Influence of fluorescence time characteristics on the spatial resolution of CW-stimulated emission depletion microscopy

As one of the most important realizations of stimulated emission depletion(STED) microscopy, the continuouswave(CW) STED system, constructed by using CW lasers as the excitation and STED beams, has been investigated and developed for nearly a decade. However, a theoretical model of the suppression factors in CW STED has not been well established. In this investigation, the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied. The full-width-at-half-maximum(FWHM) of a CW STED with a doughnut-shaped STED beam is also reanalyzed. It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities. In addition, the FWHM is highly sensitive to both the fluorescence rate(inverse of fluoresce lifetime) and the quenching rate, but insensitive to the rate of vibrational relaxation. For comparison, the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity. Our model is highly consistent with published experimental data for evaluating the spatial resolution. This investigation is important in guiding the development of new CW STED systems.     

Quantitative pupil analysis in stimulated emission depletion microscopy using phase retrieval

The resolution attainable with stimulated emission depletion (STED) microscopy greatly depends on the quality of the STED laser focus. So far, visual inspection of a measured STED focus has been the only convenient means of gauging the source of aberrations. Here we describe a method, requiring no instrument modifications, for obtaining an equivalent to the complex pupil function at the back aperture of the objective and show that it provides quantitative information about aberration sources (including aberrations induced by the objective or sample). We show the accuracy of this field representation to be sufficient for reconstructing the STED focus in three dimensions and determining corrective steps.     

Super‐resolution deep imaging with hollow Bessel beam STED microscopy

Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method, breaking the diffraction barrier for improved spatial resolution in cellular imaging, lithography, etc. Because of specimen‐induced aberrations and scattering distortion, it is a great challenge for STED to maintain consistent lateral resolution deep inside specimens. Here we report on deep imaging STED microscopy using a Gaussian beam for excitation and a hollow Bessel beam for depletion (GB‐STED). The proposed scheme shows an improved imaging depth of up to about 155 μm in a solid agarose sample, 115 μm in polydimethylsiloxane, and 100 μm in a phantom of gray matter in brain tissue with consistent super resolution, while standard STED microscopy shows a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB‐STED, paving the way for deep tissue super‐resolution imaging and three‐dimensional precise laser fabrication.

     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.     

Axial super-resolution by mirror-reflected stimulated emission depletion microscopy

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a microreflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.     

Manipulation of doughnut focal spot by image inverting interferometry

Based on image inverting interference combined with phase modulation, we theoretically demonstrate that the doughnut focal spot can readily be manipulated, and either shrinkage or expansion of size of the central dark spot is possible in a large scale (peak-to-peak value: 0.555λ-0.830λ, or 93.3%-140.8% compared with the standard one). As the interference and phase modulation can both be achieved by a double Porro prism, it is feasible to introduce this approach into optical tweezers to improve their performance. As much as 33.9% intensity of stimulated emission depletion (STED) beam can be reduced if the further optimized configuration is utilized in STED microscopy.     

受激发射损耗荧光显微镜的模型设计及参量优化

受激发射损耗荧光显微镜利用荧光饱和和激发态荧光受激损耗的非线性关系,通过限制损耗区域,可突破远场光学显微术的衍射极限分辨力并实现三维成像。基于对粒子速率方程组的修正,建立了描述荧光团各能级粒子数概率时间特性的模型,并定义了时间平均损耗效率判据。采用高斯函数模拟两束入射激光脉冲通过对模型的数值计算,模拟了激发脉冲的SIED激光脉冲的光强、脉冲宽度以及两束光的延迟时间等参量与损耗效率之间的关系,并获得了各参量的最佳值,优化了损耗效率,为提高系统分辨力提供了有效的途径。     

Saturated Förster resonance energy transfer microscopy with a stimulated emission depletion beam: a pathway toward single-molecule resolution in far-field bioimaging

We theoretically demonstrate that the spatial resolution of stimulated emission depletion (STED) microscopy can be substantially enhanced without increasing the intensity of the STED beam. In our scheme, tiny nanobeads codoped with donor and acceptor molecules are used as fluorescent probes, in which F?rster resonance energy transfer (FRET) can occur with an ~100% efficiency between the donors and acceptors. Enhancement of the depletion of acceptors in the nanobeads with the doughnut-shaped depletion beam can lead to an increase of FRET efficiency in the outer area of the excitation spot, which itself is used for deexciting donor molecules and, consequently, enhancing the optical resolution.     

Advantages of STED-Inspired 3D Direct Laser Writing for Fabrication of Hybrid Nanostructures

We present a new method of additive laser technology referred to as STED nanolithography technique. This technique provides a means for fabrication of 3D dielectric and plasmonic composite nanostructures. The new technology is of the utmost interest for the electronics manufacturing industry, in particular, for formation of specific hybrid (metal–dye) nanostructures, which can be utilized as luminescent markers in biology, medicine, criminalistics, and the trade industry. In the present study, we demonstrate the advantages of STED-inspired nanolithography for fabrication of metallic and hybrid nanostructures. The 3D-scanning setup implemented offers the possibility to form both periodic and aperiodic nanostructured arrays. We show the possibility to decrease substantially the lateral size of the lines formed with the use of STED nanolithography as compared to the direct laser writing (DLW) method. The STED nanolithography technique proposed provides a means for synthesizing metallic nanoparticles in the specified points of the volume of the studied object in vivo. In addition, we demonstrate the synthesis of metallic lines by means of STED nanolithography. Moreover, nanometer spatial precision of positioning of the synthesized nanoobjects is achieved. Therefore, it is possible to obtain significant local enhancement of the emission of luminescent markers (surface enhanced luminescence) at any desired point or area of the sample due to plasmonic enhancement of the electromagnetic fields near the surface of metallic nanostructures.     

Electron Microscopy in Polymer Science

In many fields of research in science, engineering, and medicine, electron microscopy as a method for directly imaging submicroscopic structures has become increasingly important in recent decades. Electron microscopy (EM) includes several different techniques: conventional transmission electron microscopy (TEM), high-resolution electron microscopy (HREM), highvoltage electron microscopy (HVEM), scanning electron microscopy (SEM), analytical electron microscopy (AEM), emission electron microscopy (EEM), and others. In the past the central aim of using electron microscopy was structure determination, but recently it has been of growing importance for also investigating different processes, i.e., changes in materials by interaction with several influential factors (e.g., heat, electric or mechanic fields, mechanical loading). Of particular interest is the study of the micromechanical processes of deformation and fracture. Therefore, electron microscopy is a very powerful tool for materials science. The present review reports on some capabilities and limitation of the application of electron microscopy to solid polymers. In Section II the techniques of electron microscopy are briefly reviewed, followed by a section that describes the main methods of specimen preparation of solid polymeric materials. In Section IV the results of several applications of electron microscopy are discussed to reveal the morphology as well as the micromechanical deformation processes of several polymeric materials.     

Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source

We describe a subdiffraction-resolution far-field fluorescence microscope employing stimulated emission depletion (STED) with a light source consisting of a microchip laser coupled into a standard single-mode fiber, which, via stimulated Raman scattering (SRS), yields a comb-like spectrum of seven discrete peaks extending from the fundamental wavelength at 532 nm to 620 nm. Each of the spectral peaks can be used as STED light for overcoming the diffraction barrier. This SRS light source enables the simple implementation of multicolor STED and provides a spectral output with multiple available wavelengths from green to red with potential for further expansion.     

High resolution electron spin resonance microscopy

NMR microscopy is routinely employed in fields of science such as biology, botany, and materials science to observe magnetic parameters and transport phenomena in small scale structures. Despite extensive efforts, the resolution of this method is limited (>10 microm for short acquisition times), and thus cannot answer many key questions in these fields. We show, through theoretical prediction and initial experiments, that ESR microscopy, although much less developed, can improve upon the resolution limits of NMR, and successfully undertake the 1 mum resolution challenge. Our theoretical predictions demonstrate that existing ESR technology, along with advanced imaging probe design (resonator and gradient coils), using solutions of narrow linewidth radicals (the trityl family), should yield 64 x 64 pixels 2D images (with z slice selection) with a resolution of 1 x 1 x 10 microm at approximately 60 GHz in less than 1h of acquisition. Our initial imaging results, conducted by CW ESR at X-band, support these theoretical predictions and already improve upon the previously reported state-of-the-art for 2D ESR image resolution achieving approximately 10 x 10 mum, in just several minutes of acquisition time. We analyze how future progress, which includes improved resonators, increased frequency of measurement, and advanced pulsed techniques, should achieve the goal of micron resolution.     

X-ray and electron microscopy of actinide materials

Actinide materials demonstrate a wide variety of interesting physical properties in both bulk and nanoscale form. To better understand these materials, a broad array of microscopy techniques have been employed, including transmission electron microscopy (TEM), electron energy-loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDXS), high-angle annular dark-field imaging (HAADF), scanning electron microscopy (SEM), wavelength dispersive X-ray spectroscopy (WDXS), electron back scattered diffraction (EBSD), scanning tunneling microscopy (STM), atomic force microscopy (AFM), and scanning transmission X-ray microscopy (STXM). Here these techniques will be reviewed, highlighting advances made in the physics, materials science, chemistry, and biology of actinide materials through microscopy. Construction of a spin-polarized TEM will be discussed, considering its potential for examining the nanoscale magnetic structure of actinides as well as broader materials and devices, such as those for computational magnetic memory.     

二维范德瓦尔斯异质结构的制备与物性研究

二维范德瓦尔斯材料(可简称二维材料)已发展成为备受瞩目的材料大家族,而由其衍生的二维范德瓦尔斯异质结构的集成、性能及应用是现今凝聚态物理和材料科学领域的研究热点之一.二维范德瓦尔斯异质结构为探索丰富多彩的物理效应和新奇的物理现象,以及构建新型的自旋电子学器件提供了灵活而广阔的平台.本文从二维材料的转移技术着手,介绍二维范德瓦尔斯异质结构的构筑、性能及应用.首先,依据湿法转移和干法转移的分类,详细介绍二维范德瓦尔斯异质结构的制备技术,内容包括转移技术的通用设备、常用转移方法的具体操作步骤、三维操纵二维材料的方法、异质界面清洁.随后介绍二维范德瓦尔斯异质结构的性能和应用,重点介绍二维磁性范德瓦尔斯异质结构,并列举在二维范德瓦尔斯磁隧道结和摩尔超晶格领域的应用.因此,二维材料转移技术的发展和优化将进一步助力二维范德瓦尔斯异质结构在基础科学研究和实际应用上取得突破性的成果.     

二维范德瓦尔斯异质结构的制备与物性研究

二维范德瓦尔斯材料(可简称二维材料)已发展成为备受瞩目的材料大家族,而由其衍生的二维范德瓦尔斯异质结构的集成、性能及应用是现今凝聚态物理和材料科学领域的研究热点之一.二维范德瓦尔斯异质结构为探索丰富多彩的物理效应和新奇的物理现象,以及构建新型的自旋电子学器件提供了灵活而广阔的平台.本文从二维材料的转移技术着手,介绍二维范德瓦尔斯异质结构的构筑、性能及应用.首先,依据湿法转移和干法转移的分类,详细介绍二维范德瓦尔斯异质结构的制备技术,内容包括转移技术的通用设备、常用转移方法的具体操作步骤、三维操纵二维材料的方法、异质界面清洁.随后介绍二维范德瓦尔斯异质结构的性能和应用,重点介绍二维磁性范德瓦尔斯异质结构,并列举在二维范德瓦尔斯磁隧道结和摩尔超晶格领域的应用.因此,二维材料转移技术的发展和优化将进一步助力二维范德瓦尔斯异质结构在基础科学研究和实际应用上取得突破性的成果.     

Hard x-ray Zernike microscopy reaches 30 nm resolution

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.     

扫描光声显微镜

本文简单叙述了扫描光声显微镜的原理、应用、发展以及我们现阶段的研制成果,光声信号可以用振幅或相位接收的方式成象,分辨率达到4.5μm,从已获得叉指换能器,集成电路以及材料亚表面结构等样品的光声象可以说明这种显微镜技术在固体物理、材料科学,甚至医学上均具有重要的应用前景。 关键词：     

二维原子晶体的低电压扫描透射电子显微学研究

二维原子晶体材料,如石墨烯和过渡金属硫族化合物等,具有不同于其块体的独特性能,有望在二维半导体器件中得到广泛应用.晶体中的结构缺陷对材料的物理化学性能有直接的影响,因此研究结构缺陷和局域物性之间的关联是当前二维原子晶体研究中的重要内容,需要高空间分辨率的结构研究手段.由于绝大部分二维原子晶体在高能量高剂量的电子束辐照下容易发生结构损伤,利用电子显微方法对二维原子晶体缺陷的研究面临诸多挑战.低电压球差校正扫描透射电子显微(STEM)技术的发展,一个主要目标就是希望在不损伤结构的前提下对二维原子晶体的本征结构缺陷进行研究.在STEM下,多种不同的信号能够被同步采集,包括原子序数衬度高分辨像和电子能量损失谱等,是表征二维原子晶体缺陷的有力工具,不但能对材料的本征结构进行单原子尺度的成像和能谱分析,还能记录材料结构的动态变化.通过调节电子束加速电压和电子辐照剂量,扫描透射电子显微镜也可以作为电子刻蚀二维原子晶体材料的平台,用于加工新型纳米结构以及探索新型二维原子晶体的原位制备.本综述主要以本课题组在石墨烯和二维过渡金属硫族化合物体系的研究为例,介绍低电压扫描透射电子显微学在二维原子晶体材料研究中的实际应用.     

Light–matter interaction of 2D materials:Physics and device applications

In the last decade, the rise of two-dimensional(2D) materials has attracted a tremendous amount of interest for the entire field of photonics and opto-electronics. The mechanism of light–matter interaction in 2D materials challenges the knowledge of materials physics, which drives the rapid development of materials synthesis and device applications. 2D materials coupled with plasmonic effects show impressive optical characteristics, involving efficient charge transfer, plasmonic hot electrons doping, enhanced light-emitting, and ultrasensitive photodetection. Here, we briefly review the recent remarkable progress of 2D materials, mainly on graphene and transition metal dichalcogenides, focusing on their tunable optical properties and improved opto-electronic devices with plasmonic effects. The mechanism of plasmon enhanced light–matter interaction in 2D materials is elaborated in detail, and the state-of-the-art of device applications is comprehensively described. In the future, the field of 2D materials holds great promise as an important platform for materials science and opto-electronic engineering, enabling an emerging interdisciplinary research field spanning from clean energy to information technology.