原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具,具有独特的优势.本文在介绍原子力显微镜基本原理的基础上,综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用.     

单个腺病毒颗粒的原子力显微镜液相成像及样品制备方法

原子力显微镜(Atomic force microscopy,AFM)作为一种纳米级高分辨率的探针扫描显微镜,在病毒形态学研究等领域有广泛的应用。为更好地利用AFM在接近病毒生理环境(液相)中观测单个病毒颗粒,选择合适的基底吸附病毒样品是重要的前提保障。本文介绍了一种病毒样品制备及AFM液相成像方法。首先制备疏水化处理的玻璃盖玻片作为吸附基底,再将观察样品腺病毒重组载体颗粒(Adenovirus type 5F35,Ad5F35)吸附于基底上,建立在液相下单个病毒颗粒的原子力显微镜成像方法。通过该方法获取了单个腺病毒颗粒在液相下的高分辨率纳米级高度图(height image),且病毒颗粒保持了约90nm的正确测量高度。实验结果表明疏水化处理的玻璃基底具有良好的吸附力,有助于病毒颗粒牢固地吸附在基底上,且不会引起较大的形变。本研究为AFM观测病毒等生物样品提供了一种较为合适的吸附基底和在液相中的AFM成像方法,对后期开展病毒形态结构、物理属性及其与宿主细胞受体相互作用的研究打下基础。     

原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具, 具有独特的优势。本文在介绍原子力显微镜基本原理的基础上, 综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用。     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

原子力显微镜在染色体研究中的应用

原子力显微镜(Atomic force microscopy, AFM)是一种具有超高分辨率的显微成像仪器, 可在空气、真空和液体环境下对样本的表面结构进行实时观察。文章介绍了AFM的工作原理, AFM相对于其他种类显微镜在观察生物样本方面的显著优势, 并综述了AFM在染色体研究中的应用和进展。     

原子力显微镜及其在肿瘤研究中的应用

原子力显微术是一种利用原子、分子间的相互作用力来观察物体表面超微结构的新型实验技术.介绍了原子力显微镜作为一种显微探测和操纵工具的主要特点及其在肿瘤研究中的优势,评述了国内外有关原子力显微镜在肿瘤的诊断、治疗、抗肿瘤药物开发等研究中的应用情况,展望了原子力显微镜应用于肿瘤单细胞研究的前景.     

原子力显微镜在纳米生物学研究中的应用

原子力显微镜(atomic force microscope,AFM)自从1986年发明以来,作为一种重要的单分子研究工具,在包括细胞粘附、蛋白折叠以及蛋白间的相互作用等生物学过程的研究中得到了广泛应用。本文主要介绍原子力显微镜的原理,着重于研究相互作用时能垒大小的确定,以及常用的单分子表面修饰方法。     

难溶性药用纳米微粒的原子力显微检测

建立用原子力显微镜(AFM)检测难溶性药用纳米微粒的方法。将纳米微粒配成一定浓度的悬浮液,以新鲜裂解的云母表面作为样品载体,在室温下用AFM的接触模式成像,用粒度分析软件进行粒度分析。以此方法对两种微粒(活性炭及氧化锌纳米微粒)进行了成像和测量,同时对两种样品进行透射电镜(TEM)观察。AFM检测结果显示,纳米活性炭微粒形态近似球形,表面较平滑,平均直径为(299±187)nm;纳米氧化锌微粒呈球形,平均直径(50±20)nm,与TEM检测结果具有较高一致性。该法简便可靠。     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

原子力显微镜观测血卟啉单甲醚对细菌光动力杀伤作用

[目的]探讨血卟啉单甲醚(Hematoporyrin monomethyl Ether,HMME)对革兰氏阳性(G )、阴性(G-)菌的光动力杀伤作用.[方法]通过平板菌落计数法和原子力显微镜(AFM),观察细菌与HMME作用前后形貌的变化.[结论]当HMME浓度为50 μg/mL,可见光(光功密度为200 mW/cm2)光照30min时90%以上的金黄色葡萄球菌(Staphyrlococcus aureus)能被杀死,无光照时对S.aureus杀灭效果显著.同等条件下,无论光照还是无光照,HMME对大肠杆菌(E.coli)无明显的杀伤作用.AFM图像显示,S.aureus细菌表面破坏严重,完全碎裂成鱼鳞状的片状堆积.对HMME作用后的E.coli扫描可见,菌体原来光滑的表面变成网格状的裂纹排列.[讨论]HMME对G 有明显的光失活效应,而对G-效果不明显.AFM的超微图像显示HMME对细菌细胞的攻击位点主要在细胞膜上.AFM为我们研究光敏剂对细菌的光动力损伤作用机制的可视化提供了依据.     

流感病毒识别糖链受体分子机制的研究进展

近年来,由于流感病毒(influenza virus)不可预测的局部流行和有可能引发全球大流行,其一直是研究的热点课题之一．流感病毒表面糖蛋白血凝素(hemagglutinin,HA)特异识别宿主细胞表面的糖链受体是流感病毒感染宿主、进而复制并继续传播的生物学基础．影响流感病毒宿主特异性的两个主要因素是HA自身的变化(包括基因突变、重组、糖基化位点数量和糖基化位置的变化)和宿主细胞表面糖链受体的变化(包括糖链受体的类型、分布和分子构象的改变)等．因此准确掌握这些信息有助于人们进一步加强对流感病毒的防控．本文主要从糖组学角度概述了流感病毒识别糖链受体的分子机制,重点介绍流感病毒宿主细胞表面糖链受体的研究进展．     

Localization and force analysis at the single virus particle level using atomic force microscopy

Atomic force microscopy (AFM) is a vital instrument in nanobiotechnology. In this study, we developed a method that enables AFM to simultaneously measure specific unbinding force and map the viral glycoprotein at the single virus particle level. The average diameter of virus particles from AFM images and the specificity between the viral surface antigen and antibody probe were integrated to design a three-stage method that sets the measuring area to a single virus particle before obtaining the force measurements, where the influenza virus was used as the object of measurements. Based on the purposed method and performed analysis, several findings can be derived from the results. The mean unbinding force of a single virus particle can be quantified, and no significant difference exists in this value among virus particles. Furthermore, the repeatability of the proposed method is demonstrated. The force mapping images reveal that the distributions of surface viral antigens recognized by antibody probe were dispersed on the whole surface of individual virus particles under the proposed method and experimental criteria; meanwhile, the binding probabilities are similar among particles. This approach can be easily applied to most AFM systems without specific components or configurations. These results help understand the force-based analysis at the single virus particle level, and therefore, can reinforce the capability of AFM to investigate a specific type of viral surface protein and its distributions.     

Eurasian-origin gene segments contribute to the transmissibility, aerosol release, and morphology of the 2009 pandemic H1N1 influenza virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus.     

Viral detection by electron microscopy: past, present and future

Viruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions.     

Delocalization of vaccinia virus components observed by atomic force and fluorescence microscopy

Better understanding of viral genomes is emerging as an urgent need as these genomes evolve and pandemic fears surface and for better understanding of viral infection processes. To address this need, we report a method to visualize intact, viral DNA and its interaction with viral proteins with the use of the atomic force microscope (AFM) in conjunction with fluorescence microscopy. Through a series of multifaceted experiments, we were able to visualize time-dependent progressive stages of proteolytic digestion and disassembly of extracellular enveloped vaccinia virus particles. After a 1-h treatment, the viral particles were partially digested and the viral cores showed slight disassociation in the AFM as evidenced by height analysis of individual virions. Most of the components of the virions were still intact. Further verification with florescence microscopy with nucleophilic and lipophilic stains demonstrated that viral DNA was, indeed still, co-localized within the viral core. However, with prolonged treatment with proteinase K and sodium dodecylsulfate, the AFM revealed that the viral core completely collapsed onto the substrate and had delocalized from the enclosed DNA. This process was again verified using fluorescence microscopy, the viral DNA was observed to be completely released from the viral core, in globular condensed form. These studies suggest that AFM imaging and fluorescence microscopy verification with stains specific for different constituents of viral particles is a valuable method to study the structural and mechano elastic properties of virus morphology and interactions of viral nucleoproteins with its DNA core. These authors contributed equally to the work.     

长沙A(H1N1)亚型季节性流感暴发疫情的实验室诊断及其HA基因特性分析

对2009 年长沙麓山国际学校流感暴发疫情进行实验室诊断, 并探索新分离的A(H1N1)亚型流感病毒血凝素(HA)的基因特性。对流感暴发疫情的25 份鼻/咽拭子标本进行RT-PCR检测和流感病毒分离, 然后利用CEQ?8000 Genetic Analysis System对病毒分离株(A/Yuelu/314/2009)进行测序, 测序结果提交至GenBank(登录号: FJ912843)并用ClustalX和Mega4.1软件进行序列分析。结果显示, 分离出A(H1N1)亚型流感毒株18株, 检出21份A(H1N1)亚型流感病毒核酸阳性; A/Yuelu/314/2009(H1N1) HA基因序列与2008~2009 年疫苗株(A/Brisbane/59/2007)比较显示: 核苷酸和氨基酸同源性均为99%, 有6个位点的氨基酸发生了变异(V148A、S158N、G202A、I203D、A206T、W435R), 其中一个S158N氨基酸变异位于B抗原表位, HA基因序列上共有潜在糖基化位点9 个(27、28、40、71、151、176、303、497、536), 与A/Brisbane/59/2007相同且氨基酸序列保守。本实验诊断出此次流感暴发疫情的病原体为A(H1N1)型季节性流感病毒, 研究还发现A/Yuelu/314/2009(H1N1)长沙分离株与A/Brisbane/59/2007 疫苗株基因序列比较显示并未形成一个新的变种, 推测是由于分离株与疫苗株之间基因特性的改变和人群对A(H1N1)亚型流感病毒免疫力降低导致了此次长沙麓山国际学校A(H1N1)亚型流感的暴发。     

Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×10⁴ plaque-forming units per milliliter. Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.     

A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.     

Transmembrane domain of IFITM3 is responsible for its interaction with influenza virus HA2 subunit

Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses. Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA₂ subunit but not HA₁ subunit of H5 and H7 virus. Truncated analyses showed that the transmembrane domain of the IFITM3 and HA₂ subunit might play an important role in their interaction. Finally, this interaction of IFITM3 was also verified with HA₂ subunits from other subtypes of influenza A virus and influenza B virus. Overall, our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain, providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.