一种检测蛙卵卵裂卵表面运动的荧光图像漂白法

荧光漂白恢复(Fluorescence PhotobleachingBecovery,FPR)技术是采用强激光瞬时漂白荧光标记的细胞膜微区,然后用弱激光记录被漂白区域中荧光恢复的过程,获得荧光漂白恢复动力学曲线,由此计算扩散系数或流动速度.我们曾于1981年研制了FPR装置,并用于测定了林蛙卵表面受体的运动,以了解膜运动与细胞分裂的关系.由于FPR测量范围小于10μm~2的微区而难于了解卵表面大区域的流动变化.鉴于上述原因,我们在原有     

检测细胞膜表面分子运动的一种新方法——荧光图象漂白技术

荧光图象漂白技术原理的基本要点是:在显微镜的光路中插入掩膜(光栅),经强激光瞬时漂白,使细胞表面上荧光标记分子产生不可逆的光化学漂白而形成有规则的荧光图象,随即通过SIT(硅增强靶)摄象管,用微弱的激光束监测荧光图象的变化,照相机速度摄取荧光图象的变化,由此观察和研究细胞表面分子的二维变化及其运动规律。我们研制成激光漂白荧光图象     

激光扫描共聚焦显微镜观察近紫外线辐射对兔晶体上皮细胞通讯功能的影响

目的:通过激光扫描共聚焦显微镜荧光漂白恢复技术观察近紫外线对兔晶状体上皮细胞缝隙连接通讯功能的影响。方法:体外培养兔晶状体上皮细胞,设置空白阴性对照,分为对照组和实验组,分别以近紫外线辐射5分钟,10分钟和15分钟,用荧光光漂白恢复技术检测细胞的荧光恢复率。结果:空白对照组荧光恢复率为57.357±5.610,照射5分钟,10分钟和15分钟近紫外线辐射后的荧光恢复率分别为(%)34.205±3.652,18.909±3.017,7.129±2.917。结论:近紫外线损伤可以导致晶状体上皮细胞间通讯功能的受损。     

基于可控光剂量的低荧光漂白共聚焦成像技术

激发光引起的荧光漂白限制了共聚焦成像技术在长时间观测生物样本方面的应用。提出了一种基于可控光剂量的共聚焦成像技术(CLE-CM),该技术通过预实验设置高低阈值,定时读取采样像素值并与预设阈值进行比较,根据比较结果控制每个物方像素的光照时间,以更高效地利用荧光信息,在不牺牲图像质量的情况下降低了荧光漂白。用CLE-CM和标准共聚焦对牛肺动脉内皮细胞样本连续成11幅图像,与第11幅标准共聚焦图像相比,第11幅CLE-CM图像的荧光漂白减少了52.62%,具体降漂白效果与样本中的荧光分布有关。CLE-CM通过减少光剂量大幅降低了共聚焦显微成像的荧光漂白,使共聚焦显微镜能连续成更多张高质量图像。     

超分辨显微技术在活细胞中的应用与发展

细胞是生命体的基本单位和功能单位,对活细胞内部结构及其功能的研究是了解掌握生命本质的基础之一,因此活细胞的实时观测对生命科学的发展具有重要意义。传统的光学显微技术受衍射极限的限制,无法观测200 nm以下的生物结构细节。近20年来,随着超衍射极限光学理论、技术、器件和荧光探针等方面的快速发展,超分辨显微成像技术已成为应用于生命科学研究的重要手段。然而,大多数超分辨显微方法或测量耗时长,或易引起荧光蛋白漂白/细胞损伤,在活细胞研究中受到极大限制,已成为超分辨显微领域重点攻关的方向之一。为此,文中结合作者在快速超分辨显微技术研究的基础上,介绍了基于单分子成像的光激活定位显微技术和随机光学重构显微技术、基于荧光非线性可饱和光转换的受激发射显微技术以及基于结构光照明的超分辨显微技术,并探讨了在活细胞成像中的发展应用。最后,文中展望了超分辨显微成像技术在活细胞成像中的未来发展趋势。     

Perkin Elmer公司推出激光诱导活细胞快速示踪装置

Perkin Elmer公司最近推出一种激光诱导活细胞动态过程快速示踪装置（Photo Kinesis）。该装置可附加在著名的Ultra View ERS或RS系列活细胞高速成像系统上。采用该装置，可完成活细胞样本上多达100个目标区域（ROI，region of interest）的光漂白后荧光恢复（FRAP，fluorescence recovery after photobleaching）、     

研究进展

光学显微镜揭示单个蛋白质分子美国研究人员开发了一种新型光学成像技术,可以显示细胞中纳米尺度的蛋白质分子,该技术被称为光敏化定位显微术(photoactivlated localization microscopy,PALM)。它开创性地将荧光蛋白质分子附着在目标蛋     

结合光片照明与超分辨的三维荧光显微成像

随着科学的进步,生命科学的研究对象由单个器官向组织体、离体组织切片及发育过程中的活体胚胎转变。荧光特异性标记的出现,为追踪物质在单细胞、组织体、器官甚至整个胚胎内的转移过程提供了手段。为了实现整个追踪过程,需要对活体胚胎进行无损、非侵入式的亚细胞级别成像,这就对荧光显微技术提出了更高的要求。在传统荧光显微技术基础上发展了光片照明和超分辨荧光显微技术。前者通过选择平面照明方式,只激发探测物镜焦平面附近的样品,因其具有高穿透深度、低漂白和高成像速度而广泛应用于三维活体组织成像;后者利用特殊的光调控手段将显微镜的分辨率提升至纳米水平,成为研究亚细胞水平生命活动的有力武器。通过介绍2大技术的发展、融合以及目前所遇到的问题,探究新型的、适宜观察三维厚组织样品亚细胞结构和生命过程的成像方法。     

FCS/FCCS技术及其在植物细胞生物学中的应用

荧光相关光谱(fluorescence correlation spectroscopy,FCS)是一种单分子荧光检测技术,能够灵敏地检测荧光分子的浓度、运动参数及构象变化等数据。在此技术基础上发展起来的荧光互相关光谱(fluorescence crosscorrelation spectroscopy,FCCS)突破了FCS技术只能探测单种荧光分子的限制,可以通过分析两种及以上荧光信号来研究分子间的相互作用。由于FCS和FCCS检测的快速精确性,这两种技术被越来越多的应用于生命科学的研究。本文详细阐述了荧光相关光谱和荧光互相关光谱的原理,并对其在生物学尤其是植物细胞生物学中的应用进行了介绍和总结。随着检测技术的改进和数据分析技术的完善,荧光相关光谱和荧光互相关光谱技术将在植物生物学的研究中得到越来越多的应用。     

激光共聚焦显微镜样品制备方法（一）——细胞培养样品

随着激光共聚焦显微镜在生物学研究中的广泛应用,绿色荧光融合蛋白能够提供蛋白质在细胞体内的精确时空信息。因此免疫荧光样品的制备也成为基本的细胞生物学实验方法。本文主要介绍在培养细胞中表达绿色荧光融合蛋白及免疫荧光样品的制备过程。     

Supported Lipid Bilayer Assembly on PEDOT:PSS Films and Transistors

Lipid bilayers are widely employed as a model system to investigate interactions between cells and their environment. Supported lipid bilayers (SLB) with integrated transmembrane proteins are emerging as a preferred platform for sensing applications. Challenges lie in the generation of SLB on surfaces which allow transduction of signals for characterization of lipid bilayer and incorporated transmembrane proteins. For the first time, the formation of SLBs is shown on films of the conducting polymer, poly(3,4‐ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), using traditional methods for characterizing lipid bilayer quality and function (QCM‐D, FRAP) combined with impedance spectroscopy. Further, partial formation of SLBs on PEDOT:PSS based organic electrochemical transistors (OECTs) is successfully demonstrated, as well as the ability to integrate and sense the ion pore α‐hemolysin, confirming the sensitivity of the OECT as a transducer of biological membrane function. This work represents a highly promising first step toward the use of such OECTs for functional readout of transmembrane proteins in their native environment.     

Fabrication of bifunctional membrane electrode assemblies for unitised regenerative polymer electrolyte fuel cells

Bifunctional membrane electrode assemblies have been fabricated using a screen printing technique, which demonstrate a repeatable and stable operation to cell current and voltages. This approach lends itself to a rapid, low-cost and repeatable fabrication process for bifunctional catalytic electrodes in polymer electrolyte membrane fuel cells and electrolysers     

Bionanoparticle‐Based Delivery in Antihypertensive Vaccine Mediates DC Activation through Lipid‐Raft Reorganization

Bionanoparticles (BNPs) are widely used as vaccine carriers. Compared with classic protein carriers, BNPs exhibit nanostructures that enable them to interaction with biointerface‐like cell membrane or membrane domains. An antihypertensive vaccine ATR‐NP is produced based on a virus‐like particle (VLP) Qβ carrier. Qβ shows great delivery efficiency and immunogenicity to antigen‐presenting cells (APCs) such as dendritic cells (DCs), and DC activation induced by ATR‐NP is highly dependent on membrane lipid rafts. Further studies reveal that ATR‐NP exhibits tight affinity to rafts, and interface effects between them prompt membrane phase separation and raft accumulation through NP‐induced membrane curvature change. Lipid rafts are accumulated and expanded together with a decline of their diffusion in membrane, which benefits signaling protein Src partition in rafts. The heterogeneous protein partition implies functional centralization for stronger signal transduction. In conclusion, targeting and reorganizing membrane domains such as lipid rafts suggests BNPs could be used as vaccine carriers and is a potent strategy for vaccine and other immunological agent design.     

金纳米颗粒介导的细胞光穿孔表征方法

提出了一种新的表征方法--膜电位测量。膜电位恢复时间与膜穿孔尺寸相对应,因此建立起细胞膜穿孔尺寸的大小与激光能量阈值之间的关系,可为准确地向细胞内递送不同分子量的外源物质提供理论支持。将金纳米颗粒与胃癌细胞共同培养,在保证细胞不受金纳米颗粒毒性影响的前提下,选择不同能量纳秒脉冲激光照射共孵育后的胃癌细胞,并采用碘化丙啶(PI)和钙黄绿素乙酰甲酯(Calcein-AM)对穿孔后的细胞进行染色验证。结果发现:当加入直径为100 nm的金纳米颗粒,其数量与细胞数比为400\:1,激光能量密度在20 mJ/cm^2时,可以在保证细胞活性的前提下成功实现532 nm脉冲激光的细胞膜穿孔;在穿孔条件下,采用光标测技术测量细胞膜电位,发现细胞膜电位先增加后复原,最大增量为50 mV,恢复时间为250 s。膜电位结果再次验证,光穿孔造成的细胞膜损伤是可以恢复的,而且可以用膜电位变化来表征。     

多种肿瘤细胞表面超微结构的原子力显微镜观察及药物对细胞膜表面超微结构影响的初步研究

本文应用原子力显微镜对人胃癌SGC7901、人肝癌HepG2、人肺癌AFFC-A-1、人乳腺癌MCF．7.R和人肺腺癌A549等肿瘤细胞膜表面进行了超微结构的形态学观察和比较。并研究了Nm23-Hl／NDPK-A蛋白对人肺腺癌A549等细胞膜表面超微结构的影响。发现不同肿瘤细胞膜表面超微结构存在较大的差异，药物处理和对照组A549细胞膜的表面超微结构也存在较大差异，药物处理的A549细胞膜表面出现大量的疤痕状集聚物，而药物处理的其它几种肿瘤细胞膜表面未发现明显的变化。说明原子力显微镜具有发展为一种观察和鉴别某些细胞的有力工具的潜力，并有可能应用于病理学或药理学研究，但在这些研究中这种技术只能作为一种辅助手段。     

肾组织电镜包埋后银染色技术

应用电镜包埋后银染色技术对肾活检组织进行了观察。发现此技术能清晰地呈现肾小管上皮细胞的基底褶、沿上皮细胞长轴排列的线粒体及小管顶端微绒毛的双层膜结构。我们认为此技术在评价肾小管损伤,例如肾小管坏死、间质性肾炎及移植肾的病理变化中是有价值的。     

Designing a New Generation of Proton‐Exchange Membranes Using Layer‐by‐Layer Deposition of Polyelectrolytes

All fuel cells utilizing the membrane‐electrode assembly have their ion‐conductive membrane sandwiched between bipolar plates. Unfortunately, applying conventional techniques to isolated polyelectrolyte membranes is challenging and difficult. A more practical alternative is to use the layer‐by‐layer assembly technique to fabricate a membrane‐electrode assembly that is technologically relatively simple, economic, and robust. The process presented here paves the way to fabricate ion‐conductive membranes tailored for optimum performance in terms of controlled thickness, structural morphology, and catalyst loading. Composite membranes are constructed through the layered assembly of ionically conductive multilayer thin films atop a porous polycarbonate membrane. Under ambient conditions, a fuel cell using a poly(ethylene oxide)/poly(acrylic acid) (PEO/PAA) composite membrane delivers a maximum power density of 16.5 mW cm^–2 at a relative humidity of 55 %, which is close to that of some commercial fuel cells operating under the same conditions. Further optimization of these systems may lead to new, ultrathin, flexible fuel cells for portable power and micropower applications.     

蚕豆萎蔫病毒2号VP37蛋白在BY-2细胞内的定位

将绿色荧光蛋白（GFP）连接于蚕豆萎蔫病毒2号（Broad bean wilt virus2,BBWV-2）VP37蛋白的N-端,构建融合基因GFP-VP37,用农杆菌法在BY-2悬浮细胞内进行表达,激光共聚焦显微镜观察融合蛋白的分布。结果显示：GFP-VP37主要定位于细胞核的周围呈网络状,并在细胞边缘形成点状结构;ER-tracker标记显示VP37在细胞内与内质网共定位;电镜免疫金标记显示VP37蛋白主要定位在细胞质中;用BFA处理转染细胞后,VP37在细胞质及细胞边缘的定位受到抑制。推测内质网参与VP37的细胞内转运和分布。     

Preliminary Observations with an Electromagnetic Method for the Noninvasive Analysis of Cell Suspension Physiology and Induced Pathophysiology

A technique for the electromagnetic analysis of physiological and patho-physiological states in cell suspensions is presented. The technique is based upon high speed automatic network analysis in the HF band for measurement of complex permittivity. The results demonstrate that changes in HF-band permittivity dispersion may be related to physiological and drug-induced patho-physiological states of the cell membrane. Mechanical disruption of the cell membrane by sonication obliterates the HF-band dispersion of permittivity that is present in undisrupted cells. The effect of species, suspending medium, and temperature were systematically analyzed in erythrocyte suspension in order to aid comparison between published studies of red cell preparations.     

Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain

Cellular membranes have long served as an inspiration for nanomaterial research. The preparation of ultrathin polydopamine (PDA) films with integrated protein pores containing phospholipids and an embedded domain of a membrane protein glycophorin A as simplified cell membrane mimics is reported. Large area, ultrathin PDA films are obtained by electropolymerization on gold surfaces with 10–18 nm thickness and dimensions of up to 2.5 cm². The films are transferred from gold to various other substrates such as nylon mesh, silicon, or substrates containing holes in the micrometer range, and they remain intact even after transfer. The novel transfer technique gives access to freestanding PDA films that remain stable even at the air interfaces with elastic moduli of ≈6–12 GPa, which are higher than any other PDA films reported before. As the PDA film thickness is within the range of cellular membranes, monodisperse protein nanopores, so‐called “nanodiscs,” are integrated as functional entities. These nanodisc‐containing PDA films can serve as semi‐permeable films, in which the embedded pores control material transport. In the future, these simplified cell membrane mimics may offer structural investigations of the embedded membrane proteins to receive an improved understanding of protein‐mediated transport processes in cellular membranes.