Saffarian Saveez, Kirchhausen Tomas
Department of Cell Biology and Immune Disease Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biophys J. 2008 Mar 15;94(6):2333-42. doi: 10.1529/biophysj.107.117234. Epub 2007 Nov 9.
We present a method to resolve components within a diffraction-limited object by tracking simultaneously the average axial positions of two different sets of fluorescent molecules within it. The axial positions are then subtracted from each other to determine the separation of the two sets of fluorophores. This method follows the dynamic changes in the separation of the two sets of fluorophores with freely rotating dipoles using sequential acquisitions with total internal reflection and wide-field illumination, and it can be used to measure the formation of small structures on living cells. We have verified that we can achieve a resolution of 10 nm, and we have used the method to follow the location of clathrin and its adaptor AP-2 as they are recruited to a diffraction-limited coated pit during its assembly at the plasma membrane. We find a gradually increasing axial separation between the centroids of clathrin and AP-2 distribution, up to a final value of 30 nm just before coated-pit pinching and formation of the coated vesicle.
我们提出了一种方法，通过同时跟踪衍射极限物体内两组不同荧光分子的平均轴向位置来解析该物体内的成分。然后将轴向位置相互相减，以确定两组荧光团的间距。该方法利用全内反射和宽场照明的顺序采集，跟踪两组具有自由旋转偶极子的荧光团间距的动态变化，可用于测量活细胞上小结构的形成。我们已经验证能够实现10纳米的分辨率，并且我们已经使用该方法跟踪网格蛋白及其衔接蛋白AP-2的位置，因为它们在质膜上组装期间被招募到衍射极限的包被小窝中。我们发现网格蛋白和AP-2分布质心之间的轴向间距逐渐增加，在包被小窝缢缩和包被囊泡形成前达到最终值30纳米。
