Imaging vs Nonimaging Raman Spectroscopy for High-Throughput Single-Cell Phenotyping.

Raman spectroscopy can provide nonbiased single-cell analysis based on the endogenous ensemble of biomolecules, with alterations in cellular content indicative of cell state and disease. The measurements themselves can be performed in a variety of modes: generally, full imaging takes the most time but can provide the most information. By reducing the imaging resolution and generating the most characteristic single-cell Raman spectrum in the shortest time, we optimize the utility of the Raman measurement for cell phenotyping. Here, we establish methods to compare these different measurement approaches and assess what, if any, undesired effects occur in the cell. Assuming that laser-induced damage should be apparent as a change in molecular spectra across sequential measurements, and by defining the information content as the Raman-based separability of two cell lines, we thereby establish a parameter range for optimum measurement sensitivity and single-cell throughput in single-cell Raman spectroscopic analysis. While the work here uses 532 nm irradiation, the same approach can be generalized to Raman analysis at other wavelengths.

In Situ Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope.

High-speed atomic force microscopy (HS-AFM) is an indispensable technique in the field of biology owing to its imaging capability with high spatiotemporal resolution. Furthermore, recent developments established tip-scan stand-alone HS-AFM combined with an optical microscope, drastically improving its versatility. It has considerable potential to contribute to not only biology but also various research fields. A great candidate is a photoactive material, such as an azo-polymer, which is important for optical applications because of its unique nanoscale motion under light irradiation. Here, we demonstrate the in situ observation of nanoscale azo-polymer motion by combining tip-scan HS-AFM with an optical system, allowing HS-AFM observations precisely aligned with a focused laser position. We observed the dynamic evolution of unique morphologies in azo-polymer films. Moreover, real-time topographic line profile analyses facilitated precise investigations of the morphological changes. This important demonstration would pave the way for the application of HS-AFM in a wide range of research fields.

Near-field optical microscopy toward its applications for biological studies.

Near-field scanning optical microscopy (NSOM) is a super-resolution optical microscopy based on nanometrically small near-field light at a metallic tip. It can be combined with various types of optical measurement techniques, including Raman spectroscopy, infrared absorption spectroscopy, and photoluminescence measurements, which provides unique analytical capabilities to a variety of scientific fields. In particular, to understand nanoscale details of advance materials and physical phenomena, NSOM has been often adopted in the fields of material science and physical chemistry. However, owing to the recent critical developments showing the great potential for biological studies, NSOM has also recently gained much attention in the biological field. In this article, we introduce recent developments made in NSOM, aiming at biological applications. The drastic improvement in the imaging speed has shown a promising application of NSOM for super-resolution optical observation of biological dynamics. Furthermore, stable imaging and broadband imaging were made possible owing to the advanced technologies, which provide a unique imaging method to the biological field. As NSOM has not been well exploited in biological studies to date, several rooms need to be explored to determine its distinct advantages. We discuss the possibility and perspective of NSOM for biological applications. This review article is an extended version of the Japanese article, Development of Near-field Scanning Optical Microscopy toward Its Application for Biological Studies, published in SEIBUTSU BUTSURI Vol. 62, p. 128-130 (2022).

Raman Spectroscopic and DFT Study of COA-Cl and Its Analogues.

COA-Cl is a newly synthesized adenosine analogue that exhibits various physiological activities. Its angiogenic, neurotropic, and neuroprotective potencies make it promising for the development of medicines. In this study, we show Raman spectroscopic study of COA-Cl to elucidate molecular vibrations and related chemical properties. Density functional theory calculations were combined with the Raman spectroscopic data to understand the details of each vibrational mode. Comparative analysis with adenine, adenosine, and other nucleic acid analogues enabled identification of unique Raman peaks originating from the cyclobutane moiety and chloro group of COA-Cl. This study provides fundamental knowledge and crucial insights for further development of COA-Cl and related chemical species.

Tip-enhanced Raman spectroscopy with amplitude-controlled tapping-mode AFM.

Tip-enhanced Raman spectroscopy (TERS) is a powerful tool for analyzing chemical compositions at the nanoscale owing to near-field light localized at a metallic tip. In TERS, atomic force microscopy (AFM) is commonly used for tip position control. AFM is often controlled under the contact mode for TERS, whereas the tapping mode, which is another major operation mode, has not often been employed despite several advantages, such as low sample damage. One of the reasons is the low TERS signal intensity because the tip is mostly away from the sample during the tapping motion. In this study, we quantitatively investigated the effect of the tapping amplitude on the TERS signal. We numerically evaluated the dependence of the TERS signal on tapping amplitude. We found that the tapping amplitude had a significant effect on the TERS signal, and an acceptable level of TERS signal was obtained by reducing the amplitude to a few nanometers. We further demonstrated amplitude-controlled tapping-mode TERS measurement. We observed a strong dependence of the TERS intensity on the tapping amplitude, which is in agreement with our numerical calculations. This practical but essential study encourages the use of the tapping mode for further advancing TERS and related optical techniques.

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.

Polarization Raman Imaging of Organic Monolayer Islands for Crystal Orientation Analysis.

An organic semiconductor film made of diphenyl derivative dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophene (DPh-DNTT) has high carrier mobility. However, this mobility may be greatly affected by the crystal orientation of the DPh-DNTT's first layer. Polarization Raman microscopy is widely used to quantitatively analyze the molecular orientation, and thus holds great potential as a powerful tool to investigate the crystal orientation of monolayer DPh-DNTT with high spatial resolution. In this study, we demonstrate polarization Raman imaging of monolayer DPh-DNTT islands for crystal orientation analysis. We found that the DPh-DNTT sample indicated a strong dependence of the Raman intensity on the incident polarization direction. Based on the polarization dependence, we developed an analytical method of determining the crystal orientation of the monolayer DPh-DNTT islands and experimentally confirmed that our technique was highly effective at imaging the islands' crystal orientation with a spatial resolution of a few hundred nanometers.

Label-free Raman mapping of saturated and unsaturated fatty acid uptake, storage, and return toward baseline levels in macrophages.

Macrophage uptake and metabolism of fatty acids is involved in a large number of important biological pathways including immune activation and regulation of macrophages, as well as pathological conditions including obesity, atherosclerosis, and others lifestyle diseases. There are few methods available to directly probe both the uptake and later redistribution/metabolism of fatty acids within living cells as well as the potential changes induced within the cells themselves. We use Raman imaging and analysis to evaluate the effects of different fatty acids following their uptake in macrophages. The label-free nature of the methods means that we can evaluate the fatty acid dynamics without modifying endogenous cellular behavior and metabolism.

Probing stacking configurations in a few layered MoS2 by low frequency Raman spectroscopy.

Novel two-dimensional (2D) layered materials, such as MoS2, have recently gained a significant traction, chiefly due to their tunable electronic and optical properties. A major attribute that affects the tunability is the number of layers in the system. Another important, but often overlooked aspect is the stacking configuration between the layers, which can modify their electro-optic properties through changes in internal symmetries and interlayer interactions. This demands a thorough understanding of interlayer stacking configurations of these materials before they can be used in devices. Here, we investigate the spatial distribution of various stacking configurations and variations in interlayer interactions in few-layered MoS2 flakes probed through the low-frequency Raman spectroscopy, which we establish as a versatile imaging tool for this purpose. Some interesting anomalies in MoS2 layer stacking, which we propose to be caused by defects, wrinkles or twist between the layers, are also reported here. These types of anomalies, which can severely affect the properties of these materials can be detected through low-frequency Raman imaging. Our findings provide useful insights for understanding various structure-dependent properties of 2D materials that could be of great importance for the development of future electro-optic devices, quantum devices and energy harvesting systems.

White nanolight source for optical nanoimaging.

Nanolight sources, which are based on resonant excitation of plasmons near a sharp metallic nanostructure, have attracted tremendous interest in the vast research fields of optical nanoimaging. However, being a resonant phenomenon, this ideally works only for one wavelength that resonates with the plasmons. Multiple wavelengths of light in a broad range confined to one spot within a nanometric volume would be an interesting form of light, useful in numerous applications. Plasmon nanofocusing can generate a nanolight source through the propagation and adiabatic compressions of plasmons on a tapered metallic nanostructure, which is independent of wavelength, as it is based on the propagation, rather than resonance, of plasmons. Here, we report the generation of a white nanolight source spanning over the entire visible range through plasmon nanofocusing and demonstrate spectral bandgap nanoimaging of carbon nanotubes. Our experimental demonstration of the white nanolight source would stimulate diverse research fields toward next-generation nanophotonic technologies.

One-side metal-coated pyramidal cantilever tips for highly reproducible tip-enhanced Raman spectroscopy.

Tip-enhanced Raman spectroscopy (TERS) has been recognized as a useful tool for nanoscale chemical analysis, and it can further reach down to the sub-nanometer scale in the gap-mode configuration. Using an atomic force microscopy (AFM) in gap-mode TERS for position control of a metallic tip, a unique and correlative analysis can be even realized at the single molecule level. However, one of crucial issues in AFM-based gap-mode TERS is the fabrication of reliable and reproducible cantilver metallic tips. Here, we propose a simple, cost-effective fabrication method of metal-coated tips for AFM-based gap-mode TERS by means of the physical vapor deposition technique in a reproducible way. Our plamonic tips have extremely smooth silver layers on one side of the pyramidal tip, which is totally different from the regular metallic tips that hold granular metallic structures randomly arranged on their bodies. Importantly, all fabricated tips exhibited a reasonably high enhancement factor of more than 104, which indicates that the reproducibility of our plasmonic tip is virtually 100% in the gap-mode configuration. The excellent reproducibility of gap-mode TERS measurement holds great promise for rendering AFM-based TERS as a powerful analytical technique in a broad range of fields.

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies.

BACKGROUND: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. METHODS: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. RESULTS: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. CONCLUSIONS: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. GENERAL SIGNIFICANCE: We achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

Orientation analysis of pentacene molecules in organic field-effect transistor devices using polarization-dependent Raman spectroscopy.

Pentacene, an organic molecule, is a promising material for high-performance field effect transistors due to its high charge carrier mobility in comparison to usual semiconductors. However, the charge carrier mobility is strongly dependent on the molecular orientation of pentacene in the active layer of the device, which is hard to investigate using standard techniques in a real device. Raman scattering, on the other hand, is a high-resolution technique that is sensitive to the molecular orientation. In this work, we investigated the orientation distribution of pentacene molecules in actual transistor devices by polarization-dependent Raman spectroscopy and correlated these results with the performance of the device. This study can be utilized to understand the distribution of molecular orientation of pentacene in various electronic devices and thus would help in further improving their performances.

Tapered arrangement of metallic nanorod chains for magnified plasmonic nanoimaging.

Plasmonic nanolens, a 3-dimensional tapered arrangement of metallic nanorod chains, holds a great promise as a new plasmonics-based optical nano-imaging technique. While multiple nanorod chains can transfer the near-field signal originating from a sample to an image at a distance larger than a micro-meter, where each nanorod chain contributes in forming one pixel in the image, the tapered arrangement of the nanorod chains with a certain taper angle allows image magnification. We experimentally demonstrate the feature of image formation and magnification in a nanolens by fabricating a tapered arrangement of two silver nanorod chains, which were separated by a distance smaller than the diffraction limit at one end and larger than the diffraction limit at the other end. We placed two nano-sized optical sources of quantum dots near the first ends of the chains, which served as two subwavelength objects. In the optical measurement, we demonstrated that the unresolved subwavelength optical sources could be imaged at the other ends of the chains and were well resolved in accordance with the magnification feature of a nanolens. This verification is an experimental proof of the image magnification, and an important step toward the realization of plasmonic nanolens.

Raman Spectroscopic Studies of Dinaphthothienothiophene (DNTT).

The application of dinaphthothienothiophene (DNTT) molecules, a novel organic semiconductor material, has recently increased due to its high charge carrier mobility and thermal stability. Since the structural properties of DNTT molecules, such as the molecular density distribution and molecular orientations, significantly affect their charge carrier mobility in organic field-effect transistors devices, investigating these properties would be important. Here, we report Raman spectroscopic studies on DNTT in a transistor device, which was further analyzed by the density functional theory. We also show a perspective of this technique for orientation analysis of DNTT molecules within a transistor device.

Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy.

Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates.

Highly efficient plasmonic tip design for plasmon nanofocusing in near-field optical microscopy.

Near-field scanning optical microscopy (NSOM) combined with plasmon nanofocusing is a powerful nano-analytical tool due to its attractive feature of efficient background suppression as well as light energy compression to the nanoscale. In plasmon nanofocusing-based NSOM, the metallic tip plays an important role in inducing plasmon nanofocusing. It is, however, very challenging to control plasmonic properties of tips for plasmon nanofocusing with existing tip fabrication methods, even though the plasmonic properties need to be adjusted to experimental environments such as the sample or excitation wavelength. In this study, we propose an efficient tip design and fabrication which enable one to actively control plasmonic properties for efficient plasmon nanofocusing. Because our method offers flexibility in the material and structure of tips, one can easily modify the plasmonic properties depending on the requirements. Importantly, through optimization of the plasmonic properties, we achieve almost 100% reproducibility in plasmon nanofocusing in our experiments. This new approach of tip fabrication makes plasmon nanofocusing-based NSOM practical and reliable, and opens doors for many scientists working in related fields.