突破衍射极限的成像方法综述（英文）

"衍射极限"实际上不是一个真正的障碍,除非处理远场和定位精度。这种衍射障碍并不是坚不可摧的,可以利用一些智能技术来突破光学衍射极限。讨论了四种技术,近场扫描光学显微镜(NSOM)法,受激发射损耗(STED)显微镜法,光激活定位显微镜(PALM)法或随机光学重建显微镜(STORM)法和结构照明显微镜(SIM)法,并且介绍了各自的基本原则与优劣。NSOM利用纳米级探测器检测通过光纤的极小汇聚光斑,从而获得单个像素的分辨率;PALM和STORM利用荧光探针,实现暗场和荧光的转换,从而观察到极小的荧光团;SIM则是利用栅格图案与样品叠加成像来实现。其中,STORM具有相对较高的潜力,能够更为有效地突破衍射极限。     

近场光学显微镜

近场光学显微镜根据非辐射场的探测与成像原理,能够突破普通光学显微镜所受到的衍射极限,在超高光学分辨率下进行纳米尺度光学成像与纳米尺度光谱研究,本文简介近场光学的基本原理与近场光学显微镜的仪器发展;讨论近年来近扬光学显微镜的一些进展,接近原子分辨的超高分辨率光学成像,近场光谱与半导体量子器件,生命科学中超显微观察和近场荧光以及近场原理在产业化中的应用。     

扫描近场光学显微镜对细胞超微结构的研究

扫描近场光学显微镜(SNOM)突破了光学显微镜的衍射极限,在细胞研究中具有高灵敏性、无侵入性等优点,已经广泛的应用于生物学研究中。本文综述了SNOM在细胞膜、细胞器、细胞精细结构和单分子探测等领域的研究进展,介绍了扫描近场光学显微镜结合量子点的方法,并对其应用前景做了展望,对其面临困难做了概述。作为一种研究工具,SNOM在生物领域的应用还远远不足。     

受激发射损耗显微术及其在生物医学上的应用

由于受到光学衍射的限制,均匀照明宽视场荧光显微术和激光共焦扫描显微术的分辨率约为200～300nm。近年来受激发射损耗显微术在突破衍射极限以及应用方面取得许多令人瞩目的成果。本文简要介绍受激发射损耗显微术的原理、方法及其在生物医学上的应用。     

激光直写氧化石墨烯可调光子筛

光子筛不受衍射极限限制且设计结构灵活，是一种新型衍射元件。相比于振幅型光子筛，相位型光子筛能量透过率更高，成像对比度更加尖锐，具有应用优势。然而，常见衍射光学器件面临加工复杂及难以调制等局限。石墨烯及其衍生材料具有良好的光电调制特性，被广泛用于衍射光学器件制备。利用激光直写技术可实现诱导还原氧化石墨烯，是一种简单高效的微纳加工技术。利用时域有限差分方法研究光子筛的衍射特性，并基于激光直写技术制备氧化石墨烯光子筛。通过不同激光功率加工可获得材料的折射率调制，器件实现了明显的焦距调制(1.62 mm)和聚焦效率的提高(13.6%)。该方法有望为实现可调制衍射光学元件提供简便、灵活的设计与制备手段。     

全息条纹元件在光学仪器中的应用

<正> 光学元件在光学仪器中一般起转折光线的作用。大多数光学元件是利用光学介质的界面的反射和折射来改变光束方向的。当然也可以用光衍射原理来达到上述目的。最早的衍射光学元件是在1896年瑞利发明的波带板。近代光学仪器中常用的各类衍射光栅就是利用衍射来实现其复杂功能的元件。利用全息方法记录的光束(二束或二束以上)干涉衍射全息图,其理论模型从广义上可看作是一种复杂的变间距的衍射光栅——全息光学元件(HOE)。因此HOE是一种有别于像     

微小尺寸的测量

这里所说的“微小尺寸”是指1毫米以内,直到0.01微米为止,即10~(-3)～10~(-8)米间的尺寸。这一类工件,按照传统的办法,也是最直观最有效的方法是利用几何光学的原理,将工件轮廓按比例放大,再将工件图象除以放大倍数,即得工件实际尺寸。也就是说,利用光学投影仪或工具显微镜来测量。但是在使用这类可见光光学精密机械测量工件时,由于边缘不清晰而造成的误差不能不加以注视。德国光学家阿贝早就从衍射现象推导出光学系统的极限分辨本领d为:     

半导体量子点材料在Nd:YAG激光辐照下的非线性光学效应

使用10 n,脉冲调Q Nd:YAG激光器Z-scan技术测量了化学合成的无掺杂硫化锌量子点(QDs)以及掺Mn2+硫化锌量子点(QDs)的非线性光学特性,并使用透射电镜技术(TEM)以及X射线衍射法(XRD)表征合成材料的纳米结构.在室温下,分别利用UV-VIS分光光度计和分光荧光计测量了人工合成QDs胶体溶液的线性...     

现代显微成像技术综述

概述了光学宽视场显微镜、共聚焦显微镜、超分辨率显微镜中所应用的现代显微成像技术,对各种传统和先进的显微成像原理进行了总结。光学宽视场显微镜最常用的显微技术有明场成像、暗场成像、相衬成像、偏光成像、微分干涉(DIC)成像、调制对比成像和荧光成像。相衬成像中根据不同的成像结构还有切趾相衬成像。微分干涉除了传统的偏振光照明还有圆偏振光照明(C-DIC)和专用于塑料的微分干涉(PlasDIC)。共聚焦显微镜随着计算机技术和制造技术的发展而有了巨大的发展。除了传统的共聚焦荧光显微镜以外,还有连续反斯托克斯拉曼散射(CARS)共聚焦、多光子共聚焦和白光共聚焦。超分辨率显微镜中主要介绍了受激辐射淬灭(STED)技术和紧随基态淬灭显微技术的单分子返回(GSDIM)技术。     

纳米级光学显微镜研制成功

<正>英国和新加坡研究人员3月1日报告说,他们制造出能够观测50nm大小物体的光学显微镜,这是迄今观测能力最强的光学显微镜,也是世界上第一个能在普通白光照明下直接观测纳米级物体的光学显微镜。英国曼彻斯特大学研究人员和新加坡同行当天在新一期《自然·通信》杂志上报告了这项成果。由于光的衍射特性的限制,光学显微镜的观测极限通常约为1μm。研究人员通过为光学显微镜添加一种特     

Real-time computation of subdiffraction-resolution fluorescence images

In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with ∼20 nm optical resolution can be completed in less than 10 s.     

Reduction of coherent artefacts in super‐resolution fluorescence localisation microscopy

Super‐resolution localisation microscopy techniques depend on uniform illumination across the field of view, otherwise the resolution is degraded, resulting in imaging artefacts such as fringes. Lasers are currently the light source of choice for switching fluorophores in PALM/STORM methods due to their high power and narrow bandwidth. However, the high coherence of these sources often creates interference phenomena in the microscopes, with associated fringes/speckle artefacts in the images. We quantitatively demonstrate the use of a polymer membrane speckle scrambler to reduce the effect of the coherence phenomena. The effects of speckle in the illumination plane, at the camera and after software localisation of the fluorophores, were characterised. Speckle phenomena degrade the resolution of the microscope at large length scales in reconstructed images, effects that were suppressed by the speckle scrambler, but the small length scale resolution is unchanged at ～30 nm.     

3D reconstruction of high-resolution STED microscope images

Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.     

Study of the focused laser spots generated by various polarized laser beam conditions

In this work, three‐dimensional near‐field imaging of the focused laser spot was studied theoretically and experimentally. In the theoretical simulation, we use the electromagnetic equivalent of the vectorial Kirchhoff diffraction integral to calculate the intensity distribution of the focal region, and a high depolarization is found in high numerical aperture systems (NA = 0.85). The experimental set‐up is based on a near‐field scanning optical microscope (NSOM) system. A high‐NA objective lens is used to focus incident light of various polarizations, and a tapered near‐field optical fibre probe of the NSOM system is used to determine the intensity of the focal field. The results show an asymmetric distribution of the focused intensity with the linear polarized laser beam.     

A protocol for single-source dual-pulse stimulated emission depletion setup with Bessel modulation

STimulated Emission Depletion (STED) microscopy attains super-resolution in biological imaging beyond the diffraction limit. Here, we give a concise protocol to construct a dual-pulse STED setup with one super-continuum laser. Moreover, a flexible and dismountable Bessel modulation module is introduced for potential 2D-stack STED imaging. Experiments and notices are introduced in detail, with discussion on some important check-points for STED, such as detector saturation. Finally, the results validate the system working.     

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA= 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.     

Evanescent field excitation and measurement of dye fluorescence in a metallic probe near-field scanning optical microscope

We introduce a method of dye fluorescence excitation and measurement that utilizes a near-field scanning optical microscope (NSOM). This NSOM uses an apertureless metallic probe, and an optical system that contains a high numerical aperture (NA) objective lens (NA = 1.4). When the area which satisfies NA < 1 is masked, the objective lens allows for the rejection of possible transmitted light (NA < 1) through the sample. In such conditions, the focused spot consists of only the evanescent field. We found that this NSOM system strongly reduces the background of the dye fluorescence and allows for the measurement of the fluorescence intensity below the diffraction limit of the excitation source.     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Resolution enhancing using cantilevered tip-on-aperture silicon probe in scanning near-field optical microscopy

Scanning near-field optical microscopy (SNOM) achieves a resolution beyond the diffraction limit of conventional optical microscopy systems by utilizing subwavelength aperture probe scanning. A problem associated with SNOM is that the light throughput decreases markedly as the aperture diameter decreases. Apertureless scanning near-field optical microscopes obtain a much better resolution by concentrating the light field near the tip apex. However, a far-field illumination by a focused laser beam generates a large background scattering signal. Both disadvantages are overcome using the tip-on-aperture (TOA) approach, as presented in previous works. In this study, a finite difference time domain analysis of the degree of electromagnetic field enhancement is performed to verify the efficiency of TOA probes. For plasmon enhancement, silver is deposited on commercially available cantilevered SNOM tips with 20nm thicknesses. To form the aperture and TOA in the probes, electron beam-induced deposition and focused ion beam machining were applied at the end of the sharpened tip. The results show that cantilevered TOA probes were highly efficient for improvements of the resolution of optical and topological measurement of nanostructures.