微球透镜超分辨显微成像与检测技术综述

受衍射极限的影响,传统光学显微镜的分辨率最高约为波长的一半,突破衍射极限,获得更高的成像分辨率是近年来显微成像领域的研究热点。相比于其他超分辨显微成像方式,基于微球透镜的超分辨显微成像方式具有简单直接、免标记等优点。主要介绍国内外研究团队将微球与传统的光学显微镜结合实现超分辨显微成像的研究进展,从微球透镜参数选择、成像方案、成像分辨率、成像视场及成像机理等多角度进行总结与比对;并结合课题组工作,介绍了将微球透镜与干涉显微技术相结合的三维超分辨检测技术,阐述了Linnik型与Mirau型两种检测光路原理,分析了三维超分辨检测的效果;展望了微球透镜超分辨显微技术在显微成像与显微干涉检测两个方面待解决的问题与发展方向。     

Manipulation of light at the nanoscale for high-performance spectroscopic and optical applications

In this paper, we explore the use of nanostructures for a number of fascinating applications. These applications based on nanostructures include (1) optical sensors, (2) nanopixel printing, (3) improving the resolution of imaging techniques, and (4) lithography. In the sensing field, nanostructures are exploited for advanced sensor performance, namely, the label-free and enhanced sensitivity of (1) the surface plasmon resonance sensor and (2) the extraordinary optical transmission sensor and (3) the high sensitivity and selectivity of surface-enhanced Raman spectroscopy. In addition, research using nanostructures for visual applications was introduced for (1) harnessing nanostructures for full-color pixel printing and (2) exploiting metallic nanostructures to enhance the imaging resolution under diffraction limits based on the plasmonic effect. Finally, we introduce low cost, high accuracy, and fast lithographic methods based on the plasmonic effect by exploiting metallic nanostructures.     

Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination

We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.     

Tomographic diffractive microscopy with a wavefront sensor

Tomographic diffractive microscopy is a recent imaging technique that reconstructs quantitatively the three-dimensional permittivity map of a sample with a resolution better than that of conventional wide-field microscopy. Its main drawbacks lie in the complexity of the setup and in the slowness of the image recording as both the amplitude and the phase of the field scattered by the sample need to be measured for hundreds of successive illumination angles. In this Letter, we show that, using a wavefront sensor, tomographic diffractive microscopy can be implemented easily on a conventional microscope. Moreover, the number of illuminations can be dramatically decreased if a constrained reconstruction algorithm is used to recover the sample map of permittivity.     

Wide-field high-resolution structured illumination solid immersion fluorescence microscopy

The use of aplanatic solid immersion lenses (ASILs) made of high-refractive-index optical materials provides a route to wide-field high-resolution optical microscopy. Structured illumination microscopy (SIM) can double the spatial bandwidth of a microscope to also achieve high-resolution imaging. We investigate the combination of ASILs and SIM in fluorescence microscopy, which we call structured illumination solid immersion fluorescence microscopy (SISIM), to pursue a microscopic system with very large NA and high lateral resolution. We demonstrate that the combination can produce a wide-field high-resolution microscopic system with bandwidth corresponding to an NA of 3. Future developments of the SISIM system to make it achieve even higher resolution are proposed.     

基于双螺旋点扩散函数工程的多焦点图像扫描显微

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,本文提出了一种基于双螺旋点扩散函数工程的多焦点图像扫描显微成像方法和系统.在照明光路中,利用高速数字微镜器件产生周期分布的聚焦点阵对样品进行并行激发和快速二维扫描;在探测光路中,利用双螺旋相位片将激发点荧光信号的强度分布转换为双螺旋的形式;最终,利用后期数字重聚焦处理,从单次样品扫描数据中重构出多个样品层的超分辨宽场图像.在此基础上,利用搭建的系统分别对纤维状肌动蛋白和海拉细胞线粒体进行成像实验,证明了该方法的超分辨能力和快速三维成像能力.     

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.     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

Super-resolution imaging of low-contrast periodic nanoparticle arrays by microsphere-assisted microscopy

We use the label-free microsphere-assisted microscopy to image low-contrast hexagonally close-packed polystyrene nanoparticle arrays with diameters of 300 and 250 nm.When a nanoparticle array is directly placed on a glass slide,it cannot be distinguished.If a 30-nm-thick Ag film is deposited on the surface of a nanoparticle array,the nanoparticle array with nanoparticle diameters of 300 and 250 nm can be distinguished.In addition,the Talbot effect of the 300-nm-diameter nanoparticle array is also observed.If a nanoparticle sample is assembled on a glass slide deposited with a 30-nm-thick Ag film,an array of 300-nm-diameter nanoparticles can be discerned.We propose that in microsphere-assisted microscopy imaging,the resolution can be improved by the excitation of surface plasmon polaritons(SPPs) on the sample surface or at the sample/substrate interface,and a higher near-field intensity due to the excited SPPs would benefit the resolution improvement.Our study of label-free super-resolution imaging of low-contrast objects will promote the applications of microsphere-assisted microscopy in life sciences.     

光学超振荡与超振荡光学器件

传统光学器件的衍射极限极大地制约了远场超分辨光学系统的进一步发展.如何从光学器件层面突破光学衍射极限瓶颈,实现非标记远场超分辨光学成像,是光学领域面临的巨大挑战.光学超振荡在不依靠倏逝波的条件下,可以在远场实现任意小的亚波长光场结构,这为突破光学衍射极限提供了一条崭新的途径.近年来,光学超振荡现象和超振荡光学器件的相关研究得到了快速发展,在理论和实验上成功地演示了超振荡光场的产生和多种超振荡光学器件,并在实验上展示了超振荡光学器件在非标记远场超分辨光学显微、成像以及超高密度数据存储等应用领域的巨大优势和应用潜力.本文对光学超振荡相关理论、超振荡光学器件设计理论和方法、超振荡光学器件发展现状、超振荡光场测试方法以及超振荡光学器件的应用等方面进行详细介绍和分析.     

Label-free in vivo fiber-based optical-resolution photoacoustic microscopy

In this Letter, the capability of label-free fiber-based optical-resolution photoacoustic microscopy is demonstrated. This real-time imaging system takes advantage of image-guide fibers and a unique fiber laser. The 800?μm image-guide consists of 30,000 individual single-mode fibers in a bundle and the diode-pumped, pulsed Ytterbium fiber laser is utilized to perform up to 600?kHz repetition rate. Phantom studies indicate 7?μm resolution. The proposed setup keeps many of the powerful properties of previous tabletop OR-PAM systems, but also offers a submillimeter probe footprint and high flexibility due to the nature of the image-guide. This system could have significant clinical impact for endoscopic applications where the thin fiber can be inserted into the body.     

结构光照明超分辨光学显微成像技术与展望

结构光照明显微镜(Structured Illumination Microscopy,SIM)通过结构化照明在频率域以空间混频的方式将物体高频信息载入光学系统的探测通带内实现突破衍射极限的超分辨光学显微成像。SIM凭借其较低的激发光强、对荧光染料的非特异性需求以及快速的宽场成像优势已成为活细胞超分辨光学显微成像方面应用最多的技术。本文系统回顾了SIM的技术进展,对SIM的基本原理与实现方法进了详细的分析,重点介绍了本课题组研发的基于光谱分辨的单光子激发超分辨显微镜和结合自适应光学的双光子激发超分辨显微镜这两种最新的SIM技术,最后简要讨论了SIM技术在生物成像中的应用及未来发展方向。     

Wide-field optical sizing of single nanoparticles with 10 nm accuracy

There is an increasing demand for new technologies to rapidly measure individual nanoparticles in situ for applications,including early-stage diagnosis of human diseases and environmental monitoring. Here, we demonstrate a label-free wide-field optical microscopy capable of sizing dispersed non-luminescent dielectric nanoparticles(with diameters down to 22 nm) with 10 nm accuracy. This technique utilizes enhanced nanoparticle-perturbed scattering by surface plasmons created on a gold film.In the meantime, an azimuthal rotation illumination module is installed on this microscope and a differential image processing technique is carried out. The relationship between the scattering intensity and the particle size was experimentally measured with good consistency with the theoretical prediction. The capability of precise measurement of the size of dispersed nanoparticles within a larger field of view in a label-free, non-invasive and quantitative manner may find broad applications involving single nanoparticle chemistry and physics.     

Femtosecond Kerr-gated wide-field fluorescence microscopy

We present a Kerr-gated microscope capable of collecting diffraction-limited 2D fluorescence images with sub-100 fs time resolution. The concept is based on the insertion of a solid-state optical Kerr gate into a wide-field microscope. In addition to the considerably improved temporal resolution, the wide-field design allows for simultaneous tracking of several objects and ultrafast fluorescence lifetime imaging of doped and heterogeneous surfaces. The ultrafast fluorescence dynamics of gold nanoparticles is presented as an example of the capabilities of the instrument.     

Transillumination spatially modulated illumination microscopy

The diagnostic utility of a conventional transillumination microscope, the most common imaging modality in clinical use today, is limited by the microscope's resolution. It is, however, possible to achieve lateral resolution well beyond the classical limit by using laterally structured illumination in a wide-field, nonconfocal microscope. In this method, the spatially modulated illumination (SMI) makes high-resolution information that is normally inaccessible visible in the observed image. Previously presented SMI microscopy systems operated in epifluorescence mode. We describe the design, construction, and testing of a novel transillumination SMI microscope. As transillumination is necessary for most medical applications, such as histopathologic evaluation of biopsy tissue and chromosomal analysis, such a system should have a significant diagnostic effect.     

Plasmonics-based spatially activated light microscopy for super-resolution imaging of molecular fluorescence

In this Letter, we explore plasmonics-based spatially activated light microscopy (PSALM) for sub-diffraction-limited imaging of biomolecules. PSALM is based on the spatially switched activation of local amplified electromagnetic hot spots under multiple light incidence conditions. The hot spots are associated with surface plasmons that are excited and localized by surface nanostructures. The feasibility of the concept was demonstrated by imaging fluorescent nanobeads on a two-dimensional gold nanograting of a 100-nm-wide grating ridge, the size of which is the measure of the imaging resolution. The result confirms the performance of PSALM for imaging nanobeads at a resolution below the conventional diffraction limit.     

表面等离激元结构光照明显微成像技术研究进展

结构光照明显微成像技术(SIM)因其高分辨、宽场、快速成像的优势,在生物医学成像领域发挥了不可估量的作用.结构光照明显微成像技术与动态可控的亚波长表面等离激元条纹相结合,可以在不借助非线性效应的情况下,将传统SIM的分辨率从2倍于衍射极限频率提升到3 4倍,此外还有抑制背景噪声、提升信噪比的能力,在近表面的生物医学成像应用中有重要价值.本文介绍了表面等离激元结构光照明显微成像技术的原理,并总结了近几年国内外的相关研究进展.     

Fighting against diffraction: apodization and near field diffraction structures

Diffraction is a natural phenomenon, which occurs when waves propagate or encounter an obstacle. Diffraction is also a fundamental aspect of modern optics: all imaging systems are diffraction systems. However, like a coin has two sides, diffraction also leads to some unfavorable effects, such as an increase in the size of a beam during propagation, and a limited minimal beam size after focusing. To overcome these disadvantages set by diffraction, many techniques have been developed by various groups, including apodization techniques to reduce the divergence of a laser beam and increase the resolution, and time reversal, STED microscopy, super lenses and optical antennas to obtain resolution down to nano‐scale. This review concentrates on the diffraction of electromagnetic waves, and the ways to overcome beam divergence and the diffraction limit.     

Localization microscopy (SPDM) facilitates high precision control of lithographically produced nanostructures

Nanoscale resolution in material sciences is usually restricted to scanning electron beam microscopes. Here we present a procedure that allows single molecule resolution of the sample surface with visible light. Highlighting the performance we used electron beam lithography to generate highly regular nanostructures consisting of interconnected cubes. The samples were labeled with Alexa 647 dyes. The spatial organization of the dyes on nanostructured surfaces was localized with single molecule resolution using localization microscopy. This succeeded also in an absolute spatial calibration of the localization method applied (spectral precision distance microscopy/SPDM). The findings will contribute to the field of product control for industrial applications and long-term fluorescence imaging.