HeLa细胞突起中微丝束的纳米分辨荧光成像

在Matlab编程环境下模拟了单分子定位显微的纳米分辨成像,在传统实验参数条件下,对不同间隔分子带模型进行了模拟成像.模拟结果表明,单分子定位显微方法可以区分中心相隔20 nm的两个分子带.同时,也分析了不同像元大小对单分子定位精度的影响.此外,通过单分子定位显微方法在IX71倒置荧光显微镜上实现了纳米分辨,系统极限分辨率,即半高全宽为48 nm.在该系统上,获得了HeLa细胞突起中微丝束结构的纳米分辨图像.从重构获得的图像中可以看到微丝束的直径为75—200 nm.     

相干反斯托克斯拉曼散射显微成像技术研究

基于全量子理论对相干反斯托克斯拉曼散射(CARS)过程进行了分析, 在此基础上搭建了单频CARS显微成像系统, 获得了不同尺寸聚苯乙烯微球高对比度的CARS显微图像. 为了标定成像系统的空间分辨率, 采用逐点扫描方式对直径为110 nm聚苯乙烯微球成像, 从而重构出系统的点扩展函数. 结果表明: 该CARS显微成像系统的横向空间分辨率约为600 nm, 而由阿贝衍射极限决定的理论空间分辨率约为300 nm. 分析了导致分辨率降低的原因, 并提出了解决方案. 为实现纳米分辨的CARS显微成像打下了坚实的基础.     

基于点扫描的超分辨显微成像进展

光学显微镜一直推动着现代科学技术的发展.随着科学的进步,对显微成像分辨率的要求在生物、材料等领域日渐凸显,而常规宽场显微成像一直面临着成像分辨率衍射受限的问题.1968年出现的共聚焦显微镜作为点扫描显微镜的开端第一次实现了远场下成像分辨率的突破,它具有层切性好、信噪比高等优点.在1994年出现的受激辐射荧光损耗显微镜将显微成像能力突破到2.8 nm左右,并成为目前效果最佳、应用较广泛的超分辨显微技术.荧光差分显微和饱和荧光吸收竞争等点扫描技术具有无荧光染剂限制、饱和光强低、光路简单等优势,并且能取得1/6波长的分辨能力,进而在超分辨显微领域仍有着发挥空间.Airyscan技术作为以上方法的补充可以弥补点扫描系统中由于探测小孔半径减小而带来的信号丢失,从而提高成像信噪比和分辨率,但阵列探测器成本较高.上述点扫描显微镜通过改变照明或者探测的方式实现了分辨率突破.本文详细讨论了点扫描超分辨方法的原理、成像效果及面临的瓶颈,并分析了点扫描超分辨显微镜在应用和技术上的趋势.     

基于大数值孔径环形光锥照明的超分辨光学 显微成像方法研究

提出和发展了一种基于大数值孔径环形光锥照明的远场超分辨光学显微成像新方法, 采用将发光二极管(LED)面光源、窄带滤光和环形光锥照明相结合的特种照明方式, 实现超分辨显微成像. 建立了大数值孔径环形光锥照明成像的物理模型, 根据标量衍射理论, 在不同环形光锥照明时, 推导出光学显微系统像面衍射斑光强分布的理论计算公式; 通过Matlab求解和绘图, 得到衍射斑光强的分布图样, 从理论上证明这一成像方法可以有效提高光学显微镜的分辨率; 建立了相应的显微成像系统, 通过实验验证了该方法可有效改善显微镜的成像质量, 显著提高分辨率; 在中心波长450 nm、环形光锥数值孔径1.125—1.25时, 实验获得的分辨率至少优于150 nm, 与理论研究结果相符合, 从而证明了这一方法的可行性.     

超衍射极限相干反斯托克斯拉曼散射显微成像技术及其探测极限分析

理论上提出一种突破衍射极限限制的相干反斯托克斯拉曼散射显微成像方法, 并对其探测极限进行分析.通过引入环形附加探测光与艾里斑周边的声子作用, 实现点扩展函数的改造, 提高相干反斯托克斯拉曼散射显微成像系统的横向空间分辨率. 随着分辨率的提高, 信号强度也随之降低, 尤其当应用于生物学、医学研究时, 样品分子数密度通常很低, 这将导致信号探测更加困难. 因此分析系统的探测极限, 确定超分辨体积元内的最小可探测分子数是展开超衍射极限相干反斯 托克斯拉曼散射显微成像实验研究的重要前提. 当泵浦光、斯托克斯光、探测光光强均达到极大值, 分辨率约40 nm三维空间内, 超衍射极限相干反斯托克斯拉曼散射显微成像系统的散粒噪声信噪比由曝 光时间与样品分子数密度决定. 曝光时间若取20 ms, 探测极限约为10³, 样品分子数目只有大于探测极限, 才能保证信号可以从噪声背景中提取出来. 关键词： 突破衍射极限 相干反斯托克斯拉曼散射 非线性光学 探测极限     

基于宽带立体超透镜的远场超分辨率成像

为突破传统衍射极限实现远场超分辨率成像,提出了一种微波频段宽带立体超透镜用于目标远场超分辨率成像.该透镜可将携带着目标超分辨率信息的凋落波分量转换为传播波分量辐射到远场,进而可在远场接收这些信息并用于超分辨率成像.分别从频域和时域两方面对该透镜的超分辨率特性进行验证.在频域,利用多重信号分类算法对借助于该结构的扩展目标实现了λ/12的远场超分辨率成像,大幅度提升了成像效果.在时域,结合时间反演技术,验证了带宽提升对空间超分辨率聚焦特性带来的明显优势.     

基于金刚石体系中氮-空位色心的固态量子传感

在室温下,金刚石中的氮-空位(NV)色心具有荧光强度稳定、电子自旋相干时间长以及与生俱来的原子尺寸的特点,是优良的纳米量子传感器.在成像领域中,将各种超分辨成像显微技术应用于NV色心体系,发展出多种高空间纳米分辨率的成像方法.此外,NV色心作为固态量子比特可以通过光学方法对其进行初始化和读取.NV色心电子自旋量子态还可以与电磁场、应力等进行相干耦合.基于这些耦合,科研人员在实验上实现了对相关物理量纳米级空间分辨率的高灵敏表征.目前这些量子传感技术可以应用在新材料、单个蛋白质核自旋、活体神经元等方面的测量中.本综述主要介绍金刚石中NV色心纳米量子传感器件的工作原理、实验实现和优化以及在相关领域的应用.     

随机光学重构显微成像技术及其应用

光学显微成像技术在生命科学、生物医学、临床医学诊断和材料科学等领域有着非常广泛的应用。但由于光学衍射极限的存在,传统光学显微镜无法观察到纳米尺度的物质及生命活动,极大地限制科学研究和医学的发展。近年来,随着突破光学衍射极限的超分辨成像技术的不断发展,显微成像分辨率得到不同程度的提高。目前在基于不同原理的各种超高分辨率显微镜中,随机光学重构显微镜(STORM)分辨率最高,可达几十纳米,真正实现了单分子水平检测。着重介绍了STORM超分辨显微成像技术的原理、实验方法及其应用。     

光学超分辨成像精度破极限达4.1nm

中国科大郭光灿院士领导的中科院量子信息重点实验室孙方稳研究组,利用光学超分辨成像技术实现了对单个自旋态的纳米量级空间分辨率测量和操控,其成像精度达到4.1 nm。了解微纳尺度物体的物理属性及动力学过程,需要纳米尺寸的探测器,纳米尺度的固态量子测量技术因此得到快速发展。但实现高空间分辨率的电磁场等物理量测量,不仅需要高精度的成像和分辨,还需要高精度量     

受激发射损耗显微中空心损耗光的光强分布优化研究

受激发射损耗显微技术(STED)作为一种远场超分辨显微成像技术,具有几十纳米甚至几纳米的空间分辨率,是细胞生物学等研究领域的重要成像工具。圆环形空心损耗光在物镜焦点附近的光场强度分布对STED空间分辨率起决定性作用。在高数值孔径物镜聚焦下,光场的偏振态会对聚焦光场的强度分布产生显著的影响,此外,显微系统的轴外像差会严重破坏空心损耗光焦斑的中心对称性。基于矢量衍射理论,理论模拟了在高数值孔径物镜聚焦条件下,入射涡旋光的偏振态和光学系统中的彗差和像散对空心损耗光焦场强度分布的影响。实验上使用纯相位型空间光调制器来校准光学系统相差,优化变形的损耗光,利用纳米探针扫描焦点区域,测量了其焦场强度分布。测量结果与由矢量稍微理论观测的结果一致。     

Surface-tension-confined droplet microfluidics

This article is a concise overview about the developing microfluidic systems named surface-tension-confined droplet microfluidics(STORMs). Different from traditional complexed droplet microfluidics which generated and confined the droplets by three-dimensional(3D) poly(dimethylsiloxane)-based microchannels, STORM systems provide twodimensional(2D) platforms for control of droplets. STORM devices utilize surface energy, with methods such as surface chemical modification and mechanical processing, to control the movement of fluid droplets. Various STORM devices have been readily prepared, with distinct advantages over conventional droplet microfluidics, which generated and confined the droplets by 3D poly(dimethylsiloxane)-based microchannels, such as significant reduction of energy consumption necessary for device operation, facile or even direct introduction of droplets onto patterned surface without external driving force such as a micropump, thus increased frequency or efficiency of droplets generation of specific STORM device, among others. Thus, STORM devices can be excellent alternatives for majority areas in droplet microfluidics and irreplaceable choices in certain fields by contrast. In this review, fabrication methods or strategies, manipulation methods or mechanisms,and main applications of STORM devices are introduced.     

Wavelength division multiplexed confocal microscopy using supercontinuum

We investigate both theoretically and experimentally wavelength division multiplexed confocal imaging by using white light supercontinuum. We show that with the optimized pinhole diameter an axial resolution of 0.75 μm and detection efficiency of 80% can be achieved. In addition, we applied the axial WDM confocal system to 3D surface measurement and the result agreed well with that measured by commercially available surface profilometer. The measured sensitivity of the system is 3.25 nm. Finally, we demonstrated lateral confocal imaging by using supercontinuum. An effective lateral scanning range of 130 μm was obtained.     

Aspects of nanometer scale imaging with extreme ultraviolet (EUV) laboratory sources

Imaging systems with nanometer resolution are instrumental to the development of the fast evolving field of nanoscience and nanotechnology. Decreasing the wavelength of illumination is a direct way to improve the spatial resolution in photon-based imaging systems and motivated a strong interest in short wavelength imaging techniques in the extreme ultraviolet (EUV) region. In this review paper, various EUV imaging techniques, such as 2D and 3D holography, EUV microscopy using Fresnel zone plates, EUV reconstruction of computer generated hologram (CGH) and generalized Talbot self-imaging will be presented utilizing both coherent and incoherent compact laboratory EUV sources. Some of the results lead to the imaging with spatial resolution reaching 50 nm in a very short exposure time. These techniques can be used in a variety of applications from actinic mask inspection in the EUV lithography, biological imaging to mask-less lithographic processes in nanofabrication.     

Three-dimensional ghost imaging based on periodic diffraction correlation imaging

We experimentally demonstrate a three-dimensional （3D） ghost imaging method based on period diffraction correlation imaging. Compared with conventional ghost imaging, our method can easily retrieve the images of different focal planes. Due to the correlation between the disturbed object beam and the reference beams which do not pass through any scattering, the clear images can be periodically obtained in the uncovered zones even through a scattering medium. The analysis of the 3D imaging resolution reveals that the proper resolution for actual demand can be achieved by designing our devices. The implementation of this experiment is quite simple and low-cost. It facilitates the practical applications of ghost imaging.     

High resolution 3D x-ray diffraction microscopy

We have imaged a 2D buried Ni nanostructure at 8 nm resolution using coherent x-ray diffraction and the oversampling phasing method. By employing a 3D imaging reconstruction algorithm, for the first time we have experimentally determined the 3D structure of a noncrystalline nanostructured material at 50 nm resolution. The 2D and 3D imaging resolution is currently limited by the exposure time and the computing power, while the ultimate resolution is limited by the x-ray wavelengths. We believe these results pave the way for the development of atomic resolution 3D x-ray diffraction microscopy.     

Nano‐imaging through tip‐enhanced Raman spectroscopy: Stepping beyond the classical limits

The spatial resolution in optical imaging is restricted by so‐called diffraction limit, which prevents it to be better than about half of the wavelength of the probing light. Tip‐enhanced Raman spectroscopy (TERS), which is based on the SPP‐induced plasmonic enhancement and confinement of light near a metallic nanostructure, can however, overcome this barrier and produce optical images far beyond the diffraction limit. Here in this article, the basic phenomenon involved in TERS is reviewed, and the high spatial resolution achieved in optical imaging through this technique is discussed. Further, it is shown that when TERS is combined with some other physical phenomena, the spatial resolution can be dramatically improved. Particularly, by including tip‐applied extremely localized pressure in TERS process, it has been demonstrated that a spatial resolution as high as 4 nm could be achieved.     

光源尺寸和光谱带宽对波带板成像的影响

X射线菲涅耳波带板成像能实现亚微米空间分辨能力,有可能应用于激光等离子体或聚变靶的高分辨X射线成像诊断.之前的数值模拟研究表明,成像分辨能力受光源尺寸、入射光或成像光谱带宽的影响.本文报道在632.8 nm为中心波长的可见光波段,对波带板成像的数值模拟和原理性验证实验.数值模拟表明:随着扩展光源尺寸增加,视场中央分辨能力基本不变,而像对比度下降;随着成像的光谱带宽的增加,视场中央分辨能力与像对比度同时下降.实验证实了数值模拟的结论,且实验与数值模拟结果的定量比较也符合得较好.     

基于同伦l0范数最小化重建的三维动态磁共振成像

高欠采倍数的动态磁共振图像重建具有重要意义,是同时实现高时间分辨率和高空间分辨率动态对比度增强成像的重要环节.本研究提出一种结合黄金角变密度螺旋采样、并行成像和基于同伦l₀范数最小化的压缩感知的图像重建的三维动态磁共振成像方法.黄金角变密度螺旋采样轨迹被用来连续获取k空间数据,具有数据采集效率高、对运动不敏感等优点.在重建算法中,将多线圈稀疏约束应用于时间总变分域,使用基于l₀范数最小化的非线性重建算法代替传统的l₁范数最小化算法,进一步提高了欠采样率.仿真实验和在体实验表明本文所提的方法在保持图像质量的同时,也可以实现较高的空间分辨率和时间分辨率,初步验证了基于同伦l₀范数最小化重建在三维动态磁共振成像上的优势和临床价值.     

Nanotomography

Scanning probe microscopy (SPM) can be expanded to volume imaging. As an example, the core of a dislocation within the three-dimensional (3D) spatial microdomain structure of poly(styrene-block-butadiene-block-styrene) was imaged with approximately 10 nm resolution. The specimen was eroded step by step and its chemical composition in layers beneath the original surface was imaged with SPM. Similar to computed tomography, the 3D distribution of polystyrene and polybutadiene was reconstructed from a series of images. This approach might provide a simple means for real-space volume imaging with nanometer and even atomic resolution of various materials and physical properties.     

Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy

Standing-wave total-internal-reflection fluorescence (SW-TIRF) microscopy uses a super-diffraction-limited standing evanescent wave to extract the high-spatial-frequency content of an object through a diffraction-limited optical imaging system. The effective point-spread function is better than a quarter of the emission wavelength. With a 1.45 numerical aperture objective and 532 nm excitation wavelength, a Rayleigh resolution of approximately 100 nm can be achieved, which is better than twice the resolution of conventional TIRF microscopy. This first experimental realization of SW-TIRF in an objective-launched geometry demonstrates the potential for extended resolution imaging at high speed by using wide-field microscopy.