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。     

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.     

Monitoring intracellular pH fluctuation with an excited-state intramolecular proton transfer-based ratiometric fluorescent sensor

Intracellular pH is a key parameter related to various biological and pathological processes. In this study, a ratiometric pH fluorescent sensor ABTT was developed harnessing the amino-type excited-state intramolecular proton transfer (ESIPT) process. Relying on whether the ESIPT proceeds normally or not, ABTT exhibited the yellow fluorescence in acidic media, or cyan fluorescence in basic condition. According to the variation, ABTT behaved as a promising sensor which possessed fast and reversible response to pH change without interference from the biological substances, and exported a steady ratiometric signal (I₄₇₈/I₅₄₆). Moreover, due to the ESIPT effect, large Stokes shift and high quantum yield were also exhibited in ABTT. Furthermore, ABTT was applied for monitoring the pH changes in living cells and visualizing the pH fluctuations under oxidative stress successfully. These results elucidated great potential of ABTT in understanding pH-dependent physiological and pathological processes.     

一锅法可控合成金属有机框架材料Mn-荧光素用于核磁共振/荧光成像

通过一锅法,首次将核磁共振成像试剂Mn²⁺和荧光成像试剂荧光素（FSD）自组装于一个简单的金属有机框架材料（Mn-FSD）上。实验结果表明,Mn-FSD的粒径可被控制在微纳米水平内并进行生物成像。体外和体内实验结果证实,Mn-FSD可在细胞和裸鼠中显示强绿色荧光。同时,Mn-FSD表现出较高的弛豫值（r₁=4.95 L·mmol^-1·s^-1）。     

Accessing the spatiotemporal heterogeneities of single nanocatalysts by optically imaging gas nanobubbles

Catalysts that catalyze the generation of products in the gas phase, especially those involved in the hydrogen evolution reaction (HER), hold great promise for ecofriendly and sustainable energy development. In general, gas chromatography is widely used to measure catalytic activity. Unfortunately, it gives an averaged output that washes out the heterogeneities among individuals. To assess the unique catalytic properties at the single nanoparticle level, various methods based on single particle catalysis have been proposed. Over the past fifteen years, tremendous breakthroughs have been achieved, which uncovered hidden spatial and temporal heterogeneities. Although powerful, effectively quantifying the activities of single HER nanocatalysts remains challenging because of the fast diffusion of hydrogen (H₂). In 2017, a novel approach based on a nanobubble indicator was proposed to correlate the kinetics of gas bubble evolution with the catalytic activities of individual nanoentities during the HER process. Since then, a plethora of optical microscopy techniques have been utilized to monitor dynamically evolved nanobubbles and to measure the catalytic activities of single HER catalysts. In this minireview, we summarized state-of-the-art optical microscopy for in operando imaging of dynamic nanobubbles involved in gas-generating reactions while highlighting some important discoveries, including the blinking photocatalytic activity and heterogeneous distribution of active sites. Finally, challenges and future perspectives in this promising field were identified.     

单颗粒光散射显微成像技术在生化医药分析中的应用

等离子体纳米颗粒(PNPs)具有体积小、易表面修饰、生物相容性好、毒性低等优点,在生物传感、生物成像、疾病诊断、肿瘤治疗、材料科学等领域得到了广泛的应用。PNPs的光散射光学性质可以通过调节其大小、组成、形貌和微环境来控制,可用于生化和药物分析。此外,由于单粒子散射显微技术具有高空间分辨率和高灵敏度,借助PNPs具有的独特局域表面等离子体共振(LSPR)特性,可在单颗粒水平进行实时成像。根据PNPs的大小、组成、形态、微环境或耦合变化引起的信号变化,研究人员发展了多种显微成像分析方法,主要分为4种,包括散射光谱的波长位移、单粒子散射强度的变化、高通量RGB分析和计数方法。基于纳米颗粒LSPR散射光谱位移变化的方法准确、灵敏,但需要昂贵的单颗粒散射光谱仪和复杂的操作。基于纳米颗粒散射强度变化的方法简单可行,但易受纳米颗粒粒径和曝光时间等因素的影响。高通量RGB分析方法灵敏度高、成本低,但不适用于颜色变化不明显的单颗粒分析,且重复性差。单粒子计数法灵敏度高,但有时粒子分布不均匀,背景杂质的干扰限制了方法的准确度。因此,这4种定量方法各有优缺点。此外,近年来逐渐发展了一些新的定量方法。例如,研究人员开发了新的时间分辨分析定量方法,并将暗场显微镜与偏振器、滤光片等光学器件相结合以消除背景干扰,以及与电化学、拉曼等仪器相结合以扩大应用范围。此外,为提高分析方法的准确度和灵敏度,暗场显微镜与深度学习、云计算、人工智能等现代计算机科学技术的结合,越来越受到人们的欢迎。基于以上原因,该文重点介绍了单粒子光散射显微镜在生化和药物分析领域的应用,总结了近年来的最新研究进展,讨论了单粒子光散射显微镜在定量分析中的几种主要定量方法,提出了未来的发展趋势,以期为相关研究领域的新人提供一定的学术参考。     

Donor-Acceptor Typed AIE Luminogens with Near-infrared Emission for Super-resolution Imaging

Aggregation-induced emission(AIE)luminogens(AIEgens)with high brightness in aggregates exhibit great potentials in biological imaging,but these AIEgens are seldom applied in super-resolution biological imaging,especially in the imaging by using the structural illumination microscope(SIM).Based on this consideration,we synthesized the donor-acceptor typed AIEgen of DTPA-BTN,which not only owns high brightness in the near-infrared(NIR)emission region from 600 nm to 1000 nm(photoluminescence quantum yield,PLQYs=11.35%),but also displays excellent photo-stability.In addition,AIE nanoparticles based on 4,7-ditriphenylamine-[1,2,5]-thiadiazolo[3,4-c]pyridine(DTPA-BTN)were also prepared with highly emissive features and excellent biocompatibility.Finally,the developed DTPA-BTN-based AIE nanoparticles were applied in the super-resolution cellular imaging via SIM,where much smaller full width at half-maximum values and high signal to noise ratios were obtained,indicating the superior imaging resolution.The results here imply that highly emissive AIEgens or AIE nanoparticles can be promising imaging agents for super-resolution imaging via SIM.     

Organic Nanocrystals Based on a Solid-emission-tunable AIEgen for Cell Imaging

The development of fluorescent nanocrystals based on organic small molecules is of great importance in bioimaging due to the merits of easy modification,high brightness and excellent photostability,however suffering from the emission-detrimental aggregation-caused quenching(ACQ)effect.Herein,we successfully designed and synthesized an AIE-active di(N,N-dimethylaniline)-dibenzofulvene(named as NFTPE),which exhibits the crystallization-induced emission enhancement(CIEE)effect.Interestingly,two types of yellow-and orange-emissive crystals for NFTPE were obtained,exhibiting aggregation microenvironment-dependent emission tuning in the solid state.Single-crystal analysis and density functional theory(DFT)calculations reveal that different aggregation microenvironments result in the distinct molecular conformation for various emission.Excitingly,the crystallization of NFTPE in an aqueous solution under the assistance of amphiphilic PEG polymer matrices could be monitored in situ by the fluorescence changes,facilitating the preparation of NFTPE nanocrystals(NFTPE-NCs)by adjusting the aggregation microenvironment.The obtained NFTPE-NCs exhibit the superior performance in cell imaging in respect to high brightness,photostability,and biocompatibility,thus demonstrating the potential in bioimaging applications.     

Advanced Nanomaterials for Multimodal Molecular Imaging

The development of multimodal molecular imaging contrast agents based on versatile nanomaterials has recently attracted much attention in disease diagnosis and therapeutic delivery. Contrast agents made from nanoparticles and used for multimodal imaging in vivo provide a multidimensional pathophysiological overview of diseases. This review summarizes recently developed advanced nanomaterials for multimodal molecular imaging. We comprehensively discuss these nanoparticle contrast agents in terms of their targeting modalities, limitations in clinical translation and future directions.     

Expanding the Ligand Classes Used for Mn(II) Complexation: Oxa-aza Macrocycles Make the Difference

We report two macrocyclic ligands based on a 1,7-diaza-12-crown-4 platform functionalized with acetate (tO2DO2A²⁻) or piperidineacetamide (tO2DO2AM^Pip) pendant arms and a detailed characterization of the corresponding Mn(II) complexes. The X−ray structure of [Mn(tO2DO2A)(H₂O)]·2H₂O shows that the metal ion is coordinated by six donor atoms of the macrocyclic ligand and one water molecule, to result in seven-coordination. The Cu(II) analogue presents a distorted octahedral coordination environment. The protonation constants of the ligands and the stability constants of the complexes formed with Mn(II) and other biologically relevant metal ions (Mg(II), Ca(II), Cu(II) and Zn(II)) were determined using potentiometric titrations (I = 0.15 M NaCl, T = 25 °C). The conditional stabilities of Mn(II) complexes at pH 7.4 are comparable to those reported for the cyclen-based tDO2A²⁻ ligand. The dissociation of the Mn(II) chelates were investigated by evaluating the rate constants of metal exchange reactions with Cu(II) under acidic conditions (I = 0.15 M NaCl, T = 25 °C). Dissociation of the [Mn(tO2DO2A)(H₂O)] complex occurs through both proton− and metal−assisted pathways, while the [Mn(tO2DO2AM^Pip)(H₂O)] analogue dissociates through spontaneous and proton-assisted mechanisms. The Mn(II) complex of tO2DO2A²⁻ is remarkably inert with respect to its dissociation, while the amide analogue is significantly more labile. The presence of a water molecule coordinated to Mn(II) imparts relatively high relaxivities to the complexes. The parameters determining this key property were investigated using ¹⁷O NMR (Nuclear Magnetic Resonance) transverse relaxation rates and ¹H nuclear magnetic relaxation dispersion (NMRD) profiles.