Zhang Jian, Fu Yi, Liang Dong, Zhao Richard Y, Lakowicz Joseph R
Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, Maryland 21201, USA.
Anal Chem. 2009 Feb 1;81(3):883-9. doi: 10.1021/ac801932m.
Cy5-avidin conjugate-bound silver nanoparticles were prepared as a fluorescence molecular reagent for the cell imaging. Compared with the metal-free avidin conjugate, the avidin-metal complex was observed to display a stronger emission intensity, shorter lifetime, and better photostability. The avidin-metal complexes were conjugated with the biotin-sites on the surfaces of PM1 cell lines, and the cell images were recorded using scanning confocal microscopy. It was noticed that the avidin-metal complexes bound on the cell surfaces could be identified as the isolated emission spots distinct from the cellular autofluorescence. The emission intensity over the cell image was increased with an increase of the number of avidin-metal complexes on the cell surface but the lifetime was decreased. A quantitative regression curve was achieved between the amount of avidin-metal complex on the cell surface and the emission intensity or lifetime over the entire cell image. On the basis of this curve, we expect to develop an approach that can be used to quantify the amount of target molecules on the cell surfaces using the cell intensity and lifetime images at the single cell level.
制备了Cy5-抗生物素蛋白共轭结合的银纳米颗粒作为用于细胞成像的荧光分子试剂。与无金属的抗生物素蛋白共轭物相比,观察到抗生物素蛋白-金属络合物显示出更强的发射强度、更短的寿命和更好的光稳定性。抗生物素蛋白-金属络合物与PM1细胞系表面的生物素位点共轭,并使用扫描共聚焦显微镜记录细胞图像。注意到结合在细胞表面的抗生物素蛋白-金属络合物可被识别为与细胞自发荧光不同的孤立发射点。细胞图像上的发射强度随着细胞表面抗生物素蛋白-金属络合物数量的增加而增加,但寿命缩短。在整个细胞图像上,实现了细胞表面抗生物素蛋白-金属络合物的量与发射强度或寿命之间的定量回归曲线。基于此曲线,我们期望开发一种方法,该方法可用于在单细胞水平上使用细胞强度和寿命图像来量化细胞表面上目标分子的量。