Mid-infrared Photothermal Imaging Research Articles

Mid-infrared Photothermal Imaging: Instrument and Life Science Applications.

Mid-infrared Photothermal Imaging: Instrument and Life Science Applications.

Mapping enzyme activity in living systems by real-time mid-infrared photothermal imaging of nitrile chameleons.

Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase-phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.

Label-free mid-infrared photothermal live-cell imaging beyond video rate

Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the contrary, MIR microscopy has been rarely used for live biological samples in an aqueous environment due to the lack of spatial resolution and the large water absorption background. Recently, mid-infrared photothermal (MIP) imaging has proven to be applicable to 2D and 3D single-cell imaging with high spatial resolution inherited from visible light. However, the maximum measurement rate has been limited to several frames s−1, limiting its range of use. Here, we develop a significantly improved wide-field MIP quantitative phase microscope with two orders-of-magnitude higher signal-to-noise ratio than previous MIP imaging techniques and demonstrate live-cell imaging beyond video rate. We first derive optimal system design by numerically simulating thermal conduction following the photothermal effect. Then, we develop the designed system with a homemade nanosecond MIR optical parametric oscillator and a high full-well-capacity image sensor. Our high-speed and high-spatial-resolution MIR microscope has great potential to become a new tool for life science, in particular for live-cell analysis.

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel.

By optically sensing absorption-induced photothermal effect, mid-infrared (IR) photothermal (MIP) microscope enables super-resolution IR imaging of biological systems in water. However, the speed of current sample-scanning MIP system is limited to milliseconds per pixel, which is insufficient for capturing living dynamics. By detecting the transient photothermal signal induced by a single IR pulse through fast digitization, we report a laser-scanning MIP microscope that increases the imaging speed by three orders of magnitude. To realize single-pulse photothermal detection, we use synchronized galvo scanning of both mid-IR and probe beams to achieve an imaging line rate of more than 2 kilohertz. With video-rate speed, we observed the dynamics of various biomolecules in living organisms at multiple scales. Furthermore, by using hyperspectral imaging, we chemically dissected the layered ultrastructure of fungal cell wall. Last, with a uniform field of view more than 200 by 200 square micrometer, we mapped fat storage in free-moving Caenorhabditis elegans and live embryos.

Mid-infrared chemical imaging of intracellular tau fibrils using fluorescence-guided computational photothermal microscopy

Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fibrils, an important type of amyloid protein aggregates, in their intracellular environment. Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation. Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils. 3D visualization of the β-sheet for tau fibril structure is achieved.

Video-rate Mid-infrared Photothermal Imaging by Single Pulse Photothermal Detection per Pixel

Video-rate Mid-infrared Photothermal Imaging by Single Pulse Photothermal Detection per Pixel

High-Throughput Antimicrobial Susceptibility Testing of Escherichia coli by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis

Antimicrobial resistance poses great threats to global health and economics. Current gold-standard antimicrobial susceptibility testing (AST) requires extensive culture time (36-72 h) to determine susceptibility. There is an urgent need for rapid AST methods to slow down antimicrobial resistance. Here, we present a rapid AST method based on wide-field mid-infrared photothermal imaging of protein synthesis from 13C-glucose in Escherichia coli. Our wide-field approach achieved metabolic imaging for hundreds of bacteria at the single-cell resolution within seconds. The perturbed microbial protein synthesis can be probed within 1 h after antibiotic treatment in E. coli cells. The susceptibility of antibiotics with various mechanisms of action has been probed through monitoring protein synthesis, which promises great potential of the proposed platform toward clinical translation.

Mid-Infrared Photothermal Microscopy: Principle, Instrumentation, and Applications.

Midinfrared photothermal (MIP) microscopy, also called optical photothermal infrared (O-PTIR) microscopy, is an emerging tool for bond-selective chemical imaging of living biological and material samples. In MIP microscopy, a visible probe beam detects the photothermal-based contrast induced by a vibrational absorption. With submicron spatial resolution, high spectral fidelity, and reduced water absorption background, MIP microscopy has overcome the limitations in infrared chemical imaging methods. In this review, we summarize the basic principle of MIP microscopy, the different origins of MIP contrasts, and recent technology development that pushed the resolution, speed, and sensitivity of MIP imaging to a new stage. We further emphasize its broad applications in life science and material characterization, and provide a perspective of future technical advances.

Multi-modal vibrational analysis of blend polymers using mid-infrared photothermal and Raman microscopies

Chemical characterization of polymeric samples, such as blend polymers is a fundamental analytical approach for advancement of polymer science and polymer technologies. Vibrational imaging is a promising technique to analyze chemical compositions of materials in a wide range of fields. Raman scattering and infrared (IR) absorption reflect complementary molecular vibrations; therefore, Raman and IR imaging provide rich information of the spatial distributions of chemical compositions of samples, revealing their complicated chemical structures. However, the discrepancy of spatial resolution in Raman and conventional IR imaging makes it difficult to merge these techniques. In the present study, we performed chemical characterization of polymeric samples using Raman and IR imaging enabled by mid-infrared photothermal (MIP) microscopy. The chemical compositions of blend polymer films were visualized based on both Raman scattering and IR absorption with the spatial resolution of the sub-micrometer scale. We found that the varying contrasts in Raman and MIP imaging enabled the investigation of averaged and surface-sensitive compositions of polymer blends. Raman and MIP imaging holds a promise of unique applications in a wide range of current research fields, such as polymers, two-dimensional materials, and biology.

Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering.

Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μm × 70 μm. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells.

Fluorescence-Detected Mid-Infrared Photothermal Microscopy.

Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at submicrometer spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependence of scattering on the temperature, and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed through fingerprinting a single bacterium. Finally, the photobleaching issue is successfully addressed through the development of a wide-field fluorescence-detected mid-infrared photothermal microscope which allows video rate bond-selective imaging of biological specimens.

Cytoplasmic Protein Imaging with Mid-Infrared Photothermal Microscopy: Cellular Dynamics of Live Neurons and Oligodendrocytes.

Mid-infrared photothermal microscopy has been suggested as an alternative to conventional infrared microscopy because in addition to the inherent chemical contrast available upon vibrational excitation, it can feasibly achieve spatial resolution at the submicrometer level. Furthermore, it has substantial potential for real-time bioimaging for the observation of cellular dynamics without photodamage or photobleaching of fluorescent labels. We performed real-time imaging of oligodendrocytes to investigate cellular dynamics throughout the life cycle of a cell, revealing details of cell division and apoptosis, as well as cellular migration. In the case of live neurons, we observed a photothermal contrast associated with traveling protein complexes on an axon, which correspond to the transport of vesicles from the cell body to the dendritic branches of the neuron through the cytoskeleton. We anticipate that mid-infrared photothermal imaging will be of great use for gaining insights into the field of biophysical science, especially with regard to cellular dynamics and functions.

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.

Bond-Selective Imaging of Cells by Mid-Infrared Photothermal Microscopy in High Wavenumber Region.

Using a visible beam to probe the thermal effect induced by infrared absorption, mid-infrared photothermal (MIP) microscopy allows bond-selective chemical imaging at submicron spatial resolution. Current MIP microscopes cannot reach the high wavenumber region due to the limited tunability of the existing quantum cascade laser source. We extend the spectral range of MIP microscopy by difference frequency generation (DFG) from two chirped femtosecond pulses. Flexible wavelength tuning in both C-D and C-H regions was achieved with mid-infrared power up to 22.1 mW and spectral width of 29.3 cm-1. Distribution of fatty acid in live human lung cancer cells was revealed by MIP imaging of the C-D bond at 2192 cm-1.

Mid-Infrared Photothermal Imaging of Active Pharmaceutical Ingredients at Submicrometer Spatial Resolution.

Chemical imaging with sufficient spatial resolution to resolve microparticles in tablets is essential to ensure high quality and efficacy in controlled release. The existing modalities have the following disadvantages: they are time-consuming or have poor spatial resolution or low chemical specificity. Here, we demonstrate an epi-detected mid-infrared photothermal (epi-MIP) microscope at a spatial resolution of 0.65 μm. Providing identical spectral profiles as conventional infrared spectroscopy, our epi-MIP microscope enabled mapping of both active pharmaceutical ingredients and excipients of a drug tablet.

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution.

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.