Long-term super-resolution imaging of mitochondrial dynamics

Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.     

Long-term super-resolution imaging of mitochondrial dynamics

Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.     

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.     

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

BODIPY 493 acts as a bright buffering fluorogenic probe for super-resolution imaging of lipid droplet dynamics

The need for temporal resolution and long-term stability in super-resolution fluorescence imaging has motivated research to improve the photostability of fluorescent probes. Due to the inevitable photobleaching of fluorophores, it is difficult to obtain long-term super-resolution imaging regardless of the self-healing strategy of introducing peroxide scavengers or the strategy of fluorophore structure modification to suppress TICT formation. The buffered fluorogenic probe uses the intact probes in the buffer pool to continuously replace the photobleached ones in the target, which greatly improves the photostability and enables stable dynamic super-resolution imaging for a long time. But the buffering capacity comes at the expense of reducing the number of fluorescent probes in targets, resulting in low staining fluorescence intensity. In this paper, we selected BODIPY 493, a lipid droplet probe with high fluorescence brightness, to explore the dynamic process of lipid droplet staining of this probe in cells. We found that BODIPY 493 only needs very low laser power for lipid droplet imaging due to the high molecular accumulation in lipid droplets and the high brightness, and the spatiotemporal resolution is greatly improved. More importantly, we found that BODIPY 493 also has a certain buffering capacity, which enables BODIPY 493 to be used for super-resolution imaging of lipid droplet dynamics. This work reminds researchers to coordinate the buffering capacity and brightness of fluorogenic probes.     

单分子定位超分辨显微成像有机荧光探针的研究进展

在生物医学领域,对纳米尺寸级别的微小生物目标进行精确定位研究具有非常重要的意义,而光学显微成像技术为此提供了强有力的工具。 光学显微成像技术受到光学衍射极限的限制,难以分辨尺寸在衍射极限(<200 nm)以下的生物结构,无法直接获取微小生物结构信息,阻碍了生物医学的进一步发展。 近年来,随着纳米分辨显微成像技术的出现,新型荧光探针的开发、成像系统与设备的不断发展及成像算法不断完善地深入结合,促进了光学衍射极限以下尺寸微观目标的研究。 基于单分子定位的超分辨荧光显微成像(SMLM)包括光激活定位成像(PALM)与随机光学重构超分辨成像(STORM),将有机荧光探针与超分辨光学显微成像技术紧密结合在一起,荧光探针的光物理性质直接决定着超分辨成像结果的好坏。 因此,设计不同性能的荧光探针可以实现超精细结构的不同超分辨成像,为研究其生物学功能提供了有力的工具。 本文着重围绕基于SMLM的原理、有机荧光探针的设计要求、用于SMLM的荧光探针种类及其生物应用等方面进行总结综述,指出了单分子定位成像上存在的不足,并对其发展方向进行了展望,希望为对超分辨成像研究感兴趣或初涉该领域的研究者提供成像理论与探针设计方面的帮助。     

Far Red-Shifted CdTe Quantum Dots for Multicolour Stimulated Emission Depletion Nanoscopy

Stimulated emission depletion (STED) nanoscopy is a widely used nanoscopy technique. Two-colour STED imaging in fixed and living cells is standardised today utilising both fluorescent dyes and fluorescent proteins. Solutions to image additional colours have been demonstrated using spectral unmixing, photobleaching steps, or long-Stokes-shift dyes. However, these approaches often compromise speed, spatial resolution, and image quality, and increase complexity. Here, we present multicolour STED nanoscopy with far red-shifted semiconductor CdTe quantum dots (QDs). STED imaging of the QDs is optimized to minimize blinking effects and maximize the number of detected photons. The far-red and compact emission spectra of the investigated QDs free spectral space for the simultaneous use of fluorescent dyes, enabling straightforward three-colour STED imaging with a single depletion beam. We use our method to study the internalization of QDs in cells, opening up the way for future super-resolution studies of particle uptake and internalization.     

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.     

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。     

Make them Blink: Probes for Super‐Resolution Microscopy

In recent years, a number of approaches have emerged that enable far‐field fluorescence imaging beyond the diffraction limit of light, namely super‐resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super‐resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single‐molecule compatible fluorophore can be used for super‐resolution microscopy. We present examples for super‐resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super‐resolution microscopes including AFM‐based single‐molecule assembly and DNA origami.     

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。     

Silicon-substituted rhodamines for stimulated emission depletion fluorescence nanoscopy

As an essential part in the toolbox of super-resolution microscopy, stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging. Among the applied STED fluorophores, silicon-substituted rhodamines(SiRs) belong to one of the most extensively employed fluorophores. The carboxy-SiR was favored in STED bioimaging with many advantages, including reliable photostability, cell permeability, tunable fluorogenicity, feasibl...     

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。     

A Multi‐responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super‐resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long‐standing challenge because it requires super‐resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super‐resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

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.     

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Principle has it that even the most advanced super‐resolution microscope would be futile in providing biological insight into subcellular matrices without well‐designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small‐molecule fluorescent probes that not only allow cellular‐level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle‐anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.     

亲水性二芳基乙烯荧光分子开关的研究进展

二芳基乙烯荧光分子开关因具有优良的抗疲劳性和双稳态特征而被广泛地研究与应用,亲水化成为其作为荧光开关探针走向应用的关键点之一。本文综述了亲水性二芳基乙烯荧光分子开关当前的研究进展,归纳了实现亲水性的几种重要途径和结构,分析了各种亲水化方法的优缺点,并着重介绍了亲水性二芳基乙烯荧光分子开关作为荧光开关探针在化学传感、生物传感、生物成像以及超分辨成像等领域的应用现状,并指出当前应用研究中存在的一些问题,同时也对其未来的应用前景进行了展望。     

Enhanced dSTORM imaging using fluorophores interacting with cucurbituril

Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes or proteins as fluorophores. The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy’s localization precision and imaging resolution. Fluorophores TAMRA and Atto Rho6G, which can interact with macrocyclic host cucurbit[7]uril (CB7) to form host-guest compounds, were found to improve the fluorescence intensity and lifetimes of these dyes. We enhanced the localization precision of direct stochastic optical reconstruction microscopy (dSTORM) by introducing CB7 into the imaging buffer, and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6G increase over 2 times.     

Fluorescence Nanoscopy with Optical Sectioning by Two‐Photon Induced Molecular Switching using Continuous‐Wave Lasers

During the last decade far‐field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous‐wave laser beam. This bright marker enables strict control of on/off switching and provides single‐molecule localization precision down to 15 nm in the focal plane. Two‐photon induced nonlinear photoswitching of this marker with continuous‐wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost‐effective fluorescence nanoscopy with noninvasive optical sectioning.     

Cyclo-Ketal Xanthene Dyes: A New Class of Near-Infrared Fluorophores for Super-Resolution Imaging of Live Cells

Newly emerging super-resolution imaging techniques provide opportunities for precise observations on cellular microstructures. However, they also impose severe demands on fluorophores. Here, we develop a new series of NIR xanthene dyes, named as KRh s, by replacing the 10-position O of rhodamines with a cyclo-ketal. KRh s display an intense NIR emission peak at 700 nm with fluorescence quantum yields up to 0.64. More importantly, they, without the aid of enhancing buffer, exhibit stochastic fluorescence off–on switches to support time-resolved localization of single fluorophore. KRh s are functionalized into KRh-MitoFix , KRh-Mem and KRh-Halo that demonstrate mitochondria, plasma membrane and fusion protein targeting ability, respectively. Consequently, these KRh probes demonstrate straightforward usage for super-resolution imaging of these targets in live cells. Therefore, KRh s merit future development for fluorescence labeling and super-resolution imaging in the NIR region.