diffusion. In a mid-infrared (mid-IR) photothermal microscope, the imaging contrast in a standard amplitude imaging can decrease due to thermal diffusion effects. It is shown that contrast varies for poly-methyl 2-methylpropenoate (PMMA) particles of different sizes when embedded in an absorbing medium of water (H₂O) based on levels of heat exchange under the water absorption resonance. Using time-resolved boxcar (BC) detection, analysis of the transient thermal dynamics at the bead–water interface is presented, and the time decay parameters for 500 nm and 100 nm beads are determined. Enhanced (negative) imaging contrast is observed for less heat exchange between the water and bead, as in the case for the 100 nm bead. For the 500 nm bead, boxcar imaging before heat exchange starts occurring, leads to an increase of the imaging contrast up to a factor of 1.6. 

Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals). We present that the detectability of nano-sized cell membranes is enhanced to well below the optical diffraction limit since the membranes are found to act as thermal barriers. VIPPS offers a novel combination of chemical imaging and thermal diffusion characterization that paves the way towards label-free imaging of cell models and tissues as well as the study of intracellular heat dynamics. 

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.