Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.     

Complex Polarization Response in Plasmonic Nanospirals

Archimedean nanospirals exhibit many far-field resonances that result from the lack of symmetry and strong intra-spiral plasmonic interactions. Here, we present a computational study, with corroborating experimental results, on the plasmonic response of the 4π Archimedean spiral as a function of incident polarization, for spirals in which the largest linear dimension is less than 550 nm. We discuss the modulation of the near-field structure for linearly and circularly polarized light in typical nanospiral configurations. Computational studies of the near-field distributions excited by circularly polarized light illustrate the effects of chirality on plasmonic mechanisms, while rotation of linearly polarized light provides a detailed view of the effects of broken symmetry on nanospiral fields in any given direction in the plane of the spiral. The rotational geometry exhibits a preference for circular polarization that increases near-field enhancement compared to excitation with linearly polarized light and exchanges near-field configurations and resonant modes. By analyzing the effects of polarization and wavelength on the near-field configurations, we also show how the nanospiral could be deployed in applications such as tunable near-field enhancement of nonlinear optical signals from chiral molecules.     

Enhanced Terahertz Transmission Through Bullseye Plasmonics Lenses Fabricated Using Micromilling Techniques

Imaging applications at terahertz frequencies are, in general, limited to relatively low spatial resolution due to the effects of diffraction. By using a subwavelength aperture in the near-field, however, it is possible to achieve subwavelength resolution, although low transmission through the aperture limits the sensitivity of this approach. Plasmonic lenses in the form of bullseye structures, which consist of a circular subwavelength aperture surrounded by concentric periodic corrugations, have demonstrated enhanced transmission, thereby increasing the utility of near-field imaging configurations. In this paper, the design, fabrication, and experimental performance of plasmonic lenses optimized for 300 GHz are discussed. While nanofabrication techniques are required for optical applications, microfabrication techniques are sufficient for terahertz applications. The process flow for fabricating a double-sided bullseye structure using a precision micromilling technique is described. Transmission and beam profile measurements using a customized terahertz testbed are presented.

     

Terahertz Dipole Nanoantenna Arrays: Resonance Characteristics

Resonant dipole nanoantennas promise to considerably improve the capabilities of terahertz spectroscopy, offering the possibility of increasing its sensitivity through local field enhancement, while in principle allowing unprecedented spatial resolutions, well below the diffraction limit. Here, we investigate the resonance properties of ordered arrays of terahertz dipole nanoantennas, both experimentally and through numerical simulations. We demonstrate the tunability of this type of structures, in a range (～1–2 THz) that is particularly interesting and accessible by means of standard zinc telluride sources. We additionally study the near-field resonance properties of the arrays, finding that the resonance shift observed between near-field and far-field spectra is predominantly ascribable to ohmic damping.     

人脑源性神经营养因子cDNA在COS7细胞中的表达及活性研究

本文从质粒Ｍ１３ｍｐ１８－ｈＢＤＮＦ中酶切回收人脑源性神经营养因子（ｈＢＤＮＦ）全长基因,构建真核表达载体ｐＣＭＶ４－ｈＢＤＮＦ。利用脂质体的方法转染ＣＯＳ７细胞,对转染后的ＣＯＳ７细胞提取ＲＮＡ进行狭缝杂交分析和免疫细胞化学反应,分别从转录及翻译水平上检测ＢＤＮＦ基因在ＣＯＳ７细胞中的表达。实验还证实在ＣＯＳ７细胞中表达的ｈＢＤＮＦ蛋白可分泌至胞外并可促进中脑黑质细胞的发育和生长,具有良好的生物学活性。     

Near-Field Optical Analysis of Plasmonic Nano-Probes for Top-Illumination Tip-Enhanced Raman Scattering

Top-illumination tip-enhanced Raman scattering (TI-TERS) has recently emerged as a promising near-field vibrational spectroscopy method that can be adapted on an upright optical microscope. With a relatively simplified optics, TI-TERS can probe both opaque and transparent samples making them a promising tool in nanoscale chemical analysis. One of the critical components of TI-TERS is the plasmonic nano-tip used to enhance the Raman spectroscopic signature. Herein, we numerically studied the near-field optical properties of conventional gold tip (20 nm radius of curvature) and two varieties of optical antenna-based tips in the context of TI-TERS. Optical antenna-based tips included a 40-nm gold nanoparticle attached to a dielectric tip and a 50-nm equilateral gold nanotriangle attached to a dielectric tip. We evaluated the Raman enhancement spectra as a function of experimental variables such as underlying substrate and angle of the tip with respect to substrate normal. Our analysis revealed that conventional gold tip facilitates superior enhancement and optical antenna-based tips facilitate superior spectral bandwidth and lateral resolution in TI-TERS configuration. Tips with higher enhancement can be harnessed for ultra-sensitive measurements, and tips with broader spectral bandwidth can be utilized to enhance both Stokes and anti-Stokes component of the Raman spectra.     

Plasmon Resonances in V-Shaped Gold Nanostructures

Using numerical simulations, we examine the change in plasmon resonance behavior in gold nanorod structures that have a V shape. The reduction in symmetry compared to linear rods causes two different longitudinal-type resonances to appear in a single structure, and the relative intensity and hybridization of these can be controlled by varying the angle of the arms of the ??V.?? The resonances may also be selectively excited by controlling the polarization of the incident light, thereby providing a convenient way to control a nanoscale optical electric field using far-field parameters. For example, the wavelength at which a strong resonance occurs in the V-shaped structures studied can be switched between 630 and 900?nm by a 90° rotation of the polarization of the incident light. Due to the symmetry of the targets, there will be three types of special near-field location; a location at which the electric field intensity is enhanced by either resonance, a location at which the electric field intensity is enhanced by the 630?nm resonance but not by the 890?nm resonance, and a location at which the electric field intensity is enhanced by the 890?nm resonance but not by the 630?nm one.     

Near-Field Scanning Optical Microscopy, a Siren Call to Biology

Near-field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far-field microscopes. Near-field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near-field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.     

Differential mobility of the N-terminal headpiece in the lac-repressor protein.

It is shown by resolution enhancement and relaxation studies of the 360 MHz ¹H nuclear magnetic resonance spectra of the lac-repressor of Escherichia coli and the two fragments derived from it by limited tryptic digestion (the N-terminal headpiece and remaining T-core) that the majority of the relatively mobile residues in the intact lac-repressor are located in the headpiece. Although nuclear magnetic resonance data clearly indicate that the headpiece is a highly structured entity, even when isolated, it is a more mobile part of the repressor than the T-core.     

The Influence of Element Deformation on Terahertz Mode Interaction in Split-Ring Resonator-Based Meta-Atoms

The interaction between terahertz (THz) resonance modes and element deformation in rectangular split-ring resonator (RSRR)-based meta-atoms (MAs) is investigated experimentally. Two types of RSRR-based MAs are presented: lateral-varied SRR (LV-SRR) and arm-twisted SRR (AT-SRR). When the distances from the gaps to the opposite sides of above meta-atoms increase from 10 to 40 μm, the inductive-capacitive (LC) resonance modes and dipole oscillation modes exhibit redshift behavior. The quality factor (Q factor) of LC resonance decreases while that of dipole oscillation modes increases. The THz mode interaction is subject to the distance between the gap and opposite side. An extension of lateral side contributes much more to the enhancement of Q factor of dipole oscillation mode than the twisted arms. The relationship between the near-field coupling effect and THz modes is revealed by the analysis of surface currents as well as the electric energy density distribution, as is in agreement with the experimental results.     

An array of planar apertures for near-field fluorescence correlation spectroscopy

We have developed a method of performing near-field fluorescence correlation spectroscopy via an array of planarized circular apertures of 50 nm diameter. This technique provides 1 μs and 60 nm resolution on proximal samples, including live cells, without incorporating a scanning probe or pulsed lasers or requiring penetration of the sample into the aperture. Millions of apertures are created in an array within a thin film of aluminum on a coverslip and planarized to achieve no height distinction between the apertures and the surrounding metal. Supported lipid bilayers and plasma membranes from live cells adhere to the top of this substrate. We performed fluorescence correlation spectroscopy to demonstrate the sub-diffraction-limited illumination with these devices.     

Photo-CIDNP NMR methods for studying protein folding

Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly powerful in time-resolved NMR measurements. Real-time, rapid-injection protein refolding experiments permit the observation of changes in the accessibility of specific residues during the folding process. CIDNP pulse-labeling gives information on the accessibility of residues in partially structured proteins (e.g., molten globule states) whose NMR spectra are broad and poorly resolved. Heteronuclear two-dimensional (15)N-(1)H CIDNP techniques allow identification of surface-accessible residues with improved resolution and sensitivity. These methods offer residue-specific structural and kinetic information on transient folding intermediates and other partially folded states of proteins that are not readily available from more routine NMR techniques.     

Near-Infrared Super Resolution Imaging with Metallic Nanoshell Particle Chain Array

We propose a near-infrared super resolution near field imaging system with an array of metallic nanoshell particle chain. The imaging array can plasmonically transfer the near field components of dipole sources and the super resolution images can be reconstructed in the output plane. By decreasing the metallic nanoshell’s thickness of the fixed size nanoparticle, the plasmon resonance wavelength of the isolate nanoshell particle is red-shifted to the near-infrared region. The operation wavelength of the imaging array is correspondingly red-shifted to the near-infrared region. In this paper, we study the incoherent and coherent super resolution imaging. The field intensity distributions at the different planes of imaging process are calculated using the finite element method. The simulation results demonstrate that the array has super resolution imaging capability at near-infrared wavelengths in the incoherent and coherent manners. The results also show that the image formation highly depends on the source coherence. In the same structural parameters, the reconstructed images under the illumination of incoherent light source reach to the higher image quality and spatial resolution than the images under the illumination of coherent light source of in phase. By reasonably designing parameters of the imaging array, the approximate spatial resolutions of λ/13 in incoherent case and λ/10 in coherent case are obtained at the near-infrared wavelength of 764 nm. Furthermore, the image–array distance and the chains’ spacing also affect the image reconstruction.     

Energy deposition in a model of man in the near field

The spatial distribution of the specific absorption rate (SAR) was measured in a full-scale model of man using implantable electric field probes. The model was exposed in the near-field of linear and aperture antennas at 350 MHz. Effects of the wave polarization, antenna position and antenna gain on the SAR distribution and the average SAR in the whole-body and body parts are reported.     

Defining a Superlens Operating Regime for Imaging Fluorescent Molecules

It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such “lenses” is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close ( nm) or too far ( nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating structure (such as in the far-field superlens design previously proposed [1]) or a fast near-field scanning probe, it could provide a means for fast fluorescent imaging with sub-diffraction limit resolution.     

Polarization Dependent of Plasmonic Lenses with Variant Periods on Superfocusing

A plasmonic lens with variant periods was investigated for optical behavior at near-field by means of numerical computational method. To study influence of incident light on different polarization modes, we considered linear polarization, circular polarization, elliptical polarization, radial polarization (RP), and azimuthally polarization in our computational analyses. A finite difference and time domain algorithm is employed in the numerical study. Our computational numerical calculation results demonstrate that focusing performance for the plasmonic lens illuminated under radial polarization is best in comparison to that of the illumination with the other four polarization states. The plasmonic lens with RP illumination can realize superfocusing with ultra-long depth of focus. It is possible to be used as an optical probe or a type of plasmonic lens for imaging with high resolution in the near future.     

NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K⁺ channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle. High-field DNP thus offers atomic insight into the role of molecular plasticity during the course of biomolecular function in a complex cellular environment.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Antenna-Attached Parabolic Probe for Excitation and Collection Modes in SNOM

A novel optical fiber probe with a parabola-like shape and a nano-antenna mounted on the center of its endface is proposed for simultaneous excitation and collection modes in scanning near-field optical microscopy. The working principles of the probe are demonstrated, and its optical properties are theoretically investigated and compared with the conventional tip-on-aperture probe. It shows that the probe can greatly boost both the enhancement factor for the excitation mode and the collection efficiency for the collection mode. The proposed probe is a promising tool to realize low-cost and high resolution for a wide variety of near-field measurements in biology, physics, and chemistry.     

Broad Tunable Nanoantenna Based on Graphene Log-Periodic Toothed Structure

A log-periodic toothed nanoantenna based on graphene is proposed, and its multi-resonance properties with respect to the variations of the chemical potential are investigated. The field enhancement and radar cross-section of the antenna for different chemical potentials are calculated, and the effect of the chemical potential on the resonance frequency is analyzed. In addition, the dependence of the resonance frequency on the substrate is also discussed. It is shown that large modulation of resonance intensity in log-periodic toothed nanoantenna can be achieved via turning the chemical potential of graphene. The tunability of the resonant frequencies of the antenna can be used to broad tuning of spectral features. The property of tunable multi-resonant field enhancement has great prospect in the field of graphene-based broadband nanoantenna, which can be applied in non-linear spectroscopy, optical sensor, and near-field optical microscopy.