Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Department of Optical Nanoscopy, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
ACS Nano. 2021 Jun 22;15(6):9509-9521. doi: 10.1021/acsnano.0c08207. Epub 2021 May 21.
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength that also excites fluorescence. Positive switching RSFPs have a reversed light response, where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. On the basis of the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-states of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential illumination steps.
可反复切换的荧光蛋白（RSFP）可以通过用不同波长的光照射在荧光开启和非荧光关闭状态之间反复切换。负向切换的 RSFPs 用同样激发荧光的波长从开启状态切换到关闭状态。正向切换的 RSFPs 具有相反的光响应，其中荧光激发波长诱导从关闭状态到开启状态的转变。可逆饱和光学线性（荧光）跃迁（RESOLFT）纳米显微镜利用这些切换状态实现了无衍射限制的分辨率，但迄今为止主要依赖于负向切换的 RSFPs，采用时间顺序切换方案。基于绿色荧光的 RSFP Padron，我们设计了正向切换的 RSFP Padron2。与前代产品相比，它可以进行 50 倍以上的切换循环，同时在开启和关闭状态之间显示出超过 100:1 的对比度。由于其稳健的切换行为，Padron2 支持 RESOLFT 成像方案，完全避免了顺序切换，因为它只需要对两个空间上重叠的光分布进行光束扫描。使用 Padron2，我们演示了无需顺序照明步骤的活细胞 RESOLFT 纳米显微镜。
