结合微针及AFM的单细胞精准激励与力学特性同步测量

目的 细胞力学特性与细胞生理病理变化过程及机体健康状态密切相关,研究细胞力学特性对于揭示生命活动内在机制具有重要科学意义.原子力显微镜(AFM)的出现为单细胞研究提供了新的技术手段,它不仅可以在溶液环境下对单个活细胞的形貌结构进行高分辨率成像,还能够对细胞力学特性进行定量测量.基于AFM的单细胞力学特性研究在过去数十年...     

基于AFM的细胞表面超微形貌成像与机械特性测量研究进展

原子力显微镜(AFM)以其独特的优势(纳米级空间分辨率、皮牛级力灵敏度、免标记、可在溶液下工作)成为细胞生物学的重要研究手段.AFM不仅可以对活细胞表面超微形貌进行可视化表征,同时还可通过压痕技术对细胞机械特性(如杨氏模量)进行定量测量,为原位探索纳米尺度下单个活细胞动态生理活动及力学行为提供了可行性.过去的数十年中,研究人员利用AFM在细胞超微形貌成像和机械特性测量方面开展了广泛的应用研究,展示了有关细胞生理活动的大量新认识,为生命医药学领域相关问题的解决提供了新的思路;同时AFM自身的性能也在不断得到改进和提升,进一步促进了其在生命科学领域的应用.本文结合作者在应用AFM观测纳米尺度下癌症靶向药物作用效能方面的研究工作,介绍了AFM成像与细胞机械特性测量的原理,总结了近年来AFM用于细胞表面超微形貌成像与机械特性测量所取得的进展,讨论了AFM表征与检测细胞生理特性存在的问题,并对其未来发展方向进行了展望.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

基于多参数成像AFM的细胞及分子力学特性探测研究进展

原子力显微镜(AFM)的发明为微纳尺度下高分辨率探测天然状态生物样本的物理特性提供了强大工具,是对传统生化特性检测方法的有力补充.近年来,多参数成像模式AFM的出现使得人们不仅可以获取生物样本表面形貌特征,还能同时获取生物样本多种力学特性图(如杨氏模量、黏附力、形变等),为研究生物结构、力学特性及其生理功能之间的关联提供了新的技术手段.多参数成像AFM的生物医学应用研究为细胞/分子生理活动及相关疾病内在机理带来了大量新的认识.本文结合作者在AFM细胞探测方面的研究工作,介绍了多参数成像AFM工作原理,总结了多参数成像AFM在细胞及分子力学特性探测方面的研究进展,并对其存在的问题进行了讨论和展望.     

原子力显微镜研究多西他赛对人肺腺癌A549细胞的作用机制

利用原子力显微镜(AFM)研究了化疗药物多西他赛对肺腺癌细胞A549的作用机制。选取了不同浓度多西他赛作用下的A549细胞,观察和分析了多西他赛对A549细胞形貌信息和生物力学特性的影响。结果表明细胞的峰谷差和超微结构的平均粗糙度随着药物浓度增加而减小,表面粘附力随着药物浓度增加而增大。该结果对AFM可作为一种利用纳米量级分辨率进行药物药效评估的工具进行了佐证。     

不同厚度细胞薄切片对 AFM 图像反差的影响

利用原子力显微镜（ AFM ）观察超薄切片的表面,探索表面形貌与切片厚度、朝向等因素的关系以及对图像反差的影响 . 选择三种不同类型的细胞,培养后按电镜超薄切片法固定、包埋并切片后,将不同厚度的切片区分上下表面转移到云母上, AFM 在空气中以接触模式进行观察 . 结果发现,切片表面细胞相对包埋介质的凸起与凹陷与切片本身的厚度密切相关,并随切片厚度的不同呈现有规律的变化 . 实验统计结果显示这种现象可能具有普遍性 .     

原子力显微镜在生物医学上的应用

原子力显微镜(atomic force microscope,AFM)是扫描探针显微镜(SPM)的一种,其分辨率达到纳米级,能对从原子到分子尺度的结构进行三维成像和测量,能观察任何活的生命样品及动态过程。本文概述了AFM的基本工作原理及在生物医学上对DNA、蛋白质、细胞及生物过程等方面进行的研究。     

原子力显微镜在生物学研究中的应用进展

原子力显微镜（atomic force microscope,AFM)具有原子级分辨率,能够在生理条件下对生物样品进行观察,本综述了AFM的原理及技术要点,举例说明了它在核酸,蛋白质,微生物及细胞等领域的应用进展,相信AFM必将在生物学研究中起到越来越重要的作用。     

基于AFM的细胞弹性及黏弹性研究

原子力显微镜(AFM)的发明为测量生理环境下单个活细胞的机械特性提供了新的技术手段.现有AFM单细胞机械特性研究集中在测量细胞弹性.细胞本质上是黏弹性的,但目前关于细胞黏弹性在细胞生理活动行为中作用的认知还很不足.基于AFM逼近-停留-回退实验,发展了可同时对细胞弹性及黏弹性进行测量的方法,并应用该方法首先测量了正常乳腺细胞和乳腺癌细胞的弹性(杨氏模量)及黏弹性(松驰时间),显示出正常乳腺细胞和乳腺癌细胞的杨氏模量及松弛时间均有着显著的差异.AFM成像揭示了正常乳腺细胞和乳腺癌细胞在细胞表面形态及几何特征方面的差异.随后对3种不同类型的细胞系及原代B淋巴细胞进行了测量,证明了松驰时间在辅助杨氏模量鉴定细胞状态方面的潜力.实验结果为定量测量细胞机械特性提供了新的方法,便于从多个角度研究单个细胞的生物力学行为.     

基于AFM单细胞力谱技术的淋巴瘤细胞黏附力测量

细胞黏附在细胞生理功能中起着重要的调控作用,对细胞黏附行为进行定量研究有助于理解生命活动内在机制.原子力显微镜（AFM）的出现为研究溶液环境下微纳尺度生物系统的生物物理特性提供了强大工具,特别是AFM单细胞力谱（SCFS）技术可以对单细胞黏附力进行测量.但目前利用SCFS技术进行的研究主要集中在贴壁细胞,对于动物悬浮细胞黏附行为进行的研究还较为缺乏.本文利用AFM单细胞力谱技术（SCFS）对淋巴瘤细胞黏附行为进行了定量测量.研究了淋巴瘤细胞与其单克隆抗体药物利妥昔（利妥昔单抗与淋巴瘤细胞表面的CD20结合后激活免疫攻击）之间的黏附力,分析了利妥昔浓度及SCFS测量参数对黏附力的影响,并对淋巴瘤细胞之间的黏附力进行了测量.实验结果证明了SCFS技术探测动物悬浮细胞黏附行为的能力,加深了对淋巴瘤细胞黏附作用的认识,为单细胞尺度下生物力学探测提供了新的可能.     

A method for anchoring round shaped cells for atomic force microscope imaging.

More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM). However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers. This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape. Here we propose a simple and inexpensive solution to overcome these limitations. The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell. The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.     

Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.     

Paddle cilia and discocilia — Genuine structures?

Kinocilia of epidermal sensory cells in fixed marine Turbellaria often terminate as flattened biconcave discs. The distal part of the ciliary axoneme curves back upon itself forming a 360 degree loop which is enveloped by the plasmalemma. In living animals this structure can be induced by the addition of sodium cacodylate, monobasic sodium phosphate, dibasic sodium phosphate, sucrose, calcium chloride, or formaldehyde to the sea water. Specimens treated with sodium chloride, glutaraldehyde, or osmium tetroxide do not show modified cilia. In animals prepared for EM at low temperature and with a buffered hypotonic fixative less kinocilia are modified than in animals treated with a buffered iso- or hypertonic fixative and at a higher temperature. It is assumed that the unusually shaped cilia, described as "paddle cilia" or "discocilia" in other invertebrates, do not represent a genuine but an artificial structure.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

Induction of new ADAM related proteins from treated human Chang-liver cells

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.     

Induction of new adam related protein from treated human Chang-liver cells

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.     

An Air-Drying Technique for Flattening Chromosomes in Mammalian Cells Grown In Vitro

The method described was developed to facilitate the analysis of chromosome complements in cells freshly isolated from monkey kidney cortex and grown on glass, and in “altered” monkey cells grown on glass or in suspension. Cells were treated with hypotonic solution (quarter-strength Tyrode or diluted medium) for 30 min, or with colchicine in a final concentration of 25 μg/ml (.0025%) for 12-18 hr followed by hypotonic salt solution for 5 min, then fixed in acetic alcohol (1:3) for 5 min. With cells centrifuged from suspended cultures, addition of fixative had to be gradual. Directly after fixation, films of cells on slides were air dried completely. This produces a more uniform and complete flattening of cells than can be achieved by manual pressure; yet, fragmentation of chromosome complements does not occur. Fixed and air dried slides may be stored for days without deterioration or they may be stained immediately in 2% natural orcein (G. T. Gurr, London) in 50% acetic acid. Preparations can be made permanent by a dry ice schedule, without loss, shrinkage, or distortion of cells.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary A method is presented which allows the study of the progeny of single cells during Drosophila embryogenesis. Cells from various larval anlagen of donor embryos labelled with a lineage tracer are individually transplanted from defined positions into similar, or different, positions in unlabelled hosts. The clones produced by these cells can be seen in whole mounts or in sections of fixed material, when using a histochemical marker (i.e. HRP), and/or in living embryos, when using fluorescent lineage tracers. The characteristics of the clones disclose lineage parameters, such as division patterns, morphogenetic movements and differentiation. The method is especially useful for testing the respective roles of positional information and cell lineage on the commitment of progenitor cells by transplanting these cells into heterotopic positions or into hosts of different genotypes.     

mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

Background  

The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.