Single-cell infrared vibrational analysis by optical trapping mid-infrared photothermal microscopy.

Single-cell analysis by means of vibrational spectroscopy combined with optical trapping is a reliable platform for unveiling cell-to-cell heterogeneities in vast populations. Although infrared (IR) vibrational spectroscopy provides rich molecular fingerprint information on biological samples in a label-free manner, its application with optical trapping has never been achieved due to weak gradient forces generated by the diffraction-limited focused IR beam and strong background of water absorption. Herein, we present single-cell IR vibrational analysis that incorporates mid-infrared photothermal (MIP) microscopy with optical trapping. Optically trapped single polymer particles and red blood cells (RBCs) in blood could be chemically identified owing to their IR vibrational fingerprints. This single-cell IR vibrational analysis further allowed us to probe the chemical heterogeneities of RBCs originating from the variation in the intracellular characteristics. Our demonstration paves the way for the IR vibrational analysis of single cells and chemical characterization in various fields.

Label-free visualization of photosynthetic microbial biofilms using mid-infrared photothermal and autofluorescence imaging.

The formation of photosynthetic microbial biofilms comprising multispecies biomolecules, such as extracellular polymeric substances (EPSs), and microbial cells play pivotal roles in maintaining or stimulating their biological functions. Although there are numerous studies on photosynthetic microbial biofilms, the spatial distribution of EPS components that are vital for microbial biofilm formation, such as exopolysaccharides and proteins, is not well understood. Visualization of photosynthetic microbial biofilms requires label-free methods, because labelling EPSs results in structural changes or aggregation. Raman spectroscopy is useful for label-free visualization of biofilm constituents based on chemical contrast. However, interference resulting from the bright autofluorescence of photosynthetic molecules and the low detection efficiency of Raman scattering make visualization a challenge. Herein, we visualized photosynthetic microbial biofilms in a label-free manner using a super-resolution optical infrared absorption imaging technique, called mid-infrared photothermal (MIP) microscopy. By leveraging the advantages of MIP microscopy, such as its sub-micrometer spatial resolution, autofluorescence-free features, and high detection sensitivity, the distribution of cyanobacteria and their extracellular polysaccharides in the biofilm matrix were successfully visualized. This showed that cyanobacterial cells were aligned along acidic/sulfated polysaccharides in the extracellular environment. Furthermore, spectroscopic analyses elucidated that during formation of biofilms, sulfated polysaccharides initially form linear structures followed by entrapment of cyanobacterial cells. The present study provides the foundation for further studies on the formation, structure, and biological functions of microbial biofilms.

Experimental and theoretical elucidation of catalytic pathways in TiO2-initiated prebiotic polymerization.

The tendency of glycine to form polymer chains on a rutile(110) surface under wet/dry conditions (dry-wet cycles at high temperature) is studied through a conjunction of surface sensitive experimental techniques and sequential periodic multilevel calculations that mimics the experimental procedures with models of decreasing complexity and increasing accuracy. X-ray photoemission spectroscopy (XPS) and thermal desorption spectroscopy (TDS) experimentally confirmed that the dry-wet cycles lead to Gly polymerization on the oxide support. This was supported by all the theoretical characterizations. First, classical reactive molecular dynamics (MD) simulations based on the ReaxFF approach were used to reproduce the adsorption of the experimental glycine solution droplets sprayed onto an oxide support and to identify the most probable arrangement of the molecules that triggered the polymerization mechanisms. Then, quantum chemistry density functional tight binding (DF-TB) MDs and static density functional theory (DFT) calculations were carried out to further explore favorable configurations and to evaluate the energy barriers of the most promising reaction pathways for the peptide bond-formation reactions. The results confirmed the fundamental role played by the substrate to thermodynamically and kinetically favor the process and disclosed its main function as an immobilizing agent: the molecules accommodated in the surface channels close to each other were the ones starting the key events of the dimerization process and the most favorable mechanism was the one where a water molecule acted as a proton exchange mediator in the condensation process.

Size-Dependent Affinity of Glycine and Its Short Oligomers to Pyrite Surface: A Model for Prebiotic Accumulation of Amino Acid Oligomers on a Mineral Surface.

The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on mineral surfaces, especially that of pyrite, has been proposed as an important step in many origins of life scenarios. To investigate such notions, we used AFM-based force measurements to probe possible non-covalent interactions between pyrite and amino acids, starting from the simplest amino acid, Gly. Although Gly itself interacted with the pyrite surface only weakly, progressively larger unbinding forces and binding frequencies were obtained using oligomers from di-Gly to penta-Gly. In addition to an expected increase of the configurational entropy and size-dependent van der Waals force, the increasing number of polar peptide bonds, among others, may be responsible for this observation. The effect of chain length was also investigated by performing similar experiments using l-lysine vs. poly-l-lysine (PLL), and l-glutamic acid vs. poly-l-glutamic acid. The results suggest that longer oligomers/polymers of amino acids can be preferentially adsorbed on pyrite surfaces.

Enhanced optical magnetism for reversed optical binding forces between silicon nanoparticles in the visible region.

We perform a comprehensive numerical analysis on the optical binding forces of a multiple-resonant silicon nanodimer induced by the normal illumination of a plane wave in the visible region. The silicon nanodimer provides either repulsive or attractive forces in water while providing only attractive forces in air. The enhancement of the magnetic dipole mode is attributed to the generation of repulsive forces. The sign (attractive/repulsive) and the amplitude of the optical forces are controlled by incident polarization and separation distance between the silicon nanoparticles. These optomechanical effects demonstrate a key step toward the optical sorting and assembly of silicon nanoparticles.

Fano resonant all-dielectric core/shell nanoparticles with ultrahigh scattering directionality in the visible region.

We demonstrate Si-based single core/shell (Si/SiO2) nanoparticles which exhibit the Fano resonance associated with ultrahigh scattering directionality. The SiO2 shell plays a crucial role in achieving zero backscattering at the Fano resonance wavelength along with strongly-enhanced forward scattering. As a result, the front-to-back scattering-intensity ratio is five orders of magnitude greater than that of a Si nanoparticle. Furthermore, the Fano resonance wavelength is controlled over the entire visible region by changing the core diameter. The Fano spectra also show distinctive intensity modulations depending on the index of refraction of the surrounding medium. These unique features make Si/SiO2 nanoparticles promising for the design of low-loss nano-antennas, metamaterials, and other nanophotonic devices.

Tip-enhanced THz Raman spectroscopy for local temperature determination at the nanoscale.

Local temperature of a nanoscale volume is precisely determined by tip-enhanced terahertz Raman spectroscopy in the low temperature range of several tens of degrees. Heat generated by the tip-enhanced electric field is directly transferred to single-walled carbon nanotubes by heat conduction and radiation at the nanoscale. This heating modulates the intensity ratio of anti-Stokes/Stokes Raman scattering of the radial breathing mode of the carbon nanotube based on the Boltzmann distribution at elevated temperatures. Owing to the low-energy feature of the radial breathing mode, the local temperature of the probing volume has been successfully extracted with high sensitivity. The dependence of the temperature rise underneath the tip apex on the incident power coincides with the analytical results calculated by finite element method based on the tip enhancement effect and the consequent steady-state temperature via Joule heat generation. The results show that the local temperature at the nanoscale can be controlled in the low temperature range simply by the incident laser power while exhibiting a sufficiently high tip enhancement effect as an analytical tool for thermally sensitive materials (e.g., proteins, DNA). Graphical Abstract Tip-enhanced THz Raman spectroscopy detects the low frequency Raman mode both in Stokes and anti-Stokes shifts, which precisely reflects the local temperature of the sample volume.

Fabrication of Mie-resonant silicon nanoparticles using laser annealing for surface-enhanced fluorescence spectroscopy.

Silicon nanostructures with unique Mie resonances have garnered considerable attention in the field of nanophotonics. Here, we present a simple and efficient method for the fabrication of silicon (Si) nanoparticle substrates using continuous-wave (CW) laser annealing. The resulting silicon nanoparticles exhibit Mie resonances in the visible region, and their resonant wavelengths can be precisely controlled. Notably, laser-annealed silicon nanoparticle substrates show a 60-fold enhancement in fluorescence. This tunable and fluorescence-enhancing silicon nanoparticle platform has tremendous potential for highly sensitive fluorescence sensing and biomedical imaging applications.

Metal nanoparticles for nano-imaging and nano-analysis.

Metal nanoparticles have recently emerged as ubiquitous surface-enhanced Raman scattering (SERS) agents for nano-imaging and nano-analysis. These applications make use of the unique optical properties of metal nanoparticles to enhance the efficiency of Raman scattering, whereas the small size of the nanoparticles localizes the enhanced Raman scattering at the nanoscale. In this perspective, we review the recent progress in SERS nano-imaging and nano-spectroscopy using metal nanoparticles applied especially to biological samples and nanomaterials. For biosamples, the highly sensitive molecular SERS detection capability of metal nanoparticles is used to analyze intracellular molecules. For analysis of nanomaterials, an innovative approach called tip-enhanced Raman scattering (TERS) microscopy, where metal nanoparticles are used with an AFM cantilever, has proven to be extremely powerful for nano-imaging. The precise control and analysis of the interaction between the metal and the sample are thus the key to overcome the limitations of current microscopic and spectroscopic techniques, and to guide future developments.

Real-time hybrid angular-interrogation surface plasmon resonance sensor in the near-infrared region for wide dynamic range refractive index sensing.

Herein, we integrated angle-scanning surface plasmon resonance (SPR) and angle-fixed SPR as a hybrid angular-interrogation SPR to enhance the sensing performance. Galvanometer-mirror-based beam angle scanning achieves a 100-Hz acquisition rate of both the angular SPR reflectance spectrum and the angle-fixed SPR reflectance, whereas the use of near-infrared light enhances the refractive index (RI) sensitivity, range, and precision compared with visible light. Simultaneous measurement of the angular SPR reflectance spectrum and angle-fixed SPR reflectance boosts the RI change range, RI resolution, and RI accuracy to 10-1-10-6 RIU, 2.24 × 10-6 RIU, and 5.22 × 10-6 RIU, respectively. The proposed hybrid SPR is a powerful tool for wide-dynamic-range RI sensing with various applications.

Rapid, high-sensitivity detection of biomolecules using dual-comb biosensing.

Rapid, sensitive detection of biomolecules is important for biosensing of infectious pathogens as well as biomarkers and pollutants. For example, biosensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still strongly required for the fight against coronavirus disease 2019 (COVID-19) pandemic. Here, we aim to achieve the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein antigen by enhancing the performance of optical biosensing based on optical frequency combs (OFC). The virus-concentration-dependent optical spectrum shift produced by antigen-antibody interactions is transformed into a photonic radio-frequency (RF) shift by a frequency conversion between the optical and RF regions in the OFC, facilitating rapid and sensitive detection with well-established electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation with a dual-comb configuration enables the very small change in the virus-concentration-dependent signal to be extracted from the large, variable background signal caused by temperature disturbance. The achieved performance of dual-comb biosensing will greatly enhance the applicability of biosensors to viruses, biomarkers, environmental hormones, and so on.

Subnanometric stabilization of plasmon-enhanced optical microscopy.

We have demonstrated subnanometric stabilization of tip-enhanced optical microscopy under ambient condition. Time-dependent thermal drift of a plasmonic metallic tip was optically sensed at subnanometer scale, and was compensated in real-time. In addition, mechanically induced displacement of the tip, which usually occurs when the amount of tip-applied force varies, was also compensated in situ. The stabilization of tip-enhanced optical microscopy enables us to perform long-time and robust measurement without any degradation of optical signal, resulting in true nanometric optical imaging with high reproducibility and high precision. The technique presented is applicable for AFM-based nanoindentation with subnanometric precision.

High-sensitivity hyperspectral vibrational imaging of heart tissues by mid-infrared photothermal microscopy.

Visualizing the spatial distribution of chemical compositions in biological tissues is of great importance to study fundamental biological processes and origin of diseases. Raman microscopy, one of the label-free vibrational imaging techniques, has been employed for chemical characterization of tissues. However, the low sensitivity of Raman spectroscopy often requires a long acquisition time of Raman measurement or a high laser power, or both, which prevents one from investigating large-area tissues in a nondestructive manner. In this work, we demonstrated chemical imaging of heart tissues using mid-infrared photothermal (MIP) microscopy that simultaneously achieves the high sensitivity benefited from IR absorption of molecules and the high spatial resolution down to a few micrometers. We successfully visualized the distributions of different biomolecules, including proteins, phosphate-including proteins, and lipids/carbohydrates/amino acids. Further, we experimentally compared MIP microscopy with Raman microscopy to evaluate the sensitivity and photodamage to tissues. We proved that MIP microscopy is a highly sensitive technique for obtaining vibrational information of molecules in a broad fingerprint region, thereby it could be employed for biological and diagnostic applications, such as live-tissue imaging.

Ultrastable tip-enhanced hyperspectral optical nanoimaging for defect analysis of large-sized WS2 layers.

Optical nanoimaging techniques, such as tip-enhanced Raman spectroscopy (TERS), are nowadays indispensable for chemical and optical characterization in the entire field of nanotechnology and have been extensively used for various applications, such as visualization of nanoscale defects in two-dimensional (2D) materials. However, it is still challenging to investigate micrometer-sized sample with nanoscale spatial resolution because of severe limitation of measurement time due to drift of the experimental system. Here, we achieved long-duration TERS imaging of a micrometer-sized WS2 sample for 6 hours in a reproducible manner. Our ultrastable TERS system enabled to reveal the defect density on the surface of tungsten disulfide layers in large area equivalent to the device scale. It also helped us to detect rare defect-related optical signals from the sample. The present study paves ways to evaluate nanoscale defects of 2D materials in large area and to unveil remarkable optical and chemical properties of large-sized nanostructured materials.

Highly Stable Polymer Coating on Silver Nanoparticles for Efficient Plasmonic Enhancement of Fluorescence.

Surface coating of plasmonic nanoparticles is of huge importance to suppress fluorescence quenching in plasmon-enhanced fluorescence sensing. Herein, a one-pot method for synthesizing polymer-coated silver nanoparticles was developed using a functional polymer conjugated with disulfide-containing anchoring groups. The disulfides played a crucial role in covalently bonding polymers to the surface of the silver nanoparticles. The covalent bond enabled the polymer layer to form a long-term stable coating on the silver nanoparticles. The polymer layer coated was adequately thin to efficiently achieve plasmonic enhancement of fluorescence and also thick enough to effectively suppress quenching of fluorescence, achieving a huge net enhancement of fluorescence. The polymer-coated plasmonic nanoparticles are a promising platform for demonstrating highly sensitive biosensing for medical diagnostics.

Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes.

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

Unbinding events of amino acids and peptides from water-pyrite interfaces: A case study of life's origin on mineral surfaces.

Selective binding of aqueous-phase amino acids to mineral surfaces is regarded as a plausible first step in oligopeptide formation on early Earth. To clarify the strength and underlying mechanism of amino acid binding to pyrite surfaces, we measured the unbinding (pull-off) force of ten amino acids and two oligo-peptides from water-pyrite interfaces using atomic force microscopy (AFM). The most probable unbinding force could be described by a linearly increasing function with the size of the amino acid and a characteristic offset. A good correlation was obtained between the most probable unbinding force and the residue volume, surface area and polarizability of samples suggesting at least a partial contribution of van der Waals (vdW) forces, especially the London dispersion force. These results are useful in analysis of adhesion phenomena of amino acids in the given environmental settings such as in this work.

Surface force analysis of glycine adsorption on different crystal surfaces of titanium dioxide (TiO2).

Surface force analysis with atomic force microscope (AFM) in which a single amino acid residue was mounted on the tip apex of AFM probe was carried out for the first time at the molecular level on titanium dioxide (TiO2) as a representative mineral surface for prebiotic chemical evolution reactions. The force analyses on surfaces with three different crystal orientations revealed that the TiO2 (110) surface has unique characteristics for adsorbing glycine molecules showing different features compared to those on TiO2 (001) and (100). To examine this difference, we investigated thermal desorption spectroscopy (TDS) and the interaction between the PEG cross-linker and the three TiO2 surfaces. Our data suggest that the different single crystal surfaces would provide different chemical evolution field for amino acid molecules.