Increasing contrast in water-embedded particles via time-gated mid-infrared photothermal microscopy.

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.

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

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.

Thermal transport across membranes and the Kapitza length from photothermal microscopy.

An analytical model is presented for light scattering associated with heat transport near a cell membrane that divides a complex system into two topologically distinct half-spaces. Our analysis is motivated by experiments on vibrational photothermal microscopy which have not only demonstrated remarkably high contrast and resolution, but also are capable of providing label-free local information of heat transport in complex morphologies. In the first Born approximation, the derived Green's function leads to the reconstruction of a full 3D image with photothermal contrast obtained using both amplitude and phase detection of periodic excitations. We show that important fundamental parameters including the Kapitza length and Kapitza resistance can be derived from experiments. Our goal is to spur additional experimental studies with high-frequency modulation and heterodyne detection in order to make contact with recent theoretical molecular dynamics calculations of thermal transport properties in membrane systems.

Phase-sensitive lock-in detection for high-contrast mid-infrared photothermal imaging with sub-diffraction limited resolution.

Imaging of the phase output of a lock-in amplifier in mid-infrared photothermal vibrational microscopy is demonstrated for the first time in combination with nonlinear demodulation. In general, thermal blurring and heat transport phenomena contribute to the resolution and sensitivity of mid-infrared photothermal imaging. For heterogeneous samples with multiple absorbing features, if imaged in a spectral regime of comparable absorption with their embedding medium, it is demonstrated that differentiation with high contrast is achieved in complementary imaging of the phase signal obtained from a lock-in amplifier compared to standard imaging of the photothermal amplitude signal. Specifically, by investigating the relative contribution of the out-of-phase lock-in signal, information based on changes in the rate of heat transport can be extracted, and inhomogeneities in the thermal diffusion properties across the sample plane can be mapped with high sensitivity and sub-diffraction limited resolution. Under these imaging conditions, wavenumber regimes can be identified in which the thermal diffusion contributions are minimized and an enhancement of the spatial resolution beyond the diffraction limited spot size of the probe beam in the corresponding phase images is achieved. By combining relative diffusive phase imaging with nonlinear demodulation at the second harmonic, it is demonstrated that 1-µm-size melamine beads embedded in a thin layer of 4-octyl-4'-cyanobiphenyl (8CB) liquid crystal can be detected with a 1.3-µm spatial full-width at half-maximum (FWHM) resolution. Thus, imaging with a resolving power that exceeds the probe diffraction limited spot size by a factor of 2.5 is presented, which paves the route towards super-resolution, label-free imaging in the mid-infrared.

Erratum: Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals: publisher's note.

[This corrects the article on p. 303 in vol. 12, PMID: 33520386.].

Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals.

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.