Department of Neuroscience, Columbia University, New York, New York, USA.
Biophys J. 2012 Mar 7;102(5):1204-14. doi: 10.1016/j.bpj.2012.01.031. Epub 2012 Mar 6.
Using two-photon fluorescence anisotropy imaging of actin-GFP, we have developed a method for imaging the actin polymerization state that is applicable to a broad range of experimental systems extending from fixed cells to live animals. The incorporation of expressed actin-GFP monomers into endogenous actin polymers enables energy migration FRET (emFRET, or homoFRET) between neighboring actin-GFPs. This energy migration reduces the normally high polarization of the GFP fluorescence. We derive a simple relationship between the actin-GFP fluorescence polarization anisotropy and the actin polymer fraction, thereby enabling a robust means of imaging the actin polymerization state with high spatiotemporal resolution and providing what to the best of our knowledge are the first direct images of the actin polymerization state in live, adult brain tissue and live, intact Drosophila larvae.
利用双光子荧光各向异性成像技术对 GFP 肌动蛋白进行研究，我们开发了一种适用于广泛实验系统的肌动蛋白聚合状态成像方法，从固定细胞到活体动物。表达的 GFP 肌动蛋白单体掺入到内源性肌动蛋白聚合物中，使得 GFP 之间发生能量迁移 FRET（emFRET 或 homoFRET）。这种能量迁移降低了 GFP 荧光的正常高极化。我们得出了 GFP 肌动蛋白荧光偏振各向异性和肌动蛋白聚合物分数之间的简单关系，从而能够以高时空分辨率对肌动蛋白聚合状态进行稳健的成像，并提供了我们所知的活体成年脑组织和活体完整果蝇幼虫中肌动蛋白聚合状态的首次直接图像。
