摘要
目的 如何应用激光扫描共聚焦显微镜的Ｌａｓｅｒｓｈａｒｐ 软件进行分子共存分析．方法 使用ＢｉｏＲａｄ 公司的Ｌａｓｅｒｓｈａｒｐ 软件分析肝癌和胃组织细胞中不同分子的共存．结果 仔细进行样本制备和图象采集才能得到分子共存的可靠结果． 所有抗体应严格设立对照，确保抗体的特异性，并无交叉反应． 同时进行两个分子荧光标记，应使用间接荧光标记技术． 荧光素的发射波长应无重叠，如用Ａｌｅｘａ４８８ 和丽丝胺罗丹明或Ｔｅｘａｓ Ｒｅｄ ． 发射滤光片应达到可最大采集，同时又避免与其他荧光素发射光谱的相互渗透． 通常应用的一对荧光素是ＦＩＴＣ 和ＴＲＩＴＣ 或ＦＩＴＣ 和Ｔｅｘａｓ Ｒｅｄ ． 用抗体和共聚焦显微镜进行共存研究时根据激光光源的差别分别选用不同的荧光素． 当荧光素的发射光谱存在相互渗透时，应分别采集不同荧光素标记的图象． 使用氪／ 氩激光进行图象采集时，注意仔细平衡激发强度，即使在同时激发的情况下，也可得到充分光谱分离的图象．结论 在图象采集时避免光谱渗透，并从所有图象中去除背底染色后，Ｌａｓｅｒｓｈａｒｐ 共存分析软件包可提供正确的共存系数．
AIM To analyse the molecular co localization with Lasersharp software of laser scanning confocal microscope in digestology. METHODS Molecular co localization analysis of liver cancer and stomach tissues was performed with Lasersharp software of laser scanning confocal microscope (Bio Rad Company). RESULTS Reliable results can be obtained by sufficient careful sample preparation and acquisition. All antibody controls should be established to ensure antibody specificity and no cross reactivity between antibodies. When two molecules are being labelled immunofluorescently, the indirect technique should be used. Fluorochromes should have well separated emissions, e.g. , Alexa 488 and Lissamine rhodamine or Texas red. Emission filters should be optimized to maximize emission collection whilst avoiding bleed through from other fluorochromes. Different fluorochromes should be selected in the molecular co localization analysis. Sequential image was collected when bleed through is present. With a Krypton/*!Argon laser, careful balancing of the excitation intensities will produce images with sufficient spectral separation even in simultaneous mode. CONCLUSION The Lasersharp software co localization package can provide valid coefficients if bleed through is avoided during acquisition and background staining is subtracted from all images.
出处
《世界华人消化杂志》
CAS
1999年第9期746-752,共7页
World Chinese Journal of Digestology
关键词
激光扫描
共聚焦显微镜
软件
消化学
分子共存
laser scanning confocal microscope
software
digestology
molecular co_location
