Replication of cancer cells using soft lithography bioimprint technique

Replication of biological cells for the purpose of imaging and analysis under electron and scanning probe microscopy has facilitated the opportunity to study and examine some molecular processes of living cells in a manner that was not possible before. The difficulties faced in direct cellular analysis when using and operating atomic force microscopy (AFM) in situ for morphological studies of biological cells has lead to the development of a novel method for biological cell studies based on nanoimprint lithography. The realisation of the full potential of high-resolution AFM imaging has revealed some very important biological events such as exocytosis and endocytosis. In this work, a soft lithography bioimprint replication technique, which involved simple fabrication steps, was used to form a hard replica of the cell employing a newly developed biocompatible polymer that has fast curing time at room temperature essential for this process. The structure and topography of the endometrial (Ishikawa) cancer cell was investigated in this study. Cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the Human Ethics Committee, University of Otago. An impression of the cell profile was created by applying a layer of the polymer onto the cells attached to a substrate and rapidly cured under UV-light. Fast UV radiation helps to lock cellular processes within minutes after exposure and replicas of the cancer cells exhibit ultra-cellular structures and features down to nanometer scale. Elimination of the AFM tip damping effects due to probing of the soft biological tissue allows imaging with unprecedented resolution. High-resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. Craters that resemble granules may be observed. These represent steps on a transitional series of sequential structures that indicate either an endocytotic or exocytotic processes, which were evident on the replicas. These events, together with exocytosis, play a very significant part in the tumorigenesis of these cancer cells. By forming cell replica impressions, not only have they the potential to understand biological cell conditions, but may also benefit in synthesizing three dimensional (3-D) scaffolds for natural growth of biological cells and provide an improvement over standard cell growth conditions.     

人舌鳞癌组织超薄切片的AFM成像和切割

利用一种基于电镜超薄切片法改进的制样方法，将人舌鳞状细胞癌病理组织以环氧树脂包埋并切片后，将薄片平整地贴附在云母上，用原子力显微镜(AFM)对切片表面进行研究，可以得到高分辨率的细胞超微结构图像，局部的亚细胞水平的形态结构可以与电镜下得到的图像相比拟。在此基础上，利用AFM针尖对肿瘤细胞核内特定区域进行切割和操纵，形成生物分子的堆积，从而为拾取(pjck—up)和进一步用分子生物学手段在亚细胞基因水平研究人舌鳞癌的病理学奠定了基础。     

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.     

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。     

Segmenting and tracking fluorescent cells in dynamic 3-D microscopy with coupled active surfaces.

Cell migrations and deformations play essential roles in biological processes, such as parasite invasion, immune response, embryonic development, and cancer. We describe a fully automatic segmentation and tracking method designed to enable quantitative analyses of cellular shape and motion from dynamic three-dimensional microscopy data. The method uses multiple active surfaces with or without edges, coupled by a penalty for overlaps, and a volume conservation constraint that improves outlining of cell/cell boundaries. Its main advantages are robustness to low signal-to-noise ratios and the ability to handle multiple cells that may touch, divide, enter, or leave the observation volume. We give quantitative validation results based on synthetic images and show two examples of applications to real biological data.     

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。     

Multiparameter evanescent-wave imaging in biological fluorescencemicroscopy

Wide-field optical microscopy of live cells with nearfield precision, tracking the dynamics of subcellular organelles, imaging of single-molecule reactions with millisecond time-resolution, or watching synaptic nerve terminals "in action"-these are some examples of recent work that triggered the "renaissance" of evanescent-wave (EW) spectroscopy in biological imaging. In these studies, the goal is to markedly reduce background fluorescence from locations in the sample other than the cell/substrate interface. After a brief reminder of EW generation, we discuss how the confinement of fluorescence excitation highlights cellular structure near the plasma membrane with unprecedented detail. We then illustrate how the intensity distribution and polarization of the EW can be used to study dynamic biological processes that have neither been accessible with optical (confocal or two-photon fluorescence) nor electron microscopy, and take a glimpse of what is to come in EW imaging     

Atomic force microscopy in structural biology: from the subcellular to the submolecular

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.     

用于软刻蚀复型及转印前后形貌比较的反向重定位AFM成像方法

软刻蚀技术运用聚二甲基硅氧烷（Poly dimethylsiloxane,PDMS）等聚合物材料制备弹性图章,并用制备好的图章转印图形结构至特定基底。在很多情况下需对模板和弹性图章、图章与转印图形同一位置的形貌进行表征。基于原子力显微镜观测的反向重定位技术提供了一种微观区域内对二雏平面互为镜像关系的两种样品的原位对比观测方法。本文利用坐标实时显示的程控高精度样品台系统,联合使用非对称表面标记及坐标镜像复制的定位法,实现了对样品的精确反向重定位成像。     

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.     

数字全息显微定量相位成像的实验研究

建立了一套预放大式数字全息显微成像系统,通过对样品进行显微放大,实现了高分辨率的定量相位成像;并通过计算机控制相机自动曝光记录序列的数字全息图实现了动态相位成像。用标准样品验证了系统测量的准确性;以活体洋葱表皮细胞和血红细胞为样品,获得了清晰的定量相位像;以置于水环境的草履虫为样品,实现了动态成像。实验结果表明建立的系统可以实现高分辨率的动态定量相位成像,可以应用于生物活体样品的显微研究。     

New Imaging Technologies to Enhance the Molecular Sensitivity of Positron Emission Tomography

Positron emission tomography (PET) is used in the clinic and in vivo small animal research to study certain molecular processes associated with diseases such as cancer, heart disease, and neurological disorders and guide the discovery and development of new treatments. New PET molecular probes and associated small animal imaging assays are under development to target, visualize, and quantify subtle molecular and cellular processes such as protein-protein interactions in signal transduction pathways, cancer cell trafficking, therapeutic stem cells and their progeny, interaction of the immune system and tumor cells, and gene delivery and expression in living animals. These next-generation PET molecular imaging assays require an order of magnitude increase in PET's ability to detect, visualize, and quantify low concentrations of probe interacting with its target, which we will refer to as molecular sensitivity , in order to study the subtle signatures associated with these molecular processes. The molecular sensitivity is determined by a combination of the probe and biological/physiological properties of the subject that determine its specificity for the target, and the performance capabilities of the imaging system that determine how well the resulting signal can be measured. This paper focuses on the second aspect: the challenges of advancing PET technology and some of the new imaging system technologies under investigation to substantially enhance PET's molecular sensitivity. If successful, these novel imaging system technology advances, together with new probe molecules that target specific molecular processes associated with disease, will substantially enhance the molecular sensitivity of PET and thus increase its role in preclinical and clinical research as well as evaluating and managing disease in the clinic.     

AFM观察稳恒磁场对细胞表面精细结构的影响

利用原子力显微镜(AFM)观察稳恒磁场(SMF)处理后,悬浮生长细胞(K562人白血病细胞)、贴壁生长细胞(人结肠癌SW480细胞)、小鼠肝癌细胞Hepal-6和原代小鼠肝细胞表面精细结构的变化,以了解SMF杀伤肿瘤细胞的可能机制.观察结果显示:随曝磁时间延长,SMF可在肿瘤细胞表面造成不同程度损伤,主要表现为细胞膜上出现许多大小不一的凹陷,且凹陷数量和直径随着曝磁时间延长而增加.与MTT检测相比,AFM观察到的各类细胞表面损伤远早于细胞的生长抑制.实验观察显示,悬浮生长细胞比贴壁生长细胞对磁场处理更为敏感,小鼠肝癌细胞比肝细胞对磁处理更敏感.实验结果显示,AFM能够及早观察到细胞表面因SMF作用而产生的精细结构方面的变化.     

Automated segmentation, classification, and tracking of cancer cell nuclei in time-lapse microscopy

Quantitative measurement of cell cycle progression in individual cells over time is important in understanding drug treatment effects on cancer cells. Recent advances in time-lapse fluorescence microscopy imaging have provided an important tool to study the cell cycle process under different conditions of perturbation. However, existing computational imaging methods are rather limited in analyzing and tracking such time-lapse datasets, and manual analysis is unreasonably time-consuming and subject to observer variances. This paper presents an automated system that integrates a series of advanced analysis methods to fill this gap. The cellular image analysis methods can be used to segment, classify, and track individual cells in a living cell population over a few days. Experimental results show that the proposed method is efficient and effective in cell tracking and phase identification.     

Magnetic Force Microscopy of Iron and Nickel Nanowires Fabricated by the Matrix Synthesis Technique

Iron and nickel nanowires are grown by the matrix synthesis technique in the pores of the track membrane fabricated based on polyethylene terephthalate (PET). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to image the pores on the surface of the specimens and determine the 3d metal nanowires in a polymer bulk. The magnetization curves of the arrays of the nanowires are obtained. Magnetic properties of the nanowires are studied by the magnetic force microscopy (MFM) methods. The influence of the interposition and magnetostatic interaction of the nanowires of the magnetic metals in the polymer membrane, as well as the magnitudes and orientation of the applied external magnetic field, on the obtained MFM images is shown. The simulation results of the MFM images are in good agreement with the experimental data.     

肿瘤多细胞球:一种近似实体瘤的体外肿瘤模型

本文介绍了一种简便易行的方法,用以在体外建立近似体内实体瘤的肿瘤模型—肿瘤多细胞球模型(multicellular tumor spheroids,MCTSs).采用液滴重叠法(liquid overlay method)来构建HeLa肿瘤多细胞球模型,并在光镜下对肿瘤多细胞球的生长状况进行观察描述,再应用场发射扫描电...     

Designer Biomaterials to Model Cancer Cell Invasion In Vitro: Predictive Tools or Just Pretty Pictures?

Metastasis is the leading cause of mortality in cancer patients. Underlying this process is the invasion and colonization of cancer cells into healthy tissues. Engineered hydrogel models of tumor microenvironments present an opportunity to understand the microenvironmental determinants of cellular invasion. The biochemical and mechanical cues, presented in the form of adhesion sites, degradable cues, matrix stiffness, and architecture, have significant effects on the extent of cancer cell migration, and the mechanisms employed by these cells to move through their matrix. Coculture with stromal cells such as cancer associated fibroblasts, endothelial cells, and immune cells that are associated with poor prognosis demonstrate that these cells exacerbate cancer cell invasion. With these models, researchers aim not only to recapitulate known cancer cell behaviors in a dish, but also to uncover new insights into mechanisms underlying these phenomena, paving the way for novel treatment strategies. In this perspective, the design of engineered models that are used to study cancer cell invasion and metastasis in vitro is discussed. To this end, the authors seek to understand and put into perspective: do these models reveal relevant mechanisms of cancer cell migration, or are they simply pretty pictures with little biological translatability?     

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。     

Studies of the micro- and nanostructure of polycrystalline CdTe and CuInSe2 using atomic force and scanning tunneling microscopy

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been used to study the micro- and nanostructure of CdTe and CuInSe₂ thin films used for photovoltaic cells. Topographic images are comparable with those reported previously using conventional scanning electron microscopy (SEM)—to the limit of spatial resolution of the SEM technique. For higher magnifications, nanoscale structures and features have been observed for the first time with AFM and STM, and these observations have implications for the suitability and preparation of these semiconductors for high-efficiency solar cell realization.