Micrometer Spatial Resolution Research Articles

First in vivo visualization of the human subarachnoid space and brain cortex via optical coherence tomography.

The present work explores optical coherence tomography (OCT) as a suitable in vivo neuroimaging modality of the subarachnoid space (SAS). Patients (n = 26) with frontolateral craniotomy were recruited. The temporal and frontal arachnoid mater and adjacent anatomical structures were scanned using microscope-integrated three-dimensional OCT, (iOCT). Analysis revealed a detailed depiction of the SAS (76.9%) with delineation of the internal microanatomical structures such as the arachnoid barrier cell membrane (ABCM; 96.2%), trabecular system (50.2%), internal blood vessels (96.2%), pia mater (26.9%) and the brain cortex (96.2%). Orthogonal distance measuring was possible. The SAS showed a mean depth of 570 µm frontotemporal. The ABCM showed a mean depth of 74 µm frontotemporal. These results indicate that OCT provides a dynamic, non-invasive tool for real-time imaging of the SAS and adjacent anatomical structures at micrometer spatial resolution. Further studies are necessary to evaluate the value of OCT during microsurgical procedures.

Single Layer Graphene Assisted Transmission Mode Continuous Wave Laser Desorption for Micrometer Spatial Resolution Atmospheric Pressure Mass Spectrometry Imaging

Single Layer Graphene Assisted Transmission Mode Continuous Wave Laser Desorption for Micrometer Spatial Resolution Atmospheric Pressure Mass Spectrometry Imaging

Potential of GAGG:Ce scintillation crystals for synchrotron X-Ray micro-imaging

We examined the use of cerium-doped gadolinium aluminum gallium garnet (GAGG:Ce) as a material for scintillation screens in synchrotron X-ray radiography (SXR). Highlighted for gamma-ray detection, GAGG:Ce is also an attractive material for SXR because of its high X-ray stopping power and the high light yield at around 530 nm. A thin and transparent screen prepared from our grown ingot of GAGG:Ce was compared with commonly used scintillators of CdWO4 and YAG:Ce in terms of image quality under monochromatic X-ray illumination. Micron spatial resolutions were achieved with comparable image quality, suggesting that a single crystal of GAGG:Ce is competent scintillation material for SXR.

Cell-Nanoparticle Interactions at (Sub)-Nanometer Resolution Analyzed by Electron Microscopy and Correlative Coherent Anti-Stokes Raman Scattering.

A wide variety of nanoparticles are playing an increasingly important role in drug delivery. Label-free imaging techniques are especially desirable to follow the cellular uptake and intracellular fate of nanoparticles. The combined correlative use of different techniques, each with unique advantages, facilitates more detailed investigation about such interactions. The synergistic use of correlative coherent anti-Stokes Raman scattering and electron microscopy (C-CARS-EM) imaging offers label-free, chemically-specific, and (sub)-nanometer spatial resolution for studying nanoparticle uptake into cells as demonstrated in the current study. Coherent anti-Stokes Raman scattering (CARS) microscopy offers chemically-specific (sub)micron spatial resolution imaging without fluorescent labels while transmission electron microscopy (TEM) offers (sub)-nanometer scale spatial resolution and thus visualization of precise nanoparticle localization at the sub-cellular level. This proof-of-concept imaging platform with unlabeled drug nanocrystals and macrophage cells revealed good colocalization between the CARS signal and electron dense nanocrystals in TEM images. The correlative TEM images revealed subcellular localization of nanocrystals inside membrane bound vesicles, showing multivesicular body (MVB)-like morphology typical for late endosomes (LEs), endolysosomes, and phagolysosomes. C-CARS-EM imaging has much potential to study the interactions between a wide range of nanoparticles and cells with high precision and confidence.

Measurement of water vapor diffusion in nanoscale polymer films by frequency-domain probe beam deflection.

We developed an optical pump-probe technique, frequency-domain probe beam deflection (FD-PBD) to measure the diffusion of water vapor in nanoscale polymer films with microsecond temporal resolution and micrometer spatial resolution. We describe the quantitative model of beam deflection for multilayer structures, the experimental approach, and the application of FD-PBD to measure the diffusivity (D m) of saturated water vapor in various glassy polymer films including polyimide (PI, D m = 7.0 × 10-13 m2 s-1), poly(methyl methacrylate) (PMMA, D m = 1.2 × 10-12 m2 s-1), poly-(vinylpyrrolidone) (PVP, D m = 1.7 × 10-12 m2 s-1), and cellulose acetate (CA, D m = 2.6 × 10-11 m2 s-1), and the piperazine/trimesoyl chloride (PIP/TMC, D m = 9 × 10-11 m2 s-1) nanofiltration membrane synthesized by interfacial polymerization. The uncertainty of the measurements is typically ≈8%.

Atmospheric discharge plasma in aqueous solution: Importance of the generation of water vapor bubbles for plasma onset and physicochemical evolution

Discharge plasma formed in aqueous solutions has attracted much attention for its applications in environmental purification and material syntheses. The onset and evolution of the discharge plasma in an aqueous solution and transient reactive species formed in it are successfully monitored with micrometer spatial resolution and nanosecond temporal resolution. The combination of a custom-made microscopic discharge system and a high-speed camera provides direct evidence that water vapor bubbles form before the discharge with the thermal phase transition of aqueous solution at the electrode tip. The water vapor bubbles, i.e., locally formed space in the gas phase, connect the gap between the tips of the opposed electrodes. The local gas area formed in aqueous solution plays a crucial role in the ignition and continuance of the discharge plasma. It is also found that the initially formed plasma lasts for under 100 ns and quenches rapidly. However, plasma regenerates in the water vapor bubble and successively bridges the opposing electrodes during the pulsed-voltage application (ca. 1 μs). These two temporally distinct generations of plasma, i.e., the initial plasma (IP) and the following successive plasma (SP), can be seen to correspond to the dielectric breakdown and glow-like plasma, respectively. These results provide an important picture for the proposed mechanism for plasma evolution in water and also important information for the efficient control of the discharge plasma with its applications in waste-water treatments, nanomaterial syntheses with plasma oxidation–reduction reactions, and the chemical modification of the material surfaces in aqueous solutions as a form of “green chemistry.”

Multiphoton Microscopy of π-Conjugated Copolymers and Copolymer/Fullerene Blends for Organic Photovoltaic Applications

Organic photovoltaic (OPV) cells based on π-conjugated copolymer/fullerene blends are devices with the highest power conversion efficiencies within the class of organic semiconductors. Although a number of image microscopies have been applied to films of π-conjugated copolymers and their fullerene blends, seldom have they been able to detect microscopic defects in the blend films. We have applied multiphoton microscopy (MPM) using a 65 fs laser at 1.56 μm for spectroscopy and mapping of films of various π-conjugated copolymers and their fullerene blends. All pristine copolymer films have shown third harmonic generation (THG) and two-photon or three-photon photoluminescence that could be used for mapping the films with micrometer spatial resolution. Since the fullerenes have much weaker THG efficiency than those of the copolymers, we could readily map the copolymer/fullerene blend films that showed interpenetrating micron-sized grains of the two constituents. In addition, we also found second harmonic generation from various micron-sized defects in the films that are formed during film deposition or light illumination at ambient conditions, which do not possess inversion symmetry. The MPM method is therefore beneficial for organic films and devices for investigating the properties and growth of copolymer/fullerene blends for OPV applications.

IN SITU visualization of water adsorption in cellulose nanofiber film with micrometer spatial resolution using micro-FTIR imaging

Hygroscopic behavior is an inherent characteristic of nanocellulose film which strongly affects its applications. In order to gain a better understanding of water adsorption, micro-Fourier transform infrared (FTIR) imaging was used to investigate the water adsorption in cellulose nanofiber film with a spatial resolution of 20 um. Four spectral peaks at 2905 cm−1, 1428 cm−1, 1371 cm−1, and 1317 cm−1 attributed to CH and CH2 groups were used to generate 2D micro-FTIR images of cellulose distribution, and the most intense peak at 3348 cm−1 was employed to generate 2D micro-FTIR image of OH group distribution. On this basis, difference 2D micro-FTIR images of OH group distribution at different relative humidity (RH) levels demonstrated the development of adsorbed water distribution in cellulose nanofiber film during the water adsorption process. The study results confirmed that the micro-FTIR imaging was one promising tool for in situ visualization of water adsorption with micron-scale resolution.

Analysis of the Myc-induced pancreatic β cell islet tumor microenvironment using imaging ToF-SIMS.

Solid tumors are a structurally complex system, composed of many different cell types. The tumor microenvironment includes nonmalignant cell types that participate in complex interactions with tumor cells. The cross talk between tumor and normal cells is implicated in regulating cell growth, metastatic potential, and chemotherapeutic drug resistance. A new approach is required to interrogate and quantitatively characterize cell to cell interactions in this complex environment. Here, the authors have applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze Myc-induced pancreatic β cell islet tumors. The high mass resolution and micron spatial resolution of ToF-SIMS allows detection of metabolic intermediates such as lipids and amino acids. Employing multivariate analysis, specifically, principal component analysis, the authors show that it is possible to chemically distinguish cancerous islets from normal tissue, in addition to intratumor heterogeneity. These heterogeneities can then be imaged and investigated using another modality such as sum harmonic generation microscopy. Using these techniques with a specialized mouse model, the authors found significant metabolic changes occurring within β cell tumors and the surrounding tissues. Specific alterations of the lipid, amino acid, and nucleotide metabolism were observed, demonstrating that ToF-SIMS can be utilized to identify large-scale changes that occur in the tumor microenvironment and could thereby increase the understanding of tumor progression and the tumor microenvironment.

Electronic structure of exfoliated and epitaxial hexagonal boron nitride

Hexagonal boron nitride (hBN) is an essential component in van der Waals heterostructures as it provides high quality and weakly interacting interfaces that preserve the electronic properties of adjacent materials. While exfoliated flakes of hBN have been extensively studied using electron transport and optical probes, detailed experimental measurements of the energy- and momentum- dependent electronic excitation spectrum are lacking. Here, we directly determine the full valence band (VB) electronic structure of micron-sized exfoliated flakes of hBN using angle-resolved photoemission spectroscopy with micrometer spatial resolution. We identify the {\pi}- and {\sigma}-band dispersions, the hBN stacking order and determine a total VB bandwidth of 19.4 eV. We compare these results with electronic structure data for epitaxial hBN on graphene on silicon carbide grown in situ using a borazine precursor. The epitaxial growth and electronic properties are investigated using photoe- mission electron microscopy. Our measurements show that the fundamental electronic properties of hBN are highly dependent on the fabrication strategy.

Vacuum Ultraviolet Laser Desorption/Ionization Mass Spectrometry Imaging of Single Cells with Submicron Craters

Mass spectrometry imaging (MSI) is a crucial label-free method to distinguish the localization patterns in single cells. MALDI-TOF MS and ToF-SIMS are now bearing the responsibility. However, MALDI-TOF MS is limited to micron spatial resolution and ToF-SIMS suffers from severe molecular fragmentation. Here, we proposed a new MSI methodology of vacuum ultraviolet laser desorption/ionization (VUVDI) with high spatial resolution, achieving higher ion yields and less fragmentation compared with ToF-SIMS at submicron level. The fluorescence image and mass spectrum of VUVDI were obtained simultaneously. In addition, the adjustable laser fluence acquired selective detection for different molecular and fragmental ions, thus realizing molecular identification. Furthermore, MSIs of single cells with submicron craters were presented. These results suggest VUVDI is a potential mass spectrometry method that provides a soft ionization source and submicron spatial resolution for molecular analysis in life science.

Experiments on bright-field and dark-field high-energy electron imaging with thick target material

Using a high energy electron beam for the imaging of high density matter with both high spatial-temporal and areal density resolution under extreme states of temperature and pressure is one of the critical challenges in high energy density physics . When a charged particle beam passes through an opaque target, the beam will be scattered with a distribution that depends on the thickness of the material. By collecting the scattered beam either near or off axis, so-called bright field or dark field images can be obtained. Here we report on an electron radiography experiment using 45 MeV electrons from an S-band photo-injector, where scattered electrons, after interacting with a sample, are collected and imaged by a quadrupole imaging system. We achieved a few micrometers (about 4 micrometers) spatial resolution and about 10 micrometers thickness resolution for a silicon target of 300-600 micron thickness. With addition of dark field images that are captured by selecting electrons with large scattering angle, we show that more useful information in determining external details such as outlines, boundaries and defects can be obtained.

A micro-SQUID with dispersive readout for magnetic scanning microscopy

We have designed and characterized a micro-SQUID with dispersive readout for use in low temperature scanning probe microscopy systems. The design features a capacitively shunted RF SQUID with a tunable resonance frequency from 5 to 12 GHz, micrometer spatial resolution, and integrated superconducting coils for local application of magnetic fields. The SQUID is operated as a nonlinear oscillator with a flux- and power-dependent resonance frequency. Measurements for device characterization and noise benchmarking were carried out at 4 K. The measured flux noise above 10 kHz at 4 K is 80 nΦ0 Hz−1∕2 at a bandwidth of 200 MHz. Estimations suggest that one can benefit from parametric gain based on inherent nonlinearity of the Josephson junction and reduce the flux noise to 30 nΦ0Hz–1∕2 at 100 mK, which corresponds to 10.6 μBHz–1∕2 for a magnetic moment located at the center of the pickup loop.

Micro-imaging of Brain Cancer Radiation Therapy Using Phase-contrast Computed Tomography

Experimental neuroimaging provides a wide range of methods for the visualization of brain anatomic morphology down to subcellular detail. Still, each technique-specific detection mechanism presents compromises among the achievable field-of-view size, spatial resolution, and nervous tissue sensitivity, leading to partial sample coverage, unresolved morphologic structures, or sparse labeling of neuronal populations and often also to obligatory sample dissection or other sample invasive manipulations. X-ray phase-contrast imaging computed tomography (PCI-CT) is an experimental imaging method that simultaneously provides micrometric spatial resolution, high soft-tissue sensitivity, and exvivo full organ rodent brain coverage without any need for sample dissection, staining or labeling, or contrast agent injection. In the present study, we explored the benefits and limitations of PCI-CT use for invitro imaging of normal and cancerous brain neuromorphology after invivo treatment with synchrotron-generated x-ray microbeam radiation therapy (MRT), a spatially fractionated experimental high-dose radiosurgery. The goals were visualization of the MRT effects on nervous tissue and a qualitative comparison of the results to the histologic and high-field magnetic resonance imaging findings. MRT was administered invivo to the brain of both healthy and cancer-bearing rats. At 45days after treatment, the brain was dissected out and imaged exvivo using propagation-based PCI-CT. PCI-CT visualizes the brain anatomy and microvasculature in 3 dimensions and distinguishes cancerous tissue morphology, necrosis, and intratumor accumulation of iron and calcium deposits. Moreover, PCI-CT detects the effects of MRT throughout the treatment target areas (eg, the formation of micrometer-thick radiation-induced tissue ablation). The observed neurostructures were confirmed by histologic and immunohistochemistry examination and related to the micro-magnetic resonance imaging data. PCI-CT enabled a unique 3D neuroimaging approach for exvivo studies on small animal models in that it concurrently delivers high-resolution insight of local brain tissue morphology in both normal and cancerous micro-milieu, localizes radiosurgical damage, and highlights the deep microvasculature. This method could assist experimental small animal neurology studies in the postmortem evaluation of neuropathology or treatment effects.

Front Cover: Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second (J. Biophotonics 4/2018)

SECTR is a novel multimodal imaging platform for combined volumetric optical coherence tomography (OCT) and en face spectrally encoded reflectometry (SER). The authors demonstrate three‐dimensional motion‐tracking with millisecond temporal and micron spatial resolution using complementary data from OCT and SER, and preliminary algorithms and results showing real‐time image aiming and multi‐volumetric mosaicking for reconstruction of wide‐field composites. The image shows a noninvasively imaged nine‐field mosaic of in vivo human retina and depth‐resolved visualization of tissue microstructures.Further details can be found in the article by Mohamed T. El‐Haddad, Ivan Bozic, and Yuankai K. Tao (e201700268) image

Perfusion double-channel micropipette probes for oxygen flux mapping with single-cell resolution.

Measuring cellular respiration with single-cell spatial resolution is a significant challenge, even with modern tools and techniques. Here, a double-channel micropipette is proposed and investigated as a probe to achieve this goal by sampling fluid near the point of interest. A finite element model (FEM) of this perfusion probe is validated by comparing simulation results with experimental results of hydrodynamically confined fluorescent molecule diffusion. The FEM is then used to investigate the dependence of the oxygen concentration variation and the measurement signal on system parameters, including the pipette’s shape, perfusion velocity, position of the oxygen sensors within the pipette, and proximity of the pipette to the substrate. The work demonstrates that the use of perfusion double-barrel micropipette probes enables the detection of oxygen consumption signals with micrometer spatial resolution, while amplifying the signal, as compared to sensors without the perfusion system. In certain flow velocity ranges (depending on pipette geometry and configuration), the perfusion flow increases oxygen concentration gradients formed due to cellular oxygen consumption. An optimal perfusion velocity for respiratory measurements on single cells can be determined for different system parameters (e.g., proximity of the pipette to the substrate). The optimum perfusion velocities calculated in this paper range from 1.9 to 12.5 μm/s. Finally, the FEM model is used to show that the spatial resolution of the probe may be varied by adjusting the pipette tip diameter, which may allow oxygen consumption mapping of cells within tissue, as well as individual cells at subcellular resolution.

Imaging Optical Frequencies with 100 μHz Precision and 1.1 μm Resolution.

We implement imaging spectroscopy of the optical clock transition of lattice-trapped degenerate fermionic Sr in the Mott-insulating regime, combining micron spatial resolution with submillihertz spectral precision. We use these tools to demonstrate atomic coherence for up to 15s on the clock transition and reach a record frequency precision of 2.5×10^{-19}. We perform the most rapid evaluation of trapping light shifts and record a 150mHz linewidth, the narrowest Rabi line shape observed on a coherent optical transition. The important emerging capability of combining high-resolution imaging and spectroscopy will improve the clock precision, and provide a path towards measuring many-body interactions and testing fundamental physics.

Generation of multifocal irradiance patterns by using complex Fresnel holograms.

We experimentally demonstrate Fresnel holograms able to produce multifocal irradiance patterns with micrometric spatial resolution. These holograms are assessed from the coherent sum of multiple Fresnel lenses. The utilized encoded technique guarantees full control over the reconstructed irradiance patterns due to an optimal codification of the amplitude and phase information of the resulting complex field. From a practical point of view, a phase-only spatial light modulator is used in a couple of experiments addressed to obtain two- and three-dimensional distributions of focal points to excite both linear and non-linear optical phenomena.

Cross-Scale Molecular Analysis of Chemical Heterogeneity in Shale Rocks

The organic and mineralogical heterogeneity in shale at micrometer and nanometer spatial scales contributes to the quality of gas reserves, gas flow mechanisms and gas production. Here, we demonstrate two molecular imaging approaches based on infrared spectroscopy to obtain mineral and kerogen information at these mesoscale spatial resolutions in large-sized shale rock samples. The first method is a modified microscopic attenuated total reflectance measurement that utilizes a large germanium hemisphere combined with a focal plane array detector to rapidly capture chemical images of shale rock surfaces spanning hundreds of micrometers with micrometer spatial resolution. The second method, synchrotron infrared nano-spectroscopy, utilizes a metallic atomic force microscope tip to obtain chemical images of micrometer dimensions but with nanometer spatial resolution. This chemically “deconvoluted” imaging at the nano-pore scale is then used to build a machine learning model to generate a molecular distribution map across scales with a spatial span of 1000 times, which enables high-throughput geochemical characterization in greater details across the nano-pore and micro-grain scales and allows us to identify co-localization of mineral phases with chemically distinct organics and even with gas phase sorbents. This characterization is fundamental to understand mineral and organic compositions affecting the behavior of shales.

3D topology of orientation columns in visual cortex revealed by functional optical coherence tomography.

Orientation tuning is a canonical neuronal response property of six-layer visual cortex that is encoded in pinwheel structures with center orientation singularities. Optical imaging of intrinsic signals enables us to map these surface two-dimensional (2D) structures, whereas lack of appropriate techniques has not allowed us to visualize depth structures of orientation coding. In the present study, we performed functional optical coherence tomography (fOCT), a technique capable of acquiring a 3D map of the intrinsic signals, to study the topology of orientation coding inside the cat visual cortex. With this technique, for the first time, we visualized columnar assemblies in orientation coding that had been predicted from electrophysiological recordings. In addition, we found that the columnar structures were largely distorted around pinwheel centers: center singularities were not rigid straight lines running perpendicularly to the cortical surface but formed twisted string-like structures inside the cortex that turned and extended horizontally through the cortex. Looping singularities were observed with their respective termini accessing the same cortical surface via clockwise and counterclockwise orientation pinwheels. These results suggest that a 3D topology of orientation coding cannot be fully anticipated from 2D surface measurements. Moreover, the findings demonstrate the utility of fOCT as an in vivo mesoscale imaging method for mapping functional response properties of cortex in the depth axis. NEW & NOTEWORTHY We used functional optical coherence tomography (fOCT) to visualize three-dimensional structure of the orientation columns with millimeter range and micrometer spatial resolution. We validated vertically elongated columnar structure in iso-orientation domains. The columnar structure was distorted around pinwheel centers. An orientation singularity formed a string with tortuous trajectories inside the cortex and connected clockwise and counterclockwise pinwheel centers in the surface orientation map. The results were confirmed by comparisons with conventional optical imaging and electrophysiological recordings.