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Single cell analysis inside Environmental Scanning Electron Microscope (ESEM)-nanomanipulator system

机译:环境扫描电子显微镜(ESEM)-纳米夹持器系统内部的单细胞分析

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摘要

The conventional approach to characterize cellular biology is called biochemistry. This developed science is used for studying physiological aspects, mainly genetics, by characterizing protein and other biomaterials. Since single cells are difficult to study, a collection of cells are used for characterizing cellular physiology and inturn used to describe behavior of single cell (Brehm-Stecher & Johson, 2004). However, in addition to this advance understanding of cellular genetics, information about mechanical properties of cells is also needed. The molecular structure of the cell-wall is only partially understood, and its mechanical properties are an area of “near-total darkness” (Harold, 2005). Moreover, the approximation of single cell behavior from a group used in conventional approach also requires further justification whether it can be applied to all cell types (Shapiro, 2000). The knowledge of the cell mechanics could be valuable in the future for biomedical applications, for example, variations in cell mechanics of healthy and unhealthy cells can be linked to a specific disease. Available experimental techniques to probe single cells include micropipette aspiration, optical tweezers, magnetic tweezers (Bausch et al., 1999), atomic/molecular force probes (Gueta et al., 2006), nanoindenters, microplate manipulators, optical stretchers (Thoumine et al., 1999) and nanoneedle (Obataya et al., 2005).. The functionality of nanoneedles is not limited only to the stiffness measurements but it can also be used for single cell surgery (Leary et al., 2006) which can be further applied to a novel single cell drug delivery system (Bianco et al., 2005) or as a delivery tool for nanoparticles (Brigger et al., 2002). Conventional drug therapy suffers from the problems of inefficacy or nonspecific effects; hence, nanosystems are being developed for targeted drug delivery (Stylios et al., 2005). In order to successfully deliver materials; e.g. bioactive peptide, proteins, nucleic acids or drugs inside the cell, carriers must be able to penetrate the cell wall or cell membrane without causing death or create any mechanotransduction to the cell (Goodman et al., 2004), i.e. the process of converting physical forces to biochemical signals and integrating them into cellular responses (Huang et al., 2004). Therefore, the knowledge of biomechanics of the cell is crucial in providing prior-estimation of required insertion force to deliver drug material inside the cell. Without having this information, the insertion process may be unsuccessful due to inadequate applied force or the cell may be seriously damaged due to the excessive applied force. This chapter focuses on the following two needs, i.e. the needs for the understanding of the mechanical properties of single cells and the needs for the novel nanotools for the single cells probing. The first need was fed by highlighting our findings on the effects of three factors, i.e. cell sizes, environmental conditions and growth phases, on the strength of the single W303 yeast cells. The second need was served by showing our findings on the development of nanoneedles which can be used for single cell local stiffness characterizations and single cell surgery.
机译:表征细胞生物学的常规方法称为生物化学。这项发达的科学用于通过表征蛋白质和其他生物材料来研究生理方面,主要是遗传学。由于难以研究单细胞,因此将细胞集合用于表征细胞生理学,并进而用于描述单细胞的行为(Brehm-Stecher&Johson,2004)。但是,除了对细胞遗传学的这种提前了解之外,还需要有关细胞机械特性的信息。细胞壁的分子结构仅被部分理解,其机械性质是“几乎完全黑暗”的区域(Harold,2005)。而且,从常规方法中使用的一组近似单个细胞行为还需要进一步证明其是否可以应用于所有细胞类型(Shapiro,2000)。细胞机制的知识在将来对于生物医学应用可能是有价值的,例如,健康和不健康细胞的细胞机制的变化可以与特定疾病相关。探测单细胞的可用实验技术包括微量移液管抽吸,光学镊子,磁性镊子(Bausch等,1999),原子/分子力探针(Gueta等,2006),纳米压头,微板操纵器,光学拉伸器(Thoumine等) (1999)和纳米针(Obataya等,2005)。纳米针的功能不仅限于刚度测量,还可以用于单细胞手术(Leary等,2006),并且可以进一步应用。应用于新型单细胞药物递送系统(Bianco等,2005)或作为纳米颗粒的递送工具(Brigger等,2002)。常规药物治疗存在无效或非特异性作用的问题。因此,正在开发用于靶向药物递送的纳米系统(Stylios等,2005)。为了成功交付物料;例如细胞内的生物活性肽,蛋白质,核酸或药物,载体必须能够穿透细胞壁或细胞膜而不会导致死亡或对细胞产生任何机械转导(Goodman等人,2004),即转化物理力作用于生化信号并将其整合到细胞反应中(Huang等,2004)。因此,细胞的生物力学知识对于提供将药物材料输送到细胞内所需的插入力的事先估计至关重要。在没有此信息的情况下,插入过程可能会由于施加的力不足而失败,或者由于施加的力过大而严重损坏电池。本章重点介绍以下两个需求,即了解单细胞力学性能的需求和对单细胞探测的新型纳米工具的需求。通过强调我们对三个因素(即细胞大小,环境条件和生长阶段)对单个W303酵母细胞强度的影响的发现,满足了第一个需求。通过显示我们对纳米针发展的研究结果满足了第二个需求,该研究可用于单细胞局部硬度表征和单细胞手术。

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