单分子定位超分辨显微成像技术研究进展及展望(特邀综述)

随着新型荧光探针、先进激光、高灵敏光电探测器等相关领域的不断发展,突破衍射极限的超分辨光学显微技术为现代生物医学研究提供了新的有力工具,其中的单分子定位技术利用荧光分子的光开关效应,实现了亚细胞结构的纳米精度超分辨成像.本文介绍了单分子定位超分辨显微技术的基本原理与实现,例举了其在细胞生物学、组织生物学以及神经科学等方面的应用,讨论了该技术目前的发展趋势及可能的改进方向,为相关领域科学研究提供参考.超分辨光学显微技术的不断创新将推动生命科学的新发展.     

超分辨成像及超分辨关联显微技术研究进展

光学成像系统中有限孔径对光波的衍射,使得光学显微成像技术的分辨率受到"衍射极限"限制而无法进一步提高.自1873年E.K.Abbe提出该问题以来,衍射极限就一直是学术界研究的热点.近年来,随着高强度激光、高灵敏探测器等光电器件研制技术以及新型荧光探针开发等相关领域的快速发展,光学显微技术衍射极限问题的研究迎来了新的契机,超分辨显微成像技术(super-resolution microscopy.SRM)在近十年内取得了令人瞩目的巨大成就.本文从空域和频域角度回顾了衍射极限分辨率的基本原理,并据此对目前常见的各种SRM技术"绕过"衍射极限提高分辨率的机理给予了详解,同时介绍了各类技术的发展动态和研究方向;作为SRM的一个新的重要的发展趋势,本文详细介绍了超分辨关联显微技术的最新研究进展,包括SRM与活细胞实时荧光显微、荧光寿命显微、光谱测量和成像、电子显微、原子力显微、质谱技术等的关联,着重讨论了各类超分辨关联显微技术的作用和意义;最后,对SRM技术和超分辨关联显微技术的未来发展方向进行了展望.     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

基于环形光瞳提高荧光辐射差分超分辨显微镜的成像分辨率

荧光辐射差分(FED)显微术利用实心光斑扫描的一幅共聚焦图像减去空心光斑扫描的负共聚焦图像,实现了超分辨成像。使用简单的环形光瞳滤波器提高了无变形FED的成像分辨率。在空心焦斑光路上使用合适的环形光瞳滤波器,压缩空心焦斑的尺寸;同时,在实心焦斑的光路上允许使用更高数值孔径的孔径光阑,获得更小的、与空心焦斑相匹配的实心光斑分布:两方面作用下,提高了FED的空间分辨率。     

从光学显微镜到光学“显纳镜”

光学显微镜在生物学和医学等众多科学技术以及生产领域发挥着重要作用,分辨能力已经进入纳米尺度.本文综述了光学显微镜的放大原理、结构组成、发展历史、在生物学发展中的推动作用以及超越阿贝衍射极限实现超分辨荧光显微镜——光学显纳镜的原理和方法.光学显纳镜重点介绍了2014年获得诺贝尔化学奖的两项超分辨荧光显微技术,一是以光激活定位显微技术为代表的单分子显微技术,一是通过增加一束损耗光等效减小激发光斑大小来实现超分辨的受激发射损耗显微技术.     

平面超透镜的远场超衍射极限聚焦和成像研究进展

突破瑞利衍射极限,实现纯光学的远场超衍射极限聚焦和成像在科学和工程的各个领域都有重要意义.现有光学超分辨技术都存在一些固有的限制因素,如工作距离短、适用领域窄、不利于集成等问题.平面超透镜由于理论上的创新、设计灵活、效率高、方便集成等优势,成为实现超衍射极限的有效途径.本文综述了平面超透镜的物理原理及其在超衍射极限聚焦和成像方面近年来的研究进展,并讨论了该领域面临的问题和未来的研究重点和方向.     

基于线性成像系统的光学超分辨显微术回顾

随着纳米科学技术的发展,如何打破光学衍射极限,将光学显微术的分辨本领推进到纳米尺度,已经成为光学领域的一个核心议题.在此背景下,过去的三十年间,发展了多种超分辨光学显微技术,并在生物、材料、化学领域取得了一系列令人瞩目的应用.本文以衍射理论为线索,回顾各类基于线性成像系统的超分辨光学显微技术;对以固浸物镜、结构光照明、扫描近场光学显微术、完美透镜以及超振荡透镜为代表的超分辨光学显微技术进行综述,讨论各种技术的原理,对其特点、应用与局限加以总结,并对该领域的未来发展予以展望.     

高分辨和超分辨光学成像技术在空间和生物中的应用

大到天文光学望远镜观察浩瀚的宇宙,小到光学显微镜探察细微的纳米世界,光学成像技术在人类探索和发现未知世界奥秘的活动中扮演着至关重要的角色.看得更远、看得更细、看得更清楚是人们不断追求的目标.传统光学理论已证明所有经典光学系统都是一个衍射受限系统,即光学系统空间分辨率的物理极限是由光的波长和系统的相对孔径(或数值孔径)决...     

高分辨和超分辨光学成像技术在空间和生物中的应用

大到天文光学望远镜观察浩瀚的宇宙, 小到光学显微镜探察细微的纳米世界, 光学成像技术在人类探索和发现未知世界奥秘的活动中扮演着至关重要的角色. 看得更远、看得更细、看得更清楚是人们不断追求的目标. 传统光学理论已证明所有经典光学系统都是一个衍射受限系统, 即光学系统空间分辨率的物理极限是由光的波长和系统的相对孔径(或数值孔径)决定的. 能否突破这个极限？能否不断提高光学系统的成像分辨率？围绕着这个问题, 本文综述了近年来开展的各种光学高分辨和超分辨成像技术, 及其在空间探测和生物领域中的应用.     

Resolution enhancement techniques for optical lithography and optical imaging theory

Production of a fine pattern is necessary to get a high integration degree of integrated circuits. The conventional methods which utilize high numerical aperture and short wavelength exposure are limited by designing and manufacturing of a practical lens and make the focus depth narrow. Resolution enhancement techniques (RETs) have, therefore, been required and proposed. This paper introduces a phase-shifting mask, a typical RET, points out the problems and inconsistencies of conventional optical imaging theory and explains the image formation concept of expansion of plane waves. Essentially using this concept, an attempt is also made to describe some other typical RETs with potential.     

Phase reconstruction in lensless digital in-line holographic microscopy

The phase reconstruction in a digital in-line holographic microscopy is compared using two numerical reconstruction methods. The first method uses one Fourier transform and second one uses three Fourier transforms. It is shown that the latter method gives improved object phase reconstruction as compared to the former.     

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.

     

New directions in X-ray microscopy

The development of high brightness X-ray sources and high resolution X-ray optics has led to rapid advances in X-ray microscopy. Scanning microscopes and full-field instruments are in operation at synchrotron light sources worldwide, and provide spatial resolution routinely in the 25–50 nm range using zone plate focusing elements. X-ray microscopes can provide elemental maps and/or chemical sensitivity in samples that are too thick for electron microscopy. Lensless techniques, such as diffraction microscopy, holography and ptychography are also being developed. In high resolution imaging of radiation-sensitive material the effects of radiation damage needs to be carefully considered. This article is designed to provide an introduction to the current state and future prospects of X-ray microscopy for the non-expert.     

Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

Correlation plenoptic imaging for microscopy applications

Plenoptic microscopy is a promising technique which allows refocusing and depth-of-field enhancement, in post-processing, as well as scanning free 3D imaging. However, in its conventional implementation, spatial resolution is highly sacrificed and cannot reach the diffraction limit set by the numerical aperture of the imaging system. We recently proposed a novel method, named Correlation Plenoptic Imaging (CPI), based on measuring intensity correlation of either chaotic or entangled photon light sources. However, such protocols are not well suited for microscopic purposes: they cannot be employed with scattering or fluorescent samples and are extremely sensitive to diffusive effects. Here we consider and compare novel CPI protocols which overcome these problems and enable to perform plenoptic microscopy at the diffraction limit for generic samples; we present both theory and simulations, discuss the improved robustness with respect to previous protocols against turbulence around the sample, and highlight the physical limits of the proposed technique.     

Sectional image reconstruction and three-dimensional microscopy of small particles by angular spectrum method

In this letter, we demonstrate sectional image reconstruction and three-dimensional microscopy of small particles. We demonstrate sectional image reconstruction and holographic methods to obtain 2D and 3D images of small particles. A single hologram is sufficient to obtain a section containing only the focused parts of the reconstructed image. One can obtain images of different plane sections of a specimen in addition to its 3D display. The reconstruction of a digital hologram is based on the plane-wave expansion of the diffracted wave fields using Fourier optics (this method is also known as the angular spectrum method). With this method, the object-to-hologram distance can be quite small because the minimum-distance requirement does not apply. Furthermore, numerical reconstruction of transparent objects by this method may be interesting for micro-structure measurement.     

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。