原子力显微镜磁驱动轻敲模式在活细胞成像中的应用研究

应用MI公司最新发展的磁驱动轻敲模式(MAC mode)时体外培养成纤维细胞系3T3细胞进行在位成像研究.分别用力常数为0.95 N/m及0.03 N/m的微悬臂进行磁驱动轻敲模式成像,并与接触模式进行比较.同时研究了固定细胞与活体细胞之间的形貌差异.结果显示,利用上述两种微悬臂探针,磁驱动轻敲模式均可获得高分辨像.与接触模式相比,磁驱动轻敲模式对活细胞的影响较小,在细胞膜表面微结构及细胞内亚结构成像方面,有明显优势.而接触模式由于其施力方式,使活细胞应力纤维应激性绷紧,更适合于对活体细胞应力纤维的成像研究.固定细胞与活细胞表面形貌存在较大差异,在生理环境下,进行活细胞检测更能了解细胞的真实状况.     

原子力显微镜技术在细胞研究中的进展

原子力显微镜(AFM)是生物显微技术的一个重要组成部分,可实现液体环境下活细胞高分辨率的成像与操作,为细胞生物学研究提供了新的方法。近年来,AFM已经逐渐发展成为集样品成像、力测量及操作等功能于一体的多功能生物细胞研究平台,在细胞研究中得到了广泛的应用。在简要介绍AFM组成与工作原理的基础上,详细阐述了近年来基于AFM成像与力谱技术的细胞研究的发展状况。针对AFM本身所存在的不足,介绍了AFM与其他技术相结合的研究成果,并对AFM在生物细胞研究中未来的发展方向进行了展望。     

扫描离子电导显微镜的研制与实现

在微纳米尺度上对活细胞高分辨率成像对生命科学研究具有重要的意义,其将有助于再现正在发生的生命过程、检测细胞对外界刺激做出的响应,甚至观测某些蛋白簇在细胞膜表面的运动。然而直到今天,仍然没有很好的实现上述目标。扫描离子电导显微镜（SICM）由于其真正的非接触、高分辨、无损独特成像方式,规避了扫描过程中探针与样品表面发生力的接触,得到越来越多的关注和广泛的应用。从系统的角度阐述自制SICM 系统的设计、硬件集成及跳跃模式扫描算法的实现,并通过对聚二甲基硅氧烷（PDMS）栅格成像以及与原子力显微镜（AFM）成像结果的对比,验证了系统功能的正确性和有效性;最后开展了生理环境下活体细胞的原位扫描成像实验,初步获取了活体神经细胞轴突结构的三维形貌图像。SICM 的成功搭建,将为人们深入了解生理条件下活体生物样品表面微观结构与功能机理等提供有效的研究方法与手段。     

人红细胞老化过程中膜表面唾液酸变化的免疫AFM研究

分离人外周血的轻龄和老龄红细胞,采用麦胚凝集素和胶体金对红细胞表面的唾液酸残基进行细胞化学标记,然后在AFM下成像并进行数据分析.结果显示,老龄红细胞膜表面胶体金标记的颗粒数量明显低于轻龄红细胞表面,两者之间有显著差异.推测红细胞老化过程中细胞膜表面唾液酸含量的变化可能与老化细胞的处理机制有关.免疫胶体金标记结合原子力显微镜可在纳米尺度对膜受体蛋白进行定位和定量研究,拓展了原子力显微镜在生命科学领域的应用.     

PC12细胞凋亡及其细胞膜表面超微结构的原子力显微镜形态学观察

本文采用原子力显微镜(AFM)对PC12细胞的凋亡及其膜表面超微结构进行了实验研究，研究表明AFM可直接观察PC12细胞的生长情况，凋亡细胞膜发生皱缩、凹陷，并形成微绒毛消失的凋亡小体等现象。AFM有望发展成为一种研究细胞凋亡的工具。     

海马神经元的原子力显微成像

原子力显微镜(AFM)对完整的细胞成像并同时进行微细结构观察尚有一定困难。本实验改进了标本制备的过程．用原子力显微镜对戊二醛固定的海马神经元进行扫描，建立了方便、实用的完整海马神经元自完整胞体至超微结构的原子力显微镜成像技术，并用改进的方法获得了完整海马神经元及其超微结构的清晰的三维成像，并发现了一些其它显微技术所不能发现的微细结构。这些结构包括：①海马神经元胞体的亚细胞部分及这些亚细胞部分所具有的不同功能；②神经突触的完整形态；③损伤细胞膜表面出现的孔洞；④通过神经突触所形成的神经网络结构。     

基于近场光纤探针的活细胞无损测温方法研究

高精度的单细胞温度分布检测对于研究生命活动具有重要意义。为了实现高空间分辨率、高温度灵敏度的细胞测温,本研究团队基于原子力显微镜的扫描成像原理,将对温度敏感的量子点与音叉驱动的近场光纤探针相结合,提出了一种无损伤测量活细胞表面温度分布的方法;并以人脑星形胶质母细胞瘤细胞为研究对象,利用该方法检测了吞金纳米颗粒的固定细胞和未内吞金纳米颗粒的活细胞的温度分布。结果表明：在无金纳米颗粒作为附加热源的情况下,由活细胞内部产热引起的细胞表面最大温差超过0.5℃,而细胞与其外部环境间的温差大于2.5℃。所提活细胞无损测温方法的空间分辨率小于0.2μm,温度分辨率为0.16 nm/℃,为活细胞温度分布成像提供了新思路。     

小鼠胚胎干细胞超薄切片的AFM观察

对Balb／c小鼠胚胎干(ES)细胞的超薄切片进行AFM成像，以获得ES细胞内部结构的信息。尝试把AFM作为ES细胞形态学鉴定的一种手段。方法：利用超薄切片技术，将ES细胞样品制成厚约60nm的切片，固定在盖玻片上，用AFM在大气中观察。结果：观察到不同时期的单个ES细胞以及分裂时期ES细胞核中高度盘曲和浓聚的染色体。利用AFM的高分辨成像能力，观察了线粒体亚显微结构，细胞基质和核质的超微结构。     

一种基于正负半周联合成像的AFM成像方法

本文针对原子力显微镜(AFM)的扫描成像,提出了一种扫描过程中基于正负半周联合成像的AFM成像方法,该方法分析了AFM扫描过程中探针空降现象对扫描成像精度的影响,通过正负半周获得的扫描数据进行融合补偿,有效地降低了探针空降的影响,提高了扫描精度.最后通过对人肝癌细胞SMMC7721细胞的扫描实验对该方法进行了验证.     

非接触式扫描离子电导显微镜技术在探测活体细胞表面微结构中的应用

扫描离子电导显微镜技术是在纳米尺度进行非导电的生物样品成像的一种新型扫描探针显微镜技术。通过成功制备扫描离子电导显微镜扫描探测用纳米尺度玻璃微探针,对其进行了功能性评估;而后通过绘制探针-样品接近曲线,阐述了扫描离子电导显微镜技术实现非接触高分辨率探测的工作原理;最后采用该显微镜技术对导电标准样品及活体肾上皮A6细胞进行了表面形貌扫描成像,并与A6细胞表面形貌的扫描电镜图像进行了对照。结果表明,扫描离子电导显微镜技术不仅可实现导电样品的扫描成像,而且适宜于在生理条件下、非接触式地研究活体细胞表面的三维形貌,从而为人们深入研究细胞表面微观结构与生理功能提供了全新的技术手段。     

Quantitative analyses of topography and elasticity of living and fixed astrocytes

The topography and elasticity of living and fixed astrocytes cultured from the rat cerebra were studied quantitatively by atomic force microscopy (AFM). Ridge-like structures reflecting F-actin beneath the cell membrane were prominent in the contact-mode images of living astrocytes. Many of these ridges became unclear after fixation (2% glutaraldehyde). In addition, the ridge-like structures were invisible in the topography of living cells observed at zero-loading force in the force mapping mode, which is considered to show the real cell surface not pressed down by an AFM tip. The topography of fixed cells observed both in the contact mode and at zero-loading force in the force mapping mode was similar to that of living cells observed at zero-loading force in the force mapping mode, although some deformed areas were detected in the fixed cells. The elasticity map images of living astrocytes showed that the cell membrane above the nucleus was softer (2-3 kPa) than the surroundings, and that the cell membrane above F-actin was stiffer (10-20 kPa) than the surroundings. In the elasticity map images of fixed astrocytes, on the other hand, the elasticity of the cells was found to be relatively uniform (200-700 kPa) irrespective of the inner structures of cells. These results show that images observed by AFM should be carefully examined in consideration of the force introduced to specimens and the elasticity of specimens to find out the real surface topography.     

A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells

Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.     

Three-dimensional structural changes in living hippocampal neurons imaged using magnetic AC mode atomic force microscopy

We developed the magnetic AC (MAC) mode atomic force microscopy (AFM) to image the 3D ultrastructure of living hippocampal neurons under physiological conditions. Initially, the soma, the dendrites and the growth cones of hippocampal neurons were imaged. The imaging force was adjusted to a small value for the long-term observation. The neural spines were damaged when the tip produced a large force; the spines regenerated after the force was reduced. Subsequently, we explored the relationship between structural changes in hippocampal neurons and Alzheimer's disease by employing the new imaging technique. Time-lapse image acquisition (10 min intervals) showed that the growth cone collapsed after the addition amyloid peptide fragment beta(25-35), which is thought to initiate Alzheimer's disease. In addition, we found substantial changes in mechanical properties and in the volume of individual growth cone. This study suggested that MAC mode AFM may be a powerful tool for observing long-term structural changes in living neural cells under physiological conditions.     

活体细胞骨架的原子力显微成像

为研究原子力显微术在生物医学中的应用,实现对活体细胞骨架纳米尺度的实时观察,采用轻敲模式和接触模式对体外培养的大鼠脑微血管内皮细胞进行成像。结果显示不同模式在分辨细胞超微结构及质膜下细胞骨架纤维束等方面各有特点,可从不同角度获取细胞骨架的信息。     

The application of the atomic force microscope to studies of medically important protozoan parasites

Both living and fixed specimens of the medically-important parasitic protozoa, Trypanosoma cruzi, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, and Acanthamoeba spp. were studied by atomic force microscopy (AFM). The preparation of fixed specimens was similar to methods used for either scanning or transmission electron microscopy. AFM scanning was performed using both contact and tapping modes. A classical fixation procedure utilizing glutaraldehyde followed by ethanol dehydration was not suitable for all parasite species. AFM images could not be obtained from fixed samples of T. cruzi, T. gondii or E. histolytica. However, excellent topographic images could be obtained from specimens of G. lamblia and Acanthamoeba under identical conditions. Critical point drying permitted AFM imaging of both trypomastigote and epimastigote stages of T. cruzi. Phase imaging of T. cruzi elucidated unique surface details at a level of resolution not visible using any other imaging modalities. AFM elasticity map imaging of T. cruzi-infected and T. gondii-infected cells demonstrated that both parasites were markedly firmer than the surrounding host cell cytoplasm. The parasitophorous vacuole surrounding replicating T. gondii tachyzoites was also visualized by elasticity map imaging. These data suggest that although much remains to be learned about preparing parasitic protozoa for AFM imaging, the technique has the potential of providing unique and important insights into these disease causing organisms.     

原子力显微镜观察改性p（HEMA-MMA）对角膜基质细胞表面结构的影响

利用原子力显微镜分别对在I-型胶原和bFGF表面改性的p（HEMA-MMA）及未改性的p（HEMA-MMA）材料表面对于角膜基质细胞的膜表面结构进行了分析。将角膜基质细胞接种于改性前后的材料表面,用原子力显微镜对不同表面生长的角膜细胞的三维形态和超微结构等方面进行了研究。结果表明,改性后材料表面细胞宽度更大,高度更低,并且铺展较为完全,与正常条件下培养的细胞形态相似。膜超微结构参数分析显示在改性后材料表面生长的细胞膜微区的平均粗糙度（Ra）、均方粗糙度（Rq）、平均高度（MeanHt）、中值高度（MedianHt）明显较高于改性前材料表面的细胞。     

AFM扫描参数对表面粗糙度测量的影响分析

原子力显微镜(AFM)是纳米科技研究中一个重要工具.影响AFM测量图像质量的因素很多,本文从AFM扫描参数选择的角度,研究扫描参数对样品表面均方根粗糙度的影响.实验和理论分析表明,扫描速度、反馈控制参数中的比例常数P和积分常数I的选择均对样品的表面粗糙度测量及评定产生影响.