活细胞内单个大分子的行为

以往细胞内大分子活动的研究,多数是许多分子活动的一个平均结果,即一种集群平均（ensemble averaging）的结果,随着各种技术,特别是光学及荧光检测技术的成熟,实时视见活细胞内单个大分子的时代已经到来.在现时条件下,有许多方法可供选择,如荧光共振能量转移、原子力显微镜、全内反射显微技术、荧光相关光谱法等等.“活细胞内单个大分子的行为”的研究有可能为了解活细胞内单个分子的活动带来完全新的认识,但目前还存在不少方法学上的局限性,有待进一步提高,如有效的特异标记物、细胞深部荧光的检测、新型显微镜的开发等.     

拉曼光谱检测生物大分子损伤的研究进展

拉曼光谱是基于拉曼散射效应而发展起来的一种光谱分析技术,体现的是分子的振动或转动信息。由于拉曼光谱技术与常规化学分析技术相比,具有对样品无损、样品制备简单和所需样品量少等特点,广泛用于生物大分子结构变化的研究。拉曼光谱不仅可以用于蛋白质、核酸和脂类等生物大分子损伤的快速检测,而且可以用于癌症的诊断与手术治疗。通过对比正常组织与癌变组织的拉曼光谱,可以找到两种组织特征吸收峰的差异,从而为癌症的最终确诊和确定肿瘤切除范围提供重要信息。文章综述了拉曼光谱检测生物大分子损伤的研究进展,介绍了利用表面增强拉曼光谱、傅里叶变换拉曼光谱和紫外共振拉曼光谱等技术在检测蛋白质二级结构、膜脂及DNA损伤中的应用,并展望了未来拉曼光谱技术的发展前景。     

活细胞内的单分子荧光成像方法

文章介绍近年来新发展的几种重要的活细胞内单分子荧光成像方法,如转盘式共聚焦显微术、全内反射荧光显微术、荧光共振能量转移技术等。通过介绍它们的原理、特点和在活细胞内单分子行为研究中的应用实例,展示了这些新方法在生命科学领域广阔的应用前景。     

利用三通道实时荧光成像方法研究单个活细胞凋亡与胞浆pH值变化的关系

利用双波长分光器(Dual-View)和自制滤光片滑块,在基于像增强型电荷耦合器件(ICCD)的实时/快速荧光成像系统基础上,建立了一种基于单个ICCD的三通道实时荧光成像方法,同时发展了相应的图像校准处理方法用于消除光谱串扰的影响,并将该方法应用于单个活细胞的研究。利用Annexin V-FITC和SNARF-1两种荧光探针进行双标记,在单细胞水平上对亚硝基谷胱甘肽(GSNO)诱导小鼠胸腺细胞凋亡与其胞浆pH值变化的关系进行实时研究。结果揭示了GSNO诱导的细胞凋亡与细胞发生自发凋亡时胞浆pH值有不同的变化规律,为这种三通道实时荧光成像方法在生物医学领域的应用展示了广阔的前景。     

生物大分子的共振光散射光谱

共振光散射技术是一项在普通荧光分光光度计上可实现分析领域中应用的新技术。本文阐述了共振光散射的基本原理 ,分类介绍了在生物大分子研究和测定方面的应用 ,分析了共振散射体系的新发展。     

利用单粒子追踪技术研究活细胞内肌球蛋白的运输模式

利用单个粒子追踪技术研究?1A肾上腺素受体在活细胞内的转运．?1A肾上腺素受体在细胞内沿微丝骨架的转运表现为步进式模式，平均步长与体外对肌球蛋白的研究相一致．对不同时间分辨率下数据的模拟分析结果表明，肌球蛋白在细胞内运输内涵体的单步动力学与单个肌球蛋白在体外的报道相一致．     

红外光谱法检测生物大分子损伤的研究进展

红外光谱技术由于其灵敏度高和对样品的非破坏性等优点已成为研究生物大分子损伤的重要工具。蛋白质、脂质和核酸等受到损伤时,其红外光谱特征吸收峰的峰位、峰型和峰强会发生变化,这为检测生物大分子损伤并进一步揭示相关疾病的发生、发展及早期预防提供了依据。还综述了近年来使用红外光谱法检测生物大分子损伤的研究进展,介绍了利用傅里叶变换红外光谱、衰减全反射傅里叶变换红外光谱和傅里叶红外显微等技术在蛋白质二级结构、膜脂流动性和离子通透性以及药物对DNA的作用机制等领域的应用,以及相关的定性和定量分析方法进行了评述,提出了目前红外光谱分析技术中存在的问题,并对今后红外光谱在生物医学领域中的应用前景作了展望,指出疾病早期诊断、红外光谱联用以及定量分析技术等将成为红外光谱领域未来的研究热点。     

生物大分子多尺度理论和计算方法

分子模拟是研究生物大分子的重要手段. 过去二十年来, 人们将分子模拟与实验研究相结合, 揭示出生物大分子结构和动力学方面的诸多重要性质. 传统分子模拟主要采用全原子分子模型或各种粗粒化的分子模型. 在实际应用中, 传统分子模拟方法通常存在精度或效率瓶颈, 一定程度上限制了其应用范围. 近年来, 多尺度分子模型越来越受到人们的关注. 多尺度分子模型基于统计力学原理, 将全原子模型和粗粒化模型相耦合, 有望克服传统分子模拟方法中的精度/效率瓶颈, 进而拓展分子模拟在生物大分子研究中的应用范围. 根据模型之间的耦合方式, 近年来发展起来的多尺度分子模拟方法可归纳为如下四种类型: 混合分辨多尺度模型、并行耦合多尺度模型、单向耦合多尺度模型、以及自学习多尺度模型. 本文将对上述四类多尺度模型做简要介绍, 并讨论其主要优缺点、应用范围以及进一步发展方向.     

生物有机大分子中血红素铁可见光谱特性

在实验的基础上 ,对单体血红素、血红蛋白、细胞色素C、过氧化物酶等的可见光吸收光谱进行了比较与分析。生物有机大分子中血红素铁的第五、第六个配位键影响了该有机大分子的可见光谱的特性 ,使吸收峰的形状、位置发生变化。血红素b中铁的第六个配位键的形成使二价铁血红素在红光区有二个吸收峰 ,此时铁原子处于低自旋 ,相对较稳定 ;而在无第六个配位键的高自旋二价铁状态下 ,血红素在红光区表现出只有一个大吸收峰 ,分子处于活泼态。铁第五、第六配位键的状况对含血红素有机物吸收光谱的影响主要是通过改变铁在卟啉环平面上的空间位置和自旋状态等 ,从而改变整个血红素的电子状态所致。血红素的这些光谱特征有利于对含血红素生物有机分子的性质、功能、稳定性等的研究     

电场喷射活细胞

一般人使用喷墨打印机来打印文件和图片＋这种技术将细小的彩色墨滴从喷嘴中挤压出来．然而，在电子学和医学中正越来越多地用这种技术产生小体积的液滴．例如，使用压电晶体从一根针头中挤压出含有细胞的溶液，产生活细胞的二维和三维图像．但是细胞液滴的大小受到针头直径大小的限制＋因而这种方法不能产生小于100μm同左右的液滴．这意味着很难制成带有细微特征的小的生物组织．     

Monitoring of local pH in photodynamic therapy‐treated live cancer cells using surface‐enhanced Raman scattering probes

The local pH inside individual live glioma (U‐87 MG) cancer cells was monitored after treatment by the photodynamic therapy drug 6‐methyl‐1,3,8‐trihydroxyanthraquinone (emodin). The cellular pH is tracked by the real‐time measurement of the surface‐enhanced Raman scattering (SERS) from a probe that is embedded in the cell. The probe is a micrometer‐sized silica bead that is covered by nanosized silver colloids, which enhance Raman signal, and 4‐mercaptobenzoic acid (pMBA) whose molecular vibrations and resulting Raman spectrum are sensitive to pH. Visible excitation at different light dosages is used to activate the drug. The results indicate cell maintenance of internal pH and cell death at low and high light dosage, respectively. We demonstrate that these SERS probes are an effective tool for ex vivo pH monitoring in a live cell thanks to their high optical sensitivity and noninvasive usage. Copyright © 2011 John Wiley & Sons, Ltd.     

Field-based angle-resolved light-scattering study of single live cells

We perform field-based angle-resolved light-scattering measurements from single live cells. We use a laser interferometer to acquire phase and amplitude images of cells at the image plane. The angular scattering spectrum is calculated from the Fourier transform of the field transmitted through the cells. A concurrent 3D refractive index distribution of the same cells is measured using tomographic phase microscopy. By measuring transient increases in light scattering by single cells during exposure to acetic acid, we correlate the scattering properties of single cells with their refractive index distributions and show that results are in good agreement with a model based on the Born approximation.     

Reference database of Raman spectra of biological molecules

Raman spectra of biological materials are very complex, because they consist of signals from all molecules present in cells. In order to obtain chemical information from these spectra, it is necessary to know the Raman patterns of the possible components of a cell. In this paper, we present a collection of Raman spectra of biomolecules that can serve as references for the interpretation of Raman spectra of biological materials. We included the most important components present in a cell: (1) DNA and RNA bases (adenine, cytosine, guanine, thymine and uracil), (2) amino acids (glycine, L ‐alanine, L ‐valine, L ‐serine, L ‐glutamic acid, L ‐arginine, L ‐phenylalanine, L ‐tyrosine, L ‐tryptophan, L ‐histidine, L ‐proline), (3) fatty acids and fats (lauric acid, myristic acid, palmitic acid, stearic acid, 12‐methyltetradecanoic acid, 13‐methylmyristic acid, 14‐methylpentadecanoic acid, 14‐methylhexadecanoic acid, 15‐methylpalmitic acid, oleic acid, vaccenic acid, glycerol, triolein, trilinolein, trilinolenin), (4) saccharides (β‐D ‐glucose, lactose, cellulose, D ‐(+)‐dextrose, D ‐(+)‐trehalose, amylose, amylopectine, D ‐(+)‐mannose, D ‐(+)‐fucose, D ‐(−)‐arabinose, D ‐(+)‐xylose, D ‐(−)‐fructose, D ‐(+)‐galactosamine, N‐acetyl‐D ‐glucosamine, chitin), (5) primary metabolites (citric acid, succinic acid, fumarate, malic acid, pyruvate, phosphoenolpyruvate, coenzyme A, acetyl coenzyme A, acetoacetate, D ‐fructose‐6‐phosphate) and (6) others (β‐carotene, ascorbic acid, riboflavin, glutathione). Examples of Raman spectra of bacteria and fungal spores are shown, together with band assignments to the reference products. Copyright © 2007 John Wiley & Sons, Ltd.     

Rotation of single live mammalian cells using dynamic holographic optical tweezers

We report on a method for rotating single mammalian cells about an axis perpendicular to the optical system axis through the imaging plane using dynamic holographic optical tweezers (HOTs). Two optical traps are created on the opposite edges of a mammalian cell and are continuously transitioned through the imaging plane along the circumference of the cell in opposite directions, thus providing the torque to rotate the cell in a controlled fashion. The method enables a complete 360° rotation of live single mammalian cells with spherical or near-to spherical shape in 3D space, and represents a useful tool suitable for the single cell analysis field, including tomographic imaging.     

Superheat of silicon crystals observed by live X-ray topography

In-situ observations of Si crystal growth and melting have been carried out by live X-ray diffraction topography. Superheated solid states beyond the melting point was observed for dislocation-free crystals with melting in their inside. Dislocations were found to impede superheat and to melt the crystal without an appreciable superheating. A slightly superheated state accompanying melting removes all dislocations including immobile ones by their climb motion. It is proposed that self-interstitials needed for the volume change by melting are supplied by climb of dislocations, in contrast to dislocation-free crystals creating the interstitials thermally. In real crystal growth, remelting occurs naturally by melt convection and acts to make the growing crystal dislocation-free.     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

细胞内单颗粒示踪技术的进展

单颗粒示踪（Single particle tracking,SPT）技术是应用显微镜系统对细胞内单个特定荧光或散射颗粒的定位和追踪。由于SPT能够实时监控活细胞内复杂、高度动态的组织结构的变化并提供结构—功能间的动力学关系,因此在细胞生物学上有重要的应用。本文总结了SPT的机理以及在细胞上的应用,首先介绍了SPT的动力学原理,包括单颗粒定位,轨道重建以及轨道分析,然后总结了SPT技术现阶段重点发展的光学材料及仪器,最后阐述了SPT在细胞膜、细胞内信号通路、分子转运机制、遗传信息表达以及病毒感染机制的应用。此外,本文还对SPT技术未来的发展进行了展望。     

单分子的光学探测

介绍了三能级结构单分子光子源的工作原理,研究了利用Hanbury-Brown-Twiss探测方法,记录两个单光子计数器响应的单分子光子源输出的每一个事件,分析单事件的光子统计概率P（n,n=0,1,2）与Mandel参数.研究结果表明,当信号背景比SBR＞2.41时,P（2）＜（2-√4-2n^-）^2 /2是判别单分子的充分条件,n^-为每个激发周期内探测到的平均光子数。