Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Biophys J. 2010 Oct 20;99(8):2686-94. doi: 10.1016/j.bpj.2010.08.012.
Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.
目前的远场荧光显微镜通过荧光抑制机制提供了亚衍射分辨率。这种机制的实现方式是，当同时暴露于激发光时，距离小于衍射极限的特征会分别发射。这种瞬态荧光抑制的基本机制是通过将荧光团（通过三重态）转移到暗态中来耗尽荧光团的基态，这种机制在大多数标准染料中是可行的。在这里，我们表明，基于基态耗尽随后进行单个分子返回（GSDIM）的显微镜可以有效地对免疫标记的固定细胞和 SNAP 标签标记的活细胞进行多色、无衍射限制的分辨率成像。通过使用标准的标记技术，GSDIM 被证明可以使用仅两个检测通道和一条激光线分离多达四种不同的常规荧光团。通过选择优化的二向色镜和选择具有可忽略的非均匀发射展宽的标记分子，该方法可以扩展到更多的颜色。
