Simplified Near-Degenerate Four-Wave-Mixing Microscopy

Four-wave-mixing microscopy is widely researched in both biology and medicine. In this paper, we present a simplified near-degenerate four-wave-mixing microscopy (SNDFWM). An ultra-steep long-pass filter is utilized to produce an ultra-steep edge on the spectrum of a femtosecond pulse, and a super-sensitive four-wave-mixing (FWM) signal can be generated via an ultra-steep short-pass filter. Compared with the current state-of-the-art FWM microscopy, this SNDFWM microscopy has the advantages of simpler experimental apparatus, lower cost, and easier operation. We demonstrate that this SNDFWM microscopy has high sensitivity and high spatial resolution in both nanowires and biological tissues. We also show that the SNDFWM microscopy can achieve an ultra-sensitive detection based on the electron-resonance effect. This method might find an important application in tracking of nano drugs in vivo.     

Microstructure analysis of a carbon-carbon composite using argon ion etching.

The microstructure of carbon-carbon composites obtained by chemical vapor infiltration of a carbon fiber felt was comparatively studied by reflection light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and laser scanning confocal microscopy (LSCM). Ar+ ion etching was used to reveal and distinguish structural units of the pyrolytic carbon matrix. Mechanically polished samples, polished and subsequently ion etched samples, and fractured samples were compared. The values of surface roughness and surface height after polishing or after polishing and subsequent etching determined by AFM and LSCM correlate well with the degree of texture of the matrix layers obtained by polarized light microscopy and selected area electron diffraction. The carbon matrix is composed of structural units or "cells," which contain a carbon fiber and a sequence of several differently textured layers around each fiber. Within high-textured layers columnar grains are well recognizable using polarized reflection light microscopy and confocal microscopy. The size of depressions within high-textured carbon layers found by AFM after ion etching correlates well with the size of differently tilted domains detected by both TEM and SEM.     

Far‐Red Organic Fluorophores Contain a Fluorescent Impurity

Far‐red organic fluorophores commonly used in traditional and super‐resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near‐red emission. This near‐red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super‐resolution localization microscopy.     

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy

The combination of alternating electric fields with nanometer‐sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real‐time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements.     

Synthesis and characterization of heat-stabilized albumin magnetic microspheres

Human serum albumin magnetic microspheres containing 30% iron oxide particles were synthesized by a heat-stabilization process. The average diameter, the size distribution and the morphology were characterized by scanning electron microscopy, atomic force microscopy and transmission electron microscopy. The distribution of the iron oxide nanoparticles within the microspheres was confirmed by the contrast obtained in the morphology by backscattered electron imaging in scanning electron microscopy. Energy-dispersive X-ray spectroscopy showed the presence of iron in the microspheres. The cabbage like surface structure in some of the microspheres obtained in scanning electron microscopy can be better understood by atomic force microscopy. This peculiar surface structure in the microsphere may be due to the cross-linking in the protein molecule by heat. The amount of iron oxide in the microsphere was analyzed by atomic absorption spectroscopy. The magnetic properties of the particles were measured in a superconducting quantum interference device magnetometer. Received: 12 September 2000 Accepted: 5 February 2001     

Using fluctuation microscopy to characterize structural order in metallic glasses.

We have used fluctuation microscopy to reveal the presence of structural order on length scales of 1-2 nm in metallic glasses. We compare results of fluctuation microscopy measurements with high resolution transmission electron microscopy and electron diffraction observations on a series of metallic glass samples with differing degrees of structural order. The agreement between the fluctuation microscopy results and those of the other techniques is good. In particular, we show that the technique used to make thin specimens for electron microscopy affects the structure of the metallic glass, with ion thinning inducing more structural order than electro-polishing. We also show that relatively minor changes in the composition of the alloy can have a significant effect on the medium-range order; this increased order is correlated with changes in mechanical behavior.     

Simple, one-step synthesis of gold nanowires in aqueous solution

A simple procedure to synthesize gold nanowires based on the reduction of hydrogen tetrachloroaurate by 2-mercaptosuccinic acid in aqueous solution is presented. This procedure requires no additional capping or reduction agent and produces wires with an apparent curly morphology several micrometers in length with diameters as thin as 15 nm. Some of the wires produced end in a ribbonlike structure, finally terminated by a flat triangular prism. Investigations by scanning electron microscopy, transmission electron microscopy (bright and dark field), scanning transmission electron microscopy, and atomic force microscopy as well as conductivity measurements indicate fully connected, polycrystalline gold objects.     

New Tools for Investigating Electromagnetic Hot Spots in Single‐Molecule Surface‐Enhanced Raman Scattering

Surface‐enhanced Raman scattering (SERS) is quickly growing as an analytical technique, because it offers both molecular specificity and excellent sensitivity. For select substrates, SERS can even be observed from single molecules, which is the ultimate limit of detection. This review describes recent developments in the field of single‐molecule SERS (SM‐SERS) with a focus on new tools for characterizing SM‐SERS‐active substrates and how they interact with single molecules on their surface. In particular, techniques that combine optical spectroscopy and microscopy with electron microscopy are described, including correlated optical and transmission electron microscopy, correlated super‐resolution imaging and scanning electron microscopy, and correlated optical microscopy and electron energy loss spectroscopy.     

Fabrication and characterization of stable ultrathin film micropatterns containing DNA and photosensitive polymer diazoresin

Stable, ultrathin DNA micropatterns were fabricated from photosensitive polymer diazoresin (DR) through a self-assembly technique. The micropatterns were achieved on LBL ultrathin film after UV exposure through a photomask. The patterns were characterized systematically with scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. All of the results indicate that the combined LBL self-assembly and photolithography technique is a promising method for constructing stable, well-defined micropatterns with a nanoscale thickness.     

Imaging the cardiovascular system: seeing is believing.

From the basic light microscope through high-end imaging systems such as multiphoton confocal microscopy and electron microscopes, microscopy has been and will continue to be an essential tool in developing an understanding of cardiovascular development, function, and disease. In this review we briefly touch on a number of studies that illustrate the importance of these forms of microscopy in studying cardiovascular biology. We also briefly review a number of imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET) that, although they do not fall under the realm of microscopy, are imaging modalities that greatly complement microscopy. Finally we examine the role of proper imaging system calibration and the potential importance of calibration in understanding biological tissues, such as the cardiovascular system, that continually undergo deformation in response to strain.     

Single-molecule monitoring in living cells by use of fluorescence microscopy

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.     

Nanographenes: Ultrastable,Switchable, and Bright Probes for Super‐Resolution Microscopy

Super‐resolution fluorescence microscopy has enabled important breakthroughs in biology and materials science. Implementations such as single‐molecule localization microscopy (SMLM) and minimal emission fluxes (MINFLUX) microscopy in the localization mode exploit fluorophores that blink, i.e., switch on and off, stochastically. Here, we introduce nanographenes, namely large polycyclic aromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots, as a new class of fluorophores for super‐resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties: intrinsic blinking even in air, excellent fluorescence recovery, and stability over several months. As a proof of concept for super‐resolution applications, we use nanographenes in SMLM to generate 3D super‐resolution images of silica nanocracks. Our findings open the door for the widespread application of nanographenes in super‐resolution fluorescence microscopy.     

大数值孔径光学镜约束多级衍射机理

针对基于激光照明的离轴全息显微成像系统存在散斑和寄生条纹噪声,以及基于部分相干光照明的离轴数字全息显微技术存在相干条纹对比度差的问题,本文提出了一种基于单色LED照明的衍射相位显微成像系统。该系统利用大数值孔径物镜及光栅对物光进行多级衍射,并采用4f系统和空间滤波器分离出0级和+1级信息,分别作为参考光和物光,最终两束光在CCD阵面上干涉产生离轴全息图,从而形成共光路全息成像结构。通过理论分析和计算,对实验用到的光学元器件进行选型,确保衍射光频谱信息能够分开且满足抽样条件。最后与传统激光离轴数字全息显微成像检测结果进行对比,实验结果表明,本文提出的系统能够获得较高的成像准确度和信噪比。     

Spectroscopic imaging techniques for characterization of polymer morphologies: a combination of infrared and electron microscopy

The morphological characterization of polymer blends consisting of polyamide and poly(tetrafluoroethylene) using FT-IR spectroscopy and electron microscopy is described. To enhance the lateral resolution - one of the main limits in infrared spectroscopy - a combination with scanning electron microscopy and analytical electron microscopic methods of a transmission electron microscope was made. The possibilities of electron energy loss spectroscopy and energy filtered transmission electron microscopy (EFTEM) in the area of polymer characterization are outlined.     

A NEW APPROACH TO THE OBSERVATION OF THE RESIST IN X-RAY CONTACT MICROSCOPY

X-ray microscopy is one of the useful applications of synchrotron radiation researches, and at present, X-ray contact microscopy is the best developed method in this field. The images on the resist in X-ray contact microscopy have been observed with a scanning electron microscope, and there have been some attempts to use the transmission electron microscope, but few have used the replica method. We have applied the replica method with plasma polymerization-film to observe X-ray images on the resist with a transmission electron microscope and found it to be applicable.
A long exposure time is required when we use the monochromatic synchrotron radiation with a grasshopper monochromator. Our preliminary experiments with a new light source, undulator radiation, showed that it is very intense and useful for X-ray contact microscopy.     

铂纳米空球的制备及其对甲醇氧化的电催化性能

以粒径为100nm的硒球作模板,在室温下批量合成了粒径约110nm、壳厚约5 nm的铂空球.采用扫描电子显微镜（SEM）、透射电子显微镜（TEM）、高分辨透射电子显微镜（HR-TEM）、选区电子衍射（SAED）、X射线衍射（XRD）、能量色散X射线谱（EDX）等检测技术表征了其形貌与结构;以甲醇为探针分子研究了铂纳米空球修饰玻碳电极对甲醇电氧化的催化性能.结果表明,由铂原子簇团构筑的多孔铂空球粒径均匀、分散性好、结构稳定、比表面积大、传质性能好,是甲醇氧化的理想催化材料.循环伏安（CV）结果表明:当甲醇氧化的电流密度0.10 mA·cm^-2,正扫时,铂纳米空球的氧化电位与实心铂纳米粒子及铂黑相比,分别负移了约110和64mV;负扫时,前者比后两者分别负移了约51与13 mV.经800圈循环伏安扫描后,正扫时,甲醇在铂纳米空球上氧化峰的电流密度为实心铂纳米粒子及铂黑上的13和15倍;负扫时,前者为后两者的19和38倍.表明铂纳米空球对甲醇氧化具有较好的催化活性和稳定性.     

Nonlinear Absorption Microscopy†

For the past two decades, nonlinear microscopy has been developed to overcome the scattering problem in thick tissue imaging. Owing to its increased imaging depth and high spatial resolution, nonlinear microscopy becomes the first choice for imaging living tissues. The use of nonlinear optical effects not only facilitates the signal originating from an extremely small volume defined by light focusing but also provides novel contrast mechanisms with molecular specificity. Nonlinear absorption is a nonlinear optical effect in which the absorption coefficient depends on excitation intensity. As a commonly used spectroscopy tool, nonlinear absorption measurement uncovers many photophysical and photochemical processes correlated with electronic states of molecules. Recently we have been focusing on adapting this spectroscopy method to a microscopy imaging technique. The effort leads to a novel modality in nonlinear microscopy—nonlinear absorption microscopy. This article summarizes the principles and instrumentation of this imaging technique and highlights some of the recent progress in applying it to imaging skin pigmentation and microvasculature under ex vivo or in vivo conditions.     

Quantum-yield-optimized fluorophores for site-specific labeling and super-resolution imaging

Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy.     

Correlative imaging for polymer science

The characterization of polymeric materials is key towards the understanding of structure–activity relations and therefore for the rational design of novel and improved materials for a myriad of applications. Many microscopy techniques are currently used, with electron microscopy, fluorescence microscopy, and atomic force microscopy being the most relevant. In this perspective paper, we discuss the use of correlative imaging, that is, the combination of multiple imaging methodologies on the same sample, in the field of polymeric materials. This innovative approach is emerging as a powerful tool to unveil the structure and functional properties of biological and synthetic structures. Here we discuss the possibilities of correlative imaging and highlight their potential to answer open questions in polymer science.     

High-speed X-ray microscopy by use of high-resolution zone plates and synchrotron radiation

X-ray microscopy based on synchrotron radiation has become a fundamental tool in biology and life sciences to visualize the morphology of a specimen. These studies have particular requirements in terms of radiation damage and the image exposure time, which directly determines the total acquisition speed. To monitor and improve these key parameters, we present a novel X-ray microscopy method using a high-resolution zone plate as the objective and the matching condenser. Numerical simulations based on the scalar wave field theory validate the feasibility of the method and also indicate the performance of X-ray microscopy is optimized most with sub-10-nm-resolution zone plates. The proposed method is compatible with conventional X-ray microscopy techniques, such as computed tomography, and will find wide applications in time-resolved and/or dose-sensitive studies such as living cell imaging.