第四章-荧光分析.ppt

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Two fundamentally different methods of beam scanning Single-beam scanning The more popular method at present is single-beam scanning The scanning is most commonly achieved : by computer-controlled galvanometer-driven mirrors（one frame per second） or in some systems，by an acoustooptical device or by oscillating mirrors for faster scanning rates（near-video rates） Multiple-beam scanning The alternative is to scan the specimen with multiple beams（almost real time） There are currently two viable alternatives to confocal microscopy that produce optical sections in technically different ways: Deconvolution Multiple-photon imaging As with confocal imaging, they are based on a conventional light microscope Deconvolution Deconvolution is a computer-based method It calculates and removes the out-of-focus information from a fluorescence image The deconvolution algorithms and the computers themselves are now much faster，with the result that this technique is a practical option for imaging． Multiple-photon microscopy Multiple-photon microscopy uses a scanning system that is identical to that of the LSCM but without the pinhole This is because the laser excites the fluorochrome only at the point of focus A pinhole is therefore not necessary Using this method， photobleaching is reduced，which makes it more amenable to imaging living tissues. * * * * * * * * * * * * * * * * * * * * * * * * * * 应用举例 溴脱氧尿嘧啶核苷(Bromodeoxyuridine, BrdU)是胸苷的一种类似物； 用BrdU处理细胞，BrdU将代替胸苷参与DNA合成； 用FITC等荧光探针标记的BrdU单克隆抗体与已含有BrdU的细胞一起温浴，荧光探针的荧光强度就可以反映细胞内DNA合成的情况； 可以用这种方法研究抗癌药物或细胞生长因子对细胞增殖的抑制或促进作用，从而得到有关药物药效和药理学的信息； 应用举例 细胞内pH、膜电位、细胞内钙都是细胞激活过程中重要的细胞参数； 用pH敏感的荧光探针可以测量细胞内pH值； 用cyanine(花青苷)、Rhodamine 123、Oxonol 等膜电位敏感性荧光探针标记细胞膜，可以根据荧光强度的变化与膜电位的对应关系来测量膜电位； 用Quin-2、Indo-1、Fyra-2、Fluo-3和Rhod-2等钙螯合剂EGTA的衍生物可以测量细胞内钙的浓度； 根据这些参数，可以了解更多的细胞内信号传