Mei Erwen, Gao Feng, Hochstrasser Robin M
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Phys Chem Chem Phys. 2006 May 7;8(17):2077-82. doi: 10.1039/b601670g. Epub 2006 Mar 23.
The concentration and vesicle size-controlled collisions of single molecules with target biological assemblies allow sub-diffraction limited optical images to be obtained that are not subject to the usual photobleaching problems with single molecule experiments. For example, single molecules of the probe Nile Red in aqueous solution emit a burst of fluorescence when they collide with a 50 nm hydrophobic vesicle situated on the surface in the laser focus. The bimolecular kinetics of the bursts is defined by their on- and off-time distribution functions which depend on the concentration and diffusion of the probe and the vesicle size. The mean burst frequency changes much more sharply than does the fluorescence intensity when a vesicle is raster scanned through the laser focus. This sharpness allows the spatial resolution of two objects to be improved and separations less than the diffraction limited resolution of the conventional optical microscope to be measured. The principle of this method of trajectory time distribution optical microscopy (TTDOM) could be used in a far field optical microscopic system with a resolution of several nanometers.
单分子与目标生物组件的浓度和囊泡大小控制的碰撞使得能够获得亚衍射极限光学图像，这些图像不受单分子实验中常见的光漂白问题的影响。例如，水溶液中的探针尼罗红单分子在与位于激光焦点表面的50纳米疏水囊泡碰撞时会发出一阵荧光。爆发的双分子动力学由其开启和关闭时间分布函数定义，这些函数取决于探针的浓度和扩散以及囊泡大小。当一个囊泡通过激光焦点进行光栅扫描时，平均爆发频率的变化比荧光强度的变化要尖锐得多。这种尖锐性使得两个物体的空间分辨率得以提高，并且能够测量小于传统光学显微镜衍射极限分辨率的间距。轨迹时间分布光学显微镜（TTDOM）这种方法的原理可用于分辨率为几纳米的远场光学显微镜系统。
