荧光金纳米团簇探针的生物成像应用进展（特邀）

金纳米团簇（AuNCs）因兼具优异荧光特性、超小尺寸、精确化学组成及良好生物相容性等优势，使其成为近些年备受关注的新型荧光探针。为了推动荧光AuNCs在成像领域的应用，研究者们一直致力于发展高性能荧光AuNCs的设计与制备策略。基于对AuNCs结构与发光机制的理解，诸如提高荧光量子产率和细胞摄取率等策略陆续被提出以增强AuNCs的细胞成像效果，极大提升了其作为荧光成像探针的潜力，并将AuNCs的应用推广至荧光寿命成像、多光子成像等新兴荧光成像技术。近些年发展的具有近红外二区荧光的AuNCs进一步推动了其在活体成像的应用。文中概述了AuNCs的制备方法、提高AuNCs细胞荧光成像效果的各种策略，以及AuNCs荧光成像应用的最新进展，并对该领域的挑战和未来发展进行了展望。     

光纤荧光探针

荧光光谱分析是一种重要的测试手段。近年来,基于光纤探针的荧光测试技术成为研究热点,这种方法具有高效、微观、实时、原位、体积小易集成等优势。简要综述荧光分析原理、光纤探针中激光发射与荧光收集的空间传导理论,以及光纤荧光探针的典型结构与制备,总结了应用于生物、环境和食品安全领域的研究状况。最后,对光纤荧光探针的发展趋势进行了展望。     

激光诱导荧光检测技术

激光诱导荧光(LIE)检测作为目前灵敏度最高的检测技术之一,在生物、化学、医学等领域应用广泛,其本身的技术发展也十分迅速.综述了近年来LIF检测系统、荧光探针、荧光分析技术和应用领域的进展,重点介绍了LIF激光光源、光纤光路LIF系统、多通道LIF系统以及LIF成像的最新进展和应用前景.     

具有荧光开关特性的喹啉衍生物用于高效选择性标记细胞脂滴

脂滴是一种重要的细胞器，与细胞中多种生理活动密切相关。为了观察脂滴并研究其多种多样的功能，共聚焦荧光成像技术是最有力的工具之一。然而，细胞脂滴荧光成像所需的具有高荧光亮度和高标记选择性的脂滴荧光探针却十分有限，这严重限制了脂滴的深入研究。研制了一种具有荧光开关特性的喹啉衍生物脂滴荧光探针（Lipi-QL）。该探针因其敏感的极性淬灭荧光特性，展现出了很高的脂滴标记选择性。Lipi-QL能靶向脂滴是因为其脂溶性，进入脂滴后，荧光增强是脂滴内的较低极性引起的。给受体型分子结构还赋予了该探针高的荧光亮度以及大的斯托克斯位移。将该探针用于细胞脂滴共聚焦荧光成像，在不同浓度下都实现了显著优于脂滴商用探针BODIPY 493/503的标记选择性。此外，使用该荧光探针实现了固定细胞的三维共聚焦成像和活细胞的四色共聚焦成像。该荧光探针的研制一方面为脂滴生理功能的研究提供了强有力的工具，另一方面也为新型高标记选择性荧光探针的设计提供了新的思路。     

蛋白酶荧光探针及新型显微成像技术的生物医学应用

研究上正常生理条件蛋白酶的活化情况仍然存在很多困难,将蛋白酶荧光探针技术和基于各种光学平台的显微成像技术有机地结合起来,可以最大限度地记录活细胞生理条件下蛋白酶活性变化的时空信息.综述了蛋白酶荧光探针技术及其与该类探针应用相关的新型显微成像技术在生物医学光子学领域的应用进展.     

荧光共振能量转移技术在生命科学中的应用及研究进展

荧光共振能量转移(FRET)是指两个荧光基团间能量通过偶极-偶极耦合作用以非辐射方式从供体传递给受体的现象,可被用于测定分子间的距离.FRET荧光探针主要有荧光蛋白、有机荧光染料、镧系染料和量子点,近年来有许多新的应用;测试方法上与时间分辨、单分子检测、荧光寿命和荧光相关光谱等技术联用取得了更好的成效.     

单分子三维取向超分辨成像技术进展（特邀）

超分辨显微成像技术自诞生以来,凭借其优异的纳米级空间分辨率,已成为生命科学研究中精准揭示复杂生命现象的重要成像技术。其中,基于单分子定位的超分辨成像策略,使得定位、观察、研究单个探针分子独特的理、化、光学性能成为可能。偏振作为荧光信号的一个重要特性,近年来伴随着单分子三维取向成像技术的发展,逐步在单分子成像和超分辨领域中展示出诸多新颖且重要的应用特性。本文总结了单分子三维取向超分辨成像技术的最新进展,介绍并分析了两类主要的单分子三维取向荧光显微技术——基于荧光吸收与辐射偏振调制的单分子三维取向成像方法以及利用点扩散函数工程将单个荧光分子的三维取向信息编码到荧光图像上的成像策略。此外,还探讨了应用于活细胞或单颗粒的其他类型的超分辨取向成像技术。最后,针对单分子三维取向超分辨成像技术发展与应用前景面临的挑战,进行了总结与展望。     

植物中活性氧信号转导及其检测方法研究进展

活性氧(ROS)作为一种重要信号分子在植物体生命活动的多方面发挥着关键作用。为对活细胞内的ROS分子进行研究分析,人们发展出了多种检测手段。其中,荧光探针作为一种高度灵敏、直观的标记技术,在植物ROS研究中得到广泛应用。近年来,通过蛋白质工程和基因工程技术,研究者们基于绿色荧光蛋白创造出一批新型的特异性荧光探针,为进一步探索植物体内ROS信号途径提供更有力的工具。本文概述了植物细胞中ROS信号转导通路,并总结针对不同ROS组分的多种研究方法,尤其对利用荧光探针标记技术检测胞内ROS的研究进展进行详细介绍。     

一种两亲性荧光探针体外检测牙菌斑生物膜的方法

牙菌斑生物膜与口腔疾病的发生密切相关,因此开发新型的牙菌斑生物膜检测方法具有重要应用价值。本文建立了一种基于两亲性荧光探针TR4的牙菌斑生物膜体外检测方法。该荧光探针由疏水部分四苯基乙烯（ TPE）、棕榈酸和亲水多肽R4组成,三者的功能分别是荧光生发、粒子组装与静电吸附,从而实现紧密结合牙菌斑生物膜并发光。 TR4与牙菌斑生物膜作用时间短、反应条件简单,室温孵育1 min后检测其荧光强度变化,即可实现对牙菌斑生物膜快速、便捷的检测分析。荧光光谱也证实TR4荧光强度与牙菌斑生物膜浓度正相关,有望实现牙菌斑生物膜的定量检测。进一步通过透射电镜和共聚焦显微镜观察发现TR4探针通过静电吸附作用聚集在牙菌斑生物膜表面并自组装形成纳米粒子,从而诱导荧光基团TPE发光。     

基于微球调制光场的超分辨成像及荧光增强

微球可实现光场的调制，能够将入射光束聚焦于微球背面一个极窄区域，使得出射光束半峰全宽小于光学衍射极限，且聚焦强度远远高于入射光场强度。此外，微球具有高数值孔径特性，能够提高探测信号的收集效率。基于所述优势，微球为实现光学超分辨成像以及荧光增强提供了新思路和实现途径。相比传统技术，基于光学微球的超分辨成像及荧光增强技术更简便、更直接且易于实现，其成像及增强效果均可媲美传统超分辨技术与荧光增强技术，在生物成像及医学检测方面，具有重要的研究价值和应用前景。近年来，关于微球调制光场实现荧光增强的研究取得了较大发展，但与之相关的综述论文仍较少。系统总结阐述微球增强荧光发光以及微球调制光场技术，对于该领域的未来研究发展极为重要。首先介绍基于微球的光学超分辨成像，包括明场超分辨成像和荧光超分辨成像；然后阐述基于微球的荧光增强研究，包括现象研究、机制探索以及影响因素讨论等；最后，总结微球超分辨成像及荧光增强进展和技术应用，分析并展望该技术领域的未来发展挑战和趋势。     

Functional reentry's influence on intracellular calcium in the LRd membrane equations

This paper examines relationships between transmembrane potential (Vm), [Ca2+]i dependent membrane ionic currents, and [Ca2+]i handling by the sarcoplasmic reticulum (SR) in a two-dimensional model of cardiac tissue. Luo-Rudy dynamic (LRd) membrane equations were used because they include detailed formulations for triggered SR Ca2+ release dependent on membrane Ca2+ influx (CICR) and for spontaneous SR Ca2+ release following calsequestrin buffer overload (SCR). Reentry's rapid rate (110-ms cycle length) elevated [Ca2+]i and limited CICR, which in turn promoted SCR that occurred at intervals of 320-350 ms, was preferential at sites located inside the functional center, and destabilized the reentrant activation sequence. Although adjustment of LRd parameters for SR Ca2+ modified SCR interval and peak [Ca2+]i in voltage clamp simulations with a command waveform representing Vm time course within the functional center, SCR persisted. Using the same command waveform, SCR also occurred with an alternate SR Ca2+ formulation that represented subcellular details underlying CICR. LRd parameter adjustments to promote CICR and limit SCR in subsequent reentry simulations failed to eliminate SCR completely, as they modulated SCR intervals in a manner consistent with the voltage clamp simulations. Taken together, our findings support a destabilizing influence of functional reentry on [Ca2+]i handling. However, [Ca2+]i instabilities did not always fractionate depolarization wavefronts during reentry. Fractionation depended, in part, upon CICR and SCR parameters in the LRd formulation for SR Ca2+ release.     

Functionalized Cubic Mesoporous Silica as a Non‐Chemodosimetric Fluorescence Probe and Adsorbent for Selective Detection and Removal of Bisulfite Anions along with Toxic Metal Ions

Dual signaling and remediation systems for detection and adsorption of toxic analytes have gained more attention over sensory probes only. However, most of the sensors for bisulfites are chemodosimetric probes, which are irreversible and having drawbacks of absolute selectivity, recyclability, and solubility in a pure aqueous system. To address above drawbacks a new non‐chemodosimetric probe material with a strong hydrogen bonding pocket for bisulfites is developed. Synthesis of cubic mesoporous silica by a modified Stober process followed by functionalization with 2,2′‐(((((3‐(triethoxysilyl)propyl)azanediyl)bis(methylene))bis(2,1‐phenylene))bis(oxy))bis(N‐(4‐((E)‐phenyldiazenyl)phenyl)acetamide) (AZOL) has given a fluorogenic silica probe material SiO₂@AZOL. This material shows selectivity toward bisulfite anion (limit of detection (LOD): 64 ppb) and Hg²⁺, Cd²⁺, Cu²⁺, and Zn²⁺ cations (LOD: 126, 95, 14, and 27 ppb, respectively) among various analytes. The adsorption studies for these toxic analytes (HSO₃ ^?, Hg²⁺, Cd²⁺, Cu²⁺, and Zn²⁺) show an extraction efficiency of around 99% and adsorption capacities of 873, 630, 633, 260, and 412 mg g^?1, respectively. Spectroscopic studies along with adsorption, striping, and regeneration studies reveal that this material is a recyclable sensory cum adsorbent material for these toxic analytes. Moreover, this material can be used as a sensitive probe material for determination of HSO₃ ^? levels in various sugar samples.     

A dynamic action potential model analysis of shock-inducedaftereffects in ventricular muscle by reversible breakdown of cellmembrane

To elucidate the subcellular mechanism underlying the aftereffects of high-intensity dc shocks, a small pore, which mimics reversible breakdown of the cell membrane (electroporation), was incorporated into the phase-2 Luo-Rudy (L-R) model of ventricular action potentials. The pore size was set to occupy 0.15%-4.25% of the total cell membrane during the 10-ms shock. The pore was assumed to decrease after the shock exponentially with a time constant of 100-1,400 ms to simulate resealing process. In normal myocytes, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by prolonged depolarization and oscillation of membrane potential like early afterdepolarization (EAD). Time- and voltage-dependent changes in the delayed rectifier K+ currents (IKr, IKs) in combination with those of L-type Ca2+ current (ICa,(L)) and ion flux through the pore (I(pore)) are responsible for the potential changes. Spontaneous excitation from the oscillation depends on activation of ICa,(L). In myocytes overloaded with Na+ and Ca2+ secondary to 90% inhibition of Na+-K+ pump, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by slower cyclic depolarization in response to spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). This delayed afterdepolarization-type oscillation is abolished by complete block of Ca2+ release from the SR. These findings suggest that high-intensity electric field application will cause arrhythmogenic responses through a transient rupture of sarcolemma with different subcellular events in ventricular cells under normal and pathological conditions.     

Liquid–Solid Triboelectric Probes for Real-Time Monitoring of Sucrose Fluid Status

Triboelectric probes have rapidly developed as an efficient tool for understanding contact electrification at liquid–solid interfaces. However, the liquid–solid electrification process is susceptible to interference from chemical components in mixed solutions, severely limiting the potential applications of triboelectric probes in various liquid environments. This study for the first time reports a triboelectric probe capable of sucrose solution concentration sensing, finding that the dissolution of sucrose destroys the hydrogen bond network between water molecules and forms sucrose–water hydrogen bonds, which alters the fluid mechanics characteristics of the solution and enhances its conductivity, thereby reducing the droplet size and causing an ion charge shielding effect that significantly affects the electron transfer in liquid–solid contact electrification. Owing to the feedback of the triboelectric probe on the sucrose concentration gradient-type sensing electrical signals, efficient sensing of sucrose solution was achieved (sensitivity of −0.0038%⁻¹, response time of 90 ms). The triboelectric probe is also used as a wireless smart terminal to enable real-time detection of sucrose solution. This work contributes to the understanding of the structure–function relationship between micro hydrogen bonding and macro performance, and provides a promising solution for building sustainable intelligent sensors.     

共焦荧光显微定量研究血卟啉单甲醚亚细胞定位

目的 :研究血卟啉单甲醚 (HMME)在鼠肺毛细血管内皮细胞内的亚细胞定位 ;方法 :孵育时间分别为 2小时、2 4小时 ,四种荧光探针标记四种细胞器。激光扫描共聚焦显微镜 (LSCM)探测HMME和探针荧光图像。定性比较两种荧光分布模式 ,并定量计算高探针荧光区域与细胞内HMME荧光均量比I2 /I1 ;结果 :两种孵育时间下 ,HMME荧光均呈胞浆弥散分布 ,且在核周附近的高尔基体灰度较高 ,细胞核几乎无荧光分布。I2 /I1 高低顺序 2小时组 :高尔基体 >内质网 >溶酶体 线粒体 ;2 4小时组 :高尔基体 >线粒体 内质网 >溶酶体 ,且高尔基体组显著下降伴随线粒体组大幅上升 ;结论 :此定量分析方法能有效用于光敏剂亚细胞定位研究 ,对弥散分布光敏剂尤其适合 ;两种孵育时间下 ,高尔基体为HMME主要定位点 ,内质网有一定分布 ,溶酶体分布很少 ,细胞核几乎无分布 ;短孵育时间线粒体分布很少 ,长孵育时间由高尔基体到线粒体有一定再分布     

Gd3+‐Ion‐Doped Upconversion Nanoprobes: Relaxivity Mechanism Probing and Sensitivity Optimization

Paramagnetic gadolinium (Gd‐III)‐ion‐doped upconversion nanoparticles (UCNPs) are attractive optical‐magnetic molecule imaging probes and are a highly promising nanoplatform for future theranostic nanomedicine design. However, the related relaxivity mechanism of this contrast agent is still not well understood and no significant breakthrough in relaxivity enhancement has been achieved. Here, the origin and optimization of both the longitudinal (r₁) and transverse (r₂) relaxivities are investigated using models of water soluble core@shell structured Gd³⁺‐doped UCNPs. The longitudinal relaxivity enhancement of the nanoprobe is demonstrated to be co‐contributed by inner‐and outer‐sphere mechanisms for ligand‐free probes, and mainly by outer‐sphere mechanism for silica‐shielded probes. The origin of the transverse relaxivity is inferred to be mainly from an outer‐sphere mechanism regardless of surface‐coating, but with the r₂ values highly related to the surface‐state. Key factors that influence the observed relaxivities and r₂/r₁ ratios are investigated in detail and found to be dependent on the thickness of the NaGdF₄ interlayer and the related surface modifications. A two orders of magnitude (105‐fold) enhancement in r₁ relaxivity and 18‐fold smaller r₂/r₁ ratio compared to the first reported values are achieved, providing a new perspective for magnetic resonance (MR) sensitivity optimization and multimodality biological imaging using Gd³⁺‐doped UCNPs.     

Multiscale modeling of calcium dynamics in ventricular myocytes with realistic transverse tubules

Spatial-temporal Ca(2+) dynamics due to Ca(2+) release, buffering, and reuptaking plays a central role in studying excitation-contraction (E-C) coupling in both normal and diseased cardiac myocytes. In this paper, we employ two numerical methods, namely, the meshless method and the finite element method, to model such Ca(2+) behaviors by solving a nonlinear system of reaction-diffusion partial differential equations at two scales. In particular, a subcellular model containing several realistic transverse tubules (or t-tubules) is investigated and assumed to reside at different locations relative to the cell membrane. To this end, the Ca(2+) concentration calculated from the whole-cell modeling is adopted as part of the boundary constraint in the subcellular model. The preliminary simulations show that Ca(2+) concentration changes in ventricular myocytes are mainly influenced by calcium release from t-tubules.     

血卟啉单甲醚在鼠肺毛细血管内皮细胞内的亚细胞定位

目的 :研究血卟啉单甲醚 (HMME)在鼠肺毛细血管内皮细胞内的亚细胞定位 ;方法 :孵育时间分别为 2小时、12小时、2 4小时 ,四种荧光探针标记四种细胞器。倒置荧光显微镜和致冷CCD探测HMME和探针荧光图像。定性比较两种荧光分布模式 ,并定量计算高探针荧光区域与细胞内HMME荧光均量比I2 /I1;结果 :三种孵育时间下 ,HMME荧光均呈胞浆弥散分布 ,且在核周附近的高尔基体灰度较高 ,细胞核几乎无荧光分布。I2 /I1高低顺序 2小时组 :高尔基体 >内质网 >线粒体 /溶酶体 ;12小时组 :高尔基体 >溶酶体 >内质网 /线粒体 ,且内质网组显著下降与高尔基体组略有下降伴随溶酶体组显著上升 ;2 4小时组 :高尔基体 >线粒体 /内质网 >溶酶体 ,且溶酶体组显著下降与高尔基体组一定下降伴随线粒体组与内质网组显著上升 ;结论 :倒置荧光显微镜和致冷CCD能有效应用于光敏剂亚细胞定位研究 ,定量分析方法对弥散分布光敏剂尤其适合 ;三种孵育时间下 ,高尔基体为HMME主要定位点 ,细胞核几乎无分布。短孵育时间 ,内质网有一定分布 ;中孵育时间 ,由内质网(主要 )和高尔基体到溶酶体再分布 ;长孵育时间 ,由溶酶体和高尔基体到线粒体和内质网再分布。     

两种薄层电阻测试系统探针游移误差的对比分析

从理论上分析方形四探针和直线四探针薄层电阻测试方法中探针游移所造成的系统偏差,推导出计算游移偏差的公式,并作图展示探针游移后的电阻与理想值之比的分布情况,分析了两种四探针测试方法出现最大误差的情况。用方形四探针测试方法不仅比普通直线四探针测试方法所测量的微区小,而且方形四探针测量的游移偏差小于直线四探针测量所产生的偏差。经试验发现,实际测试过程中,方形四探针只要在合理压力范围内,探针游移完全在合理范围内,能够保证测试的准确性。     

Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons

The subcellular localization of biomolecules at high resolution has traditionally been investigated by combining transmission electron microscopy (TEM) and chemical staining with heavy metals or immuno-based labeling with gold-conjugated antibodies. Here, we employ genetically encoded tags to examine the localization of proteins in transfected cultured cells by TEM. We purified a fusion protein of postsynaptic density-95 (PSD-95) coupled to three tandem repeats of metallothionein (MT) (PDS-95-3MT) from COS7 cells grown in the presence of Cd2+. PSD-95-3MT was detected as black particles by TEM. To visualize the subcellular localization of PSD-95-3MT, expression constructs encoding this fusion protein were transfected into primary hippocampal neurons cultured in medium containing Cd2+. The subcellular accumulation of PSD-95-3MT and Cd2+ provided excellent contrast in TEM micrographs. To address if genetically encoded tags affect the function of the target proteins, we found that the conjugation of 3MT to PSD-95 did not alter its association with known binding partners. These results demonstrate that 3MT coordinating Cd2+ is a valuable genetically encoded tag to study the localization of proteins by TEM.