Applied Laser Physics and Laser Spectroscopy and Bielefeld Institute for Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Photochem Photobiol Sci. 2010 Feb;9(2):213-20. doi: 10.1039/b9pp00118b. Epub 2010 Jan 15.
Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.
光学显微镜使用可见光和透镜排列来为我们提供小样本的放大图像。结合高效的荧光探针和高灵敏度的荧光检测技术，它们允许对亚细胞结构进行非侵入性的 3D 研究，即使在活细胞或组织中也是如此。然而，光学显微镜受到光的衍射限制，使得在成像平面中的光学分辨率约为 200nm。在最近的过去，出现了强大的方法，可以实现具有亚衍射光学分辨率的荧光显微镜。由于这些方法中的大多数都是基于荧光团荧光发射的时间控制，因此可以在荧光开和非荧光关之间可逆切换的光致变色分子是超分辨率成像方法的关键。在这里，我们提出了使用螺吡喃-荧光团缀合物作为高效分子光学开关（光开关）的方法。在这些光致变色缀合物中，荧光团的荧光发射由螺吡喃的状态控制，螺吡喃可以在照射光下可逆地在无色螺吡喃和有色开环体之间切换。因此，荧光团向螺吡喃的能量转移效率可以通过照射条件来调节。我们展示了各种螺吡喃和螺吡喃-荧光团缀合物的开关过程的集合数据，并在单分子水平上证明了光开关。我们的数据表明，为了稳定开环体形式，以便在基于单个发射器的精确定位的超分辨率荧光成像中有用，螺吡喃必须固定在聚合物中。特别强调了由于 UV 照射导致的供体荧光团的光漂白，即光致变色受体的光开关。此外，我们还介绍了一种水溶性可切换的螺吡喃衍生物，并在聚合物中展示了第一个分子间单分子光开关实验。
