6.Point out the importance, application and data interpretation of the following techniques, such as negative staining, shadowing, and immunolabelling, etc.,for Molecular and Cell Biology NEGATIVE STAINING IMPORTANCE: 1.可以直接观察物体的外型 2.不破坏样品的表面. 3.样品处理制作技术较简单, 所要的样品浓度很少, 却有很高的解像力和对比度 APPLICATION: 观察微小颗粒状结构的样品, 例如细菌, 病毒, 粒线体, 和蛋白质等, 而 样品最好是一层膜或是分开的颗粒, 以防重叠到. DATA INTERPRETATTION 要知道如何解释经负染色後的玻片, 首先要知道其原理. . 在负染色中, 重金属染剂沉积在物体外面, 使物体和其背景产生强烈的对比度. 由於生物 体组成元素多为分子量较小之, 因此电子穿透力较强. 而沉积於外面的重金属有较大的原 子量, 对於电子的散射力较强, 电子不易透过. 所以电子多直接打到样品. 而穿透电子的 多寡就直接代表其原子密度. 而且当电子穿透时并不会破坏染剂, 所以跟观察结果不会影 响. SHADOW CASTING： IMPORTANCE: 是一种可以观察物体立体结构的技术. 以此法制作的样品, 除了可以显现物体三度空间的 构造外, 更可判断颗粒的形状, 大小及高低. Application: 投影又有分三种, 单向投影可使物体某一边缘呈现阴影, 故在观察时较有立体感, 但在 判断物体之长宽或大小时较不易做精确之测量. 而另一种旋转投影由於没有明显的阴影形 成, 可精确测量微细颗粒的直径, 但相对损失了立体感, 很难判定物体高低的层次. 此法是将载有标本的铜网置於真空蒸着器，将金属蒸发而由一个角度投射到标本上，如此 标本一边有一层金属，另一边则无。在穿透式电子显微镜下，有金属的一边把电子束分散 而成暗背景，将标本衬出。如果知道金属投射的角度及高度，则可计算出标本的厚度。另 外一方法是金属蒸发投射到标本时，标本不断作三百六十度旋转，则金属堆积於标本四周 ，而使标本浮现出来。DNA分子的观察就用此法，因为核酸分子宽度只有2nm，经此种旋转 式金属投影可增加其宽度，而能在电子显微镜下观察 http://cryoem.berkeley.edu/~nieder/em_for_dummies/negative_stain.html Immunolabeling IMPORTANCE 让动物组织与特定性抗体结合, 然後用非?的标记物与抗体结合, 以检测样品内有无特定 抗体. APPLICATION: Immunolabeling provides an added dimension over conventional immunofluorescence staining techniques by allowing the investigator to unambiguously identify the localization and distribution of proteins, antigens, and other macromolecules of interest at an ultrastructural level. DATA INTERPRETATION: Using various techniques, dependent on the type of sample and antigen to be immunolabeled, the primary antibody is exposed to the sample, thus adhering to the target protein or antigen. After washing steps, a secondary gold conjugate is exposed to the sample allowing for the adherence of the gold particles to the primary antibody. After further processing, the sample is examined in the electron microscope where the electron-dense gold particles are easily seen, showing the location and distribution of the target antigen or protein. Cells, tissues, and biologics in suspension can be immunolabeled using various techniques. Because of the uniqueness of each specimen and the specialized techniques involved with Immuno-Electron Microscopy, the electron microscopy staff works closely with the investigator to define the parameters of each study. Contents http://web.uct.ac.za/depts/mmi/stannard/negstain.html http://www.arches.uga.edu/~howie/negative%20staining.htm http://www.abionline.com/services/microscopy3FAQ.htm 恩~~~内容顺序映该与网站顺序不依样~~~ㄏ~~~ --

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