【摘要】 目的利用原子力显微镜(AFM)进行人舌鳞癌组织和细胞切片原位纳米尺度的操纵,并发展样品制备及处理的方法,提高舌鳞癌切片AFM图像的反差和分辨率,以满足纳米定位、切割、分离和拾取等一系列操纵的需求。方法人舌鳞癌组织和培养细胞按常规的电镜超薄切片法和石蜡切片法制样后,将切片分别转移至云母表面或载玻片表面,在空气中以接触模式AFM观察。选择合适的针尖并控制力的大小和扫描范围,进行纳米级成像、定位、分离和拾取等操纵。结果选择合适厚度的超薄切片,AFM观察显示,细胞轮廓清晰,细胞膜、核膜等结构明显,核内可见核仁,围绕核膜有颗粒状的细胞器。双氧水和二甲苯处理切片提高反差及AFM成像质量。对石蜡切片能分辨细胞轮廓、核内结构以及细胞间的结构。经上述处理的舌癌切片,通过控制扫描方向和针尖力的大小,实现了范围在20nm~1μm之间的操纵,并利用针尖获取核内遗传物质。结论以人舌鳞癌组织和细胞切片AFM高分辨率成像和结构分辨为基础,实现了AFM原位纳米尺度的操纵,获得特定细胞核内部的遗传物质,进一步开展生化分析,用于肿瘤早期诊断。更多还原

【Abstract】 Objective To investigate the appropriate nanomanipulation of slides of human tongue squamous cell carcinoma tissue and cultured cells by atomic force microscopy (AFM), and develop methods to improve the resolution and contrast of AFM images. Methods Human tongue squamous carcinoma cells of the line Tca8113 were cultured. Cell masses were isolated and collected, and sectioned. Then the white lead and silvery white sections were transferred on mica slides, dried. Eighty slides were used and divided into 2 groups to be immersed into hydrogen dioxide of the concentrations 20% and 30% for 0, 5, 10, 15, 20, 25, 30, and 40 min respectively. Samples of human tongue squamous carcinoma were collected from 15 cases. Every sample was divided into 2 halves. Half of every sample underwent routine paraffin preparation of slide and adhered onto glass, dissolving of paraffin with dimethylbenzene for 0, 10, 15, 30, 45, or 60 min; and half of the sample underwent routine preparation of slides for electron microscopy, the white lead and silvery white sections were transferred on mica slides, dried. The slides were observed by contact mode AFM in air. Results In the ultrathin sections the metastructure of the cell such as the cell membrane, nuclear membrane and nucleolus could be distinct from each other clearly. The profile of the cell and the nucleus inside could be revealed by AFM after the paraffin sections were treated in dimethylbenzene for 15~30 min. AFM manipulation could be achieved within nanometer range by precise modulation of scanning force, area, angle and so on. The appropriate needle point should contain elastic coefficient >5.0 N/m. The force exerted on the needle point should be within the range 50~300 nN. After location the scanning direction should be within the range of 20 nm~1 μm. Conclusion A series of modified techniques of preparing and treating AFM samples, such as adhering the sections onto mica slide, dissolving of epoxy resin by hydrogen dioxide and removal of paraffin by dimethylbenzene, were developed, thus realizing high resolving power AFM imaging, especially for nuclear membrane, and nucleolus, and providing a base of AFM location, dissection, isolation and obtainment at nanometer scale.更多还原