以三维荧光反卷积显微技术研究活体细胞中分泌囊泡的空间分布  

Use of Three-dimensional Fluorescence Deconvolution Microscopy for Study of Spatial Distribution of Secretory Vesicles in Living Cells

作　　者：李栋栋[1] 郭学彬[1] 瞿安连[1] 徐涛[1] 

机构地区：[1]华中科技大学生命科学与技术学院生物物理与生物化学研究所,武汉430074

出　　处：《生物化学与生物物理学报》2003年第7期671-676,共6页

基　　金：国家自然科学基金资助项目(No .60 0 710 3 0);国家自然科学基金重点项目(No .3 0 13 0 2 3 0 );国家杰出青年基金资助项目(No .3 0 0 2 5 0 2 3 )~~

摘　　要：获得活体细胞三维图像以观察细胞内分泌囊泡的空间分布有助于细胞分泌机制的研究。三维荧光反卷积显微技术可以为活体细胞观察提供低荧光漂白 ,低毒副作用的快速三维成像。研究了显微成像系统实验测定和理论计算点扩展函数之间的关系 ,并且实验验证了NA 1.6 5物镜条件下 ,理论计算点扩展函数可以较好地反映显微成像系统的特性。然后使用已知物理结构的三维样本对反卷积算法的有效性进行了研究。进而对使用吖啶橙(acridineorange)标记的大鼠胰腺 β细胞分泌囊泡进行观察。结果显示 ,反卷积算法可以有效地去除原始图像中因为焦外光影响产生的模糊 。A three dimensional image of a living cell is helpful for cell secretion study. In this report , the three dimensional fluorescence deconvolution microscopy for observing living cells was studied, because this technique can obtain a quick three dimensional imaging with minimal fluorescence quenching and cytotoxicity for living cell observation. The property of three dimensional point spread function (PSF) of imaging system was analyzed. The relationship between experimental and theoretical PSF was illustrated, and the theoretical PSF was proved that it could reflect the principle of imaging system with NA 1.65 objective in use. Three dimensional deconvolution algorithm in this report was proved effective by well defined three dimensional specimens. Furthermore, the rat pancreatic β cell secretory vesicles labeled by acridine orange was observed by using this technique. Results showed that the blurring induced by out of focus light was removed by the deconvolution algorithm effectively, under current experiment conditions (with NA 1.65 objective) the experimental PSF approximated the theoretical PSF very well, and deconvolved living cell images exhibited the spatial distribution of the secretory vesicles clearly.

关 键 词：观察 细胞内 内分泌 显微技术 胰腺Β细胞 毒副作用 大鼠 活体细胞 囊泡 反卷积 

分 类 号：Q24[生物学—细胞生物学]