可用于活细胞线粒体随机光学重构超分辨成像的分子内可逆环化五甲川菁染料探针

基于单分子定位的随机光学重构超分辨成像作为一种先进的光学成像方法,可用于尺寸小于光学衍射极限的生物结构的超清晰成像,为在单分子层面研究疾病的发病机制及寻找精准的治疗策略提供有力研究工具,在生物医学领域有着广泛的应用前景.随机光学重构超分辨成像技术依赖于标记探针的光物理性质,探针需要在大量缓冲试剂及含巯基试剂存在下才能产生稳定光致闪烁进行超分辨成像,获得理想的超分辨成像结果,但是大量缓冲试剂与巯基试剂对活细胞伤害较大,使得其在活细胞的超分辨成像应用上存在困难,而限制了其在生物医学成像领域的进一步应用,因此,需要开发可用于活细胞的单分子定位超分辨成像的新型光学探针.本工作提出了一种新的可用于单分子定位超分辨成像的五甲川菁染料探针,不需要外加成像缓冲液及巯基试剂就可以产生光致闪烁变化.基于此,开发了一种分子内自发开、关环反应的新型五甲川菁染料探针,具有活细胞膜通透性.探针不需要使用缓冲液体系及对细胞有害的含巯基试剂,在低功率单束激光直接照射下产生光致闪烁,探针对活细胞没有产生明显毒性,适合活细胞的超分辨成像.进入活细胞后探针选择性定位于细胞线粒体上,在激光照射下产生光致闪烁,电子倍增电荷耦合器相机（EMCCD）在采样频率60 Hz下收集不同条件下的光致闪烁图像,设置不同参数进行结果分析,使用ImageJ进行图像预处理后再使用Falcon算法重构获得活细胞线粒体的超分辨成像图像,相比宽场成像,成像分辨率明显提高,为生物医学光学成像提供新的研究手段.     

多种超分辨荧光成像技术比较和进展评述

所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。     

浅谈2014年诺贝尔化学奖:超高分辨率荧光显微成像

超分辨显微成像技术是近些年来发展最快、受关注度最高的光学成像技术之一。这类技术突破了光学衍射极限,将显微镜的分辨率从几百纳米提高到几十纳米,为生命科学研究提供了一个强大工具。目前主流的超分辨率显微技术主要基于点扩散函数调制和单分子定位的原理来实现。其主要贡献者也成为2014年诺贝尔化学奖的获得者。本文简要讲述超分辨显微技术的发展历程并对其发展趋势进行展望。     

受激辐射耗尽超分辨显微成像的研究进展及应用

荧光显微镜凭借其非接触、无损伤、可实时探测生物样品内部等优点,成为生命科学研究中不可或缺的工具,但是Abbe光学衍射极限的存在限制了其对亚细胞尺度生化过程的进一步研究。作为突破光学衍射极限的远场光学显微镜,受激辐射耗尽(Stimulated Emission Depletion,STED)超分辨荧光显微术,因其超高时空分辨率和三维层析能力,成为备受关注的新型成像和分析表征工具。本文结合我们课题组在STED显微成像研究方面的工作,综述了近年来STED显微成像技术的研究进展及其在细胞成像中的应用。     

光激活定位超分辨荧光成像的罗丹明分子设计

光激活定位显微技术(PALM)等超分辨成像技术的发展为高水平生命科学研究提供了突破光学衍射极限的研究工具.实现超分辨荧光成像的核心要素之一是高性能荧光染料,而罗丹明染料由于其优良的光学性质已成为设计PALM染料的热门选择.本文通过对PALM超分辨成像特点分析,给出PALM罗丹明染料的设计要求,并对已报道的PALM超分辨罗丹明染料的设计策略及其优缺点进行比较分析,希望通过对分子设计策略的综述,为研究者设计PALM超分辨罗丹明染料提供了一些帮助.     

单个量子点的光学性质与应用

量子点(QDs)是一种具有诸多优良光学特性的荧光纳米颗粒,已在化学分析、生物传感、分子影像等领域得到了广泛应用.单个量子点的光学性质研究有望发现一些宏观方法不能发现的实验现象,可以为改善其光学性能提供思路,有助于更好的应用于各领域.本文评述了单个量子点的检测与判定方法,单个量子点的荧光增强、漂白、眨眼(blinking)、蓝移等光学性质及其在单分子示踪、生物化学传感、超分辨定位技术等方面的应用.总结了目前量子点作为荧光探针在实际应用中遇到的问题,并提出未来量子点将朝着合成能同时满足尺寸小、量子产率高、 "non-blinking"、蓝移幅度大、无生物毒性的量子点及能同时为成像/检测提供荧光探针与散射探针的等离子体量子点等研究方向发展.     

基于散射信号的超分辨光学相减显微镜

现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力。因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率。不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响。随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ, 1λ = 650 nm),并且通过探测散射信号得到更多的样品细节信息。     

基于抑制扭转的分子内电荷转移(TICT)提升有机小分子荧光染料亮度及光稳定性※

有机小分子荧光染料研究已有170余年历史, 其结构和性能随着合成方法和应用需求的发展而不断革新, 已被广泛应用于荧光标记、探针和生物成像中. 近年来发展起来的超分辨荧光成像技术对有机小分子荧光染料的亮度、稳定性和开关性能等均提出了更高的要求, 这为染料发展带来了新的机遇. 当前, 化学工作者也将更多精力聚焦在染料结构改造提升有机小分子荧光染料的亮度与光稳定性. 激发态扭转的分子内电荷转移(TICT)是有机小分子荧光染料中主要的非辐射衰减途径之一. 因而, 抑制TICT能够很好地提升染料的亮度和光稳定性, 并成为目前针对超分辨成像技术发展高亮度和光稳定性的有机小分子荧光染料的主要方法. 本综述首先简要回顾了TICT的机制和发展过程, 而后重点介绍近些年通过抑制TICT策略来提升不同结构有机小分子荧光染料光谱性能方面的进展.     

单分子宽场光学显微成像技术

单分子宽场光学显微成像技术是单分子检测技术的一种,具有通量高、参数多样、可实时动态监测等优点。本文评述了单分子宽场光学显微成像的技术方法、标记探针、判定原则、检测参数及其在分析化学、生物物理学等领域的应用,指出单分子成像技术正在向仪器设备的实用化、简易化,测量参数的精确化、可视化,研究范围的广泛化、复杂化等方面发展。未来几年单分子成像的研究重点可能会集中在实用定量、突破衍射极限的距离测量、重要生物过程的机理探索和纳米目标物的表征等方面。     

半导体共轭聚合物光学探针的设计及在自发光成像和光声成像中的应用

光学成像因其无侵袭性、高时空分辨率和高灵敏度在生物医学领域得到迅速发展.光学成像中自发光成像包括化学发光成像和长余辉成像不需实时光激发,避免了自发荧光的影响,可以得到较高的灵敏度和信噪比.光声成像则是将光信号通过热膨胀转化为声信号,避免了光散射的影响,具有较高的组织穿透深度.本文针对半导体共轭聚合物光学探针在自发光成像和光声成像技术中的应用进行综述,重点介绍了半导体共轭聚合物光学探针用于增强自发光成像、光声成像的信号强度的设计策略,以及响应型光声探针的设计原理.阐述了通过降低光学探针与发光底物之间的能隙等策略增强自发光成像信号强度,通过淬灭荧光或加速热扩散等策略放大光声信号,以及通过特异性生物分子识别或相互作用激活的响应型光声探针的具体研究成果.最后,对半导体共轭聚合物光学探针在光学成像领域存在的挑战和前景进行了展望.     

Photochemical Mechanisms of Fluorophores Employed in Single-Molecule Localization Microscopy

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super-resolution imaging technologies has empowered biomedical researchers with the ability to answer long-standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super-resolution imaging experiments. Here, we introduce key super-resolution imaging technologies, with a brief discussion on single-molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next-generation fluorescent probes amenable to single-molecule imaging.     

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super‐resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of super‐resolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene‐based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.     

In Situ Visualization of Block Copolymer Self‐Assembly in Organic Media by Super‐Resolution Fluorescence Microscopy

Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

Nanoprobes for super-resolution fluorescence imaging at the nanoscale

Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of<200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.     

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.     

Improved resolution in single-molecule localization microscopy using QD-PAINT

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.Subject terms: Fluorescence imaging, Quantum dots, Oligonucleotide probes, Fluorescent dyes, Super-resolution microscopy     

Universal Super‐Resolution Multiplexing by DNA Exchange

Super‐resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time‐consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super‐resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.     

Fluorescence Microscopy with 6 nm Resolution on DNA Origami

Resolution of emerging superresolution microscopy is commonly characterized by the width of a point‐spread‐function or by the localization accuracy of single molecules. In contrast, resolution is defined as the ability to separate two objects. Recently, DNA origamis have been proven as valuable scaffold for self‐assembled nanorulers in superresolution microscopy. Here, we use DNA origami nanorulers to overcome the discrepancy of localizing single objects and separating two objects by resolving two docking sites at distances of 18, 12, and 6 nm by using the superresolution technique DNA PAINT(point accumulation for imaging in nanoscale topography). For the smallest distances, we reveal the influence of localization noise on the yield of resolvable structures that we rationalize by Monte Carlo simulations.     

Reductively Caged,Photoactivatable DNA‐PAINT for High‐Throughput Super‐resolution Microscopy

In DNA points accumulation in nanoscale topography (DNA‐PAINT), capable of single‐molecule localization microscopy with sub‐10‐nm resolution, the high background stemming from the unbound fluorescent probes in solution limits the imaging speed and throughput. Herein, we reductively cage the fluorescent DNA probes conjugated with a cyanine dye to hydrocyanine, acting as a photoactivatable dark state. The additional dark state from caging lowered the fluorescent background while enabling optically selective activation by total internal reflection (TIR) illumination at 405 nm. These benefits from “reductive caging” helped to increase the localization density or the imaging speed while preserving the image quality. With the aid of high‐density analysis, we could further increase the imaging speed of conventional DNA‐PAINT by two orders of magnitude, making DNA‐PAINT capable of high‐throughput super‐resolution imaging.