Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy
Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy
复制标题
用于活细胞超分辨率显微镜的高光稳定性、可逆光开关荧光蛋白，具有高对比度
DOI：
10.1073/pnas.1611038113
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发表时间：
2016-09-13
影响因子：
11.1
通讯作者：
Xu, Pingyong
中科院分区：
文献类型：
--
作者：
Zhang, Xi;Zhang, Mingshu;Xu, Pingyong
超分辨(SR)荧光显微镜长期存在的两个问题是高照度和长采集时间，这严重阻碍了其在活细胞成像中的应用。可逆光开关荧光蛋白(RSFP)使饱和耗尽非线性结构照明显微镜(NL-SIM)和可逆饱和光学荧光跃迁显微镜等基于饱和耗尽的SR技术中的光照强度显著降低。对于SR活细胞成像来说，RSFP的最关键的特征包括：第一，每个开关周期的积分荧光信号，这取决于吸收截面、有效量子产率和从荧光“开”到“关”的特征转换时间；第二，开/关状态的荧光对比度；第三，激发和耗尽下的光稳定性。到目前为止，Dronpa和rsEGFP(可逆可切换EGFP)家族的RSFP已被用于SR成像。然而，它们有限的切换周期、相对较低的荧光信号以及在生理条件下较差的对比度最终限制了它们在时移活细胞成像中的应用以及在合理的信噪比下达到所需分辨率的能力。在这里，我们展示了一种真正的单体RSFP，Skylan-NS，其特性针对最近开发的图案化激活NL-SIM进行了优化，使低强度(类似于100 W/cm(2))活细胞SR成像能够在亚秒采集时间内以类似于60 nm的分辨率在大视野内进行数十个时间点的成像。
Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (similar to 100 W/cm(2)) live-cell SR imaging at similar to 60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.