Nanoscale Optical Imaging Research Articles

Nanoscale Optical Imaging, Reconstruction, and Spatial Analysis of Whole Mouse Glomeruli.

Renal glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate the ultrastructure of thin sections. While these approaches are powerful, they have been limited in their ability to obtain detailed views of the glomeruli as holistic 3D functional units. To fill this knowledge gap, we have developed a novel pipeline for imaging, reconstructing, and analyzing whole mouse glomeruli at 100 nm resolution using super-resolution fluorescence microscopy. This pipeline integrates both manual and machine learning approaches to annotate and analyze glomerular structures. Using this method, we created 18 detailed glomerulus models, from a range of healthy, aged, and model diseased mice, that outline all major structures and cell types. These models have been made publicly accessible in an online repository, providing a valuable resource for further studies. Our results also uncovered a diverse set of novel phenotypes including nuclear enlargement in all glomerular cell types in aging and disease, as well as an aging-related pattern of regional thickening of the Bowman's capsule basement membrane near the tubular-glomerular junction.

Nanoscale Optical Conductivity Imaging of Double-Moiré Twisted Bilayer Graphene.

A central paradigm of moiré materials relies on the formation of superlattices that yield enlarged effective crystal unit cells. While a critical consequence of this phenomenon is the celebrated flat electronic bands that foster strong interaction effects, the presence of superlattices has further implications. Here we explore the advantages of moiré superlattices in twisted bilayer graphene (TBG) aligned with hexagonal boron nitride (hBN) for passively enhancing optical conductivity in the low-energy regime. To probe the local optical response of TBG/hBN double-moiré lattices, we use infrared (IR) nano-imaging in conjunction with nanocurrent imaging to examine local optical conductivity over a wide range of TBG twist angles. We show that interband transitions associated with the multiple moiré flat and dispersive bands produce tunable transparent IR responses even at finite carrier densities, which is in stark contrast to the previously limited metallic near transparency observed only in undoped pristine graphene.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Expansion Microscopy-based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells.

Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

Nanoscale Optical Imaging of 2D Semiconductor Stacking Orders by Exciton‐Enhanced Second Harmonic Generation

AbstractSecond harmonic generation (SHG) is a nonlinear optical response arising exclusively from broken inversion symmetry in the electric‐dipole limit. Recently, SHG has attracted widespread interest as a versatile and noninvasive tool for characterization of crystal symmetry and emerging ferroic or topological orders in quantum materials. However, conventional far‐field optics is unable to probe local symmetry at the deep subwavelength scale. Here, near‐field SHG imaging of 2D semiconductors and heterostructures with the spatial resolution down to 20 nm is demonstrated using a scattering‐type nano‐optical apparatus. It is shown that near‐field SHG efficiency is greatly enhanced by excitons in atomically thin transition metal dichalcogenides. Furthermore, by correlating nonlinear and linear scattering‐type nano‐imaging, nanoscale variations of interlayer stacking order in bilayer WSe2 are resolved, and the stacking‐tuned excitonic light–matter interactions are revealed. This work demonstrates nonlinear optical interrogation of crystal symmetry and structure–property relationships at the nanometer length scales relevant to emerging properties in quantum materials.

Optical Trapping, Sensing, and Imaging by Photonic Nanojets

The optical trapping, sensing, and imaging of nanostructures and biological samples are research hotspots in the fields of biomedicine and nanophotonics. However, because of the diffraction limit of light, traditional optical tweezers and microscopy are difficult to use to trap and observe objects smaller than 200 nm. Near-field scanning probes, metamaterial superlenses, and photonic crystals have been designed to overcome the diffraction limit, and thus are used for nanoscale optical trapping, sensing, and imaging. Additionally, photonic nanojets that are simply generated by dielectric microspheres can break the diffraction limit and enhance optical forces, detection signals, and imaging resolution. In this review, we summarize the current types of microsphere lenses, as well as their principles and applications in nano-optical trapping, signal enhancement, and super-resolution imaging, with particular attention paid to research progress in photonic nanojets for the trapping, sensing, and imaging of biological cells and tissues.

Near-field optical imaging and spectroscopy of 2D-TMDs

Abstract Two-dimensional transition metal dichalcogenides (2D-TMDs) are atomically thin semiconductors with a direct bandgap in monolayer thickness, providing ideal platforms for the development of exciton-based optoelectronic devices. Extensive studies on the spectral characteristics of exciton emission have been performed, but spatially resolved optical studies of 2D-TMDs are also critically important because of large variations in the spatial profiles of exciton emissions due to local defects and charge distributions that are intrinsically nonuniform. Because the spatial resolution of conventional optical microscopy and spectroscopy is fundamentally limited by diffraction, near-field optical imaging using apertured or metallic probes has been used to spectrally map the nanoscale profiles of exciton emissions and to study the effects of nanosize local defects and carrier distribution. While these unique approaches have been frequently used, revealing information on the exciton dynamics of 2D-TMDs that is not normally accessible by conventional far-field spectroscopy, a dedicated review of near-field imaging and spectroscopy studies on 2D-TMDs is not available. This review is intended to provide an overview of the current status of near-field optical research on 2D-TMDs and the future direction with regard to developing nanoscale optical imaging and spectroscopy to investigate the exciton characteristics of 2D-TMDs.

Waveguide-Based Platform for Large-FOV Imaging of Optically Active Defects in 2D Materials

Single-molecule localization microscopy (SMLM) is a powerful tool that is routinely used for nanoscale optical imaging of biological samples. Recently, this approach has been applied to study optic...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale optical imaging of perovskite nanocrystals directly embedded in polymer fiber

A polymer fiber film embedded with light-emitting inorganic perovskite nanocrystals (IPNCs) was prepared by multi-nozzle electrospinning of the solutions containing the inorganic perovskite (IP) precursors (CsX and PbX2) and polystyrene (PS). This is a simple and versatile method for direct confinement of nano-sized functional materials inside continuous fibers. The PL emission color of the hybrid fibers can be modulated using combinations of red (R), green (G), and blue (B) precursor solutions in one-step multi-nozzle electrospinning, leading to seven colors: R, G, B, cyan, yellow, magenta, and white. Intriguingly, nanoscale optical spectral imaging of fibers revealed the existence of local emission centers with different emission wavelengths in R and B emission fibers due to rapid formation of IPNCs with mixed halogen contents. These PL imaging results provide a fundamental understanding of the optical properties of the directly formed IPNCs in the polymer matrix.

Fabrication and physical measurement accuracy of nanoscale standard samples for vector beams confocal laser scanning microscopy

Various kinds of super-resolution optical microscope techniques have been developed to break the diffraction barrier in the past decades. Confocal laser scanning microscopy is the super-resolution microscopy. It is widely used due to high resolution and depth selectivity in obtaining images. However, there are neither accurate nor rigorous measurement methods with a nanoscale resolution. In order to measure the resolution of vector beam confocal laser scanning microscopy accurately and rigorously, a nanoscale resolution standard sample is proposed and experimentally realized. This sample is composed of a series of accurate measure patterns and a couple of arrays of triangle finding structures. It allows a wide measurement range between 40 nm to 1000 nm, and provides appropriate measurement steps and high measurement accuracy. The measurement patterns can be efficiently figured out by using the found structures, and their structure line width can be easily calculated. The first standard sample is produced on a piece of amorphous silicon by electron beam lithography and inductive coupled plasma etching technology, and measured by the scanning electron microscopy. According to the test, the sample meets the requirements of accuracy for nanoscale resolution measurement. Optical testing is applied to the sample by a vector beam confocal laser scanning microscope. And the sample shows that the resolution is 96 nm (oil immersion, refractive index 1.52) under the irradiation of 405 nm radially polarized beams, which is far beyond the diffraction barrier. Furthermore, a metal structure standard sample, which is based on a piece of indium tin oxide glass, is produced to improve the signal contrast ratio of the silicon standard sample. The measurement patterns are fabricated by electron beam lithography and electron beam evaporation and made of 10 nm titanium and 100 nm gold. It works for both reflective and transmissive confocal laser scanning microscopy, and would obtain high resolution images with a better contrast ratio. These standard samples are able to test the performance of microscope system efficiently, and provide a more rigorous way to make sub-100 nm resolution measurement and a calibration guidance for point scanning super-resolution microscope. In the meantime, we find that nanoscale opticalimaging is affected not only by sample morphology, but also by the photoelectron property of the sample. Further study is required to understand the underlying mechanism.

Nanoscale Imaging of Kidney Glomeruli Using Expansion Pathology.

Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.

Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy

Although light microscopy is a powerful tool for the assessment of kidney physiology and pathology, it has traditionally been unable to resolve structures separated by less than the ~250 nm diffraction limit of visible light. Here, we report on the optimization, validation, and application of a recently developed super-resolution fluorescence microscopy method, called expansion microscopy (ExM), for volumetric interrogation of mouse and human kidney tissue with 70–75 nm lateral and ~250 nm axial spatial resolution. Using ExM with a standard confocal microscope, we resolve fine details of structures that have traditionally required visualization by electron microscopy, including podocyte foot processes, the glomerular basement membrane, and the cytoskeleton. This inexpensive and accessible approach to volumetric, nanoscale imaging enables visualization of fine structural details of kidney tissues that were previously difficult or impossible to measure by conventional methodologies.

Silver nanowires for highly reproducible cantilever based AFM-TERS microscopy: towards a universal TERS probe.

Tip-enhanced Raman scattering (TERS) microscopy is a unique analytical tool to provide complementary chemical and topographic information of surfaces with nanometric resolution. However, difficulties in reliably producing the necessary metallized scanning probe tips has limited its widespread utilisation, particularly in the case of cantilever-based atomic force microscopy. Attempts to alleviate tip related issues using colloidal or bottom-up engineered tips have so far not reported consistent probes for both Raman and topographic imaging. Here we demonstrate the reproducible fabrication of cantilever-based high-performance TERS probes for both topographic and Raman measurements, based on an approach that utilises noble metal nanowires as the active TERS probe. The tips show 10 times higher TERS contrasts than the most typically used electrochemically-etched tips, and show a reproducibility for TERS greater than 90%, far greater than found with standard methods. We show that TERS can be performed in tapping as well as contact AFM mode, with optical resolutions around or below 15 nm, and with a maximum resolution achieved in tapping-mode of 6 nm. Our work illustrates that superior TERS probes can be produced in a fast and cost-effective manner using simple wet-chemistry methods, leading to reliable and reproducible high-resolution and high-sensitivity TERS, and thus renders the technique applicable for a broad community.

Nanoscale optical imaging of lithium-ion distribution on a LiCoO2 cathode surface

Nanoscale analysis of the local lithium-ion concentration in lithium-ion battery cathode materials is essential for achieving high-performance batteries. We developed a near-field optical technique to visualize the lithium-ion distribution in a lithium-ion battery cathode on a nanometer scale. A plasmonic metallic tip was employed to locally enhance and probe the electric field in close proximity to the LiCoO2 thin film, since it is correlated with the lithium-ion concentration at the surface. We also utilized this technique to map the electrochemically induced ion diffusion at the grain boundaries under the application of a DC voltage to the surface through the plasmonic metallic tip.

Enhancement of light-matter interaction and photocatalytic efficiency of Au/TiO2 hybrid nanowires.

Metal/TiO2 hybrid nanostructures offer more efficient charge separation and a broader range of working wavelengths for photocatalytic reactions. The sizes and shapes of such hybrid nanostructures can affect the charge separation performance when the structures interact with light, but assessments of the interaction of light with these metal-TiO2 nanostructures have only been carried out on ensemble averages, hindering both systematic descriptions of such hybrid structures and the design of new ones. Here, we fabricated TiO2 nanotubes (NTs) with and without core Au nanowires (NWs), and used spectroscopy and calculations to assess their scattering and absorption of light at the single NW level. According to the results of spectral imaging and numerical calculations, the Au/TiO2 NWs scattered and absorbed light substantially more strongly than did the plain TiO2 NTs. Measurements of the degradation of the AO7 dye to assess the photocatalytic performance of the Au/TiO2 NWs were consistent with optical measurements demonstrating a two-fold improvement over plain TiO2 NTs under 360-nm-wavelength UV illumination. Our results suggests that nanoscale optical imaging can be used to visualize the performance of the photocatalytic reaction at the single nano-object level.

Optical imaging beyond the diffraction limit by SNEM: effects of AFM tip modifications with thiol monolayers on imaging quality.

Tip-enhanced nanoscale optical imaging techniques such as apertureless scanning near-field optical microscopy (a-SNOM) and scanning near-field ellipsometric microscopy (SNEM) applications can suffer from a steady degradation in performance due to adhesion of atmospheric contaminants to the metal coated tip. Here, we demonstrate that a self-assembled monolayer (SAM) of ethanethiol (EtSH) is an effective means of protecting gold-coated atomic force microscopy (AFM) probe tips from accumulation of surface contaminants during prolonged exposure to ambient air. The period over which they yield consistent and reproducible results for scanning near-field ellipsometric microscopy (SNEM) imaging is thus extended. SNEM optical images of a microphase separated polystyrene-block-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer film, which were captured with bare and SAM-protected gold-coated AFM probes, both immediately after coating and following five days of storage in ambient air, were compared. During this period the intensity of the optical signals from the untreated gold tip fell by 66%, while those from the SAM protected tip fell by 14%. Additionally, gold coated AFM probe tips were modified with various lengths of alkanethiols to measure the change in intensity variation in the optical images with SAM layer thickness. The experimental results were compared to point dipole model calculations. While a SAM of 1-dodecanethiol (DoSH) was found to strongly suppress field enhancement we find that it can be locally removed from the tip apex by deforming the molecules under load, restoring SNEM image contrast.

Nanoscope based on nanowaveguides

The far field spatial resolution of conventional optical lenses is of the order of the wavelength of light, due to loss in the far field of evanescent, near electromagnetic field components. We show that subwavelength details can be restored in the far field with an array of divergent nanowaveguides, which map the discretized, subwavelength image of an object into a magnified image observable with a conventional optical microscope. We demonstrate in simulations that metallic nanowires, nanocoaxes, and nanogrooves can be used as such nanowaveguides. Thus, an optical microscope capable of subwavelength resolution - a nanoscope - can be produced, with possible applications in a variety of fields where nanoscale optical imaging is of value.

Nanoscale Live Cell Optical Imaging of the Dynamics of Intracellular Microvesicles in Neural Cells

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Nanoscale optical imaging by atomic force infrared microscopy

This review outlines progress in atomic force infrared microscopy, reviewing the methodology and its application in nanoscale infrared absorption imaging of both biological and functional materials, including an outline of where this emerging method has been applied to image cellular systems in aqueous environments.