超解像機能イメージングを可能にする新規蛍光タンパク質プローブの開発

由于缺乏合适的基于荧光蛋白（FP）的探针，在纳米水平上对生命系统进行功能成像是困难的。基于可逆光开关荧光蛋白（RSFPs）的探针是最好的选择。然而，由于传统的RSFP的性能差，我们试图开发一种RSFP，具有快速的光开关，高光稳定性和上级亮度。经过定点诱变和几个随机诱变循环，最后，我们开发了一个RSFP，Kohinoor，它在其亲本RSFP Padron中含有七个突变。Kohinoor具有最快的开关速度和最高的光稳定性，其开关周期比其亲本Padron RSFP多25倍。Kohinoor的量子产率为0.71，是所有RSFP中最高的。使用Kohinooor，我们通过PALM方法成功地观察了纳米桩蛋白在粘着斑中的动力学。在可逆饱和光学荧光跃迁纳米显微镜中，Kohinoor可以使用比以前更简单的光学设置和更低的激光功率获得波形蛋白的超分辨图像。我们现在已经开始使用Kohinoor开发基于RSFP的Ca+2指示剂，用于功能性Ca+2成像。

Functional imaging in a living system at nanoscopic level is difficult due to unavailability of suitable fluorescent protein (FP)-based probe. Reversibly photoswitchable fluorescent proteins (RSFPs)-based probe is the best option. However due to the poor performance of conventional RSFPs, we tried to develop an RSFP that has fast photoswitching, high photostability and superior brightness. After site-directed mutagenesis and several random mutagenesis cycles, finally, we developed an RSFP, Kohinoor that contains seven mutations in its parental RSFP Padron. Kohinoor has the fastest switching speed and the highest photostability, which produce ~25 times more switching on-off cycles than its parental Padron RSFP. The quantum yield of Kohinoor was 0.71, which is highest among all RSFPs. Using kohinooor, we successfully observed nanoscale paxillin dynamics in focal adhesion by PALM method. In reversible saturable optical fluorescence transition nanoscopy, Kohinoor could obtain super-resolved images of vimentin using a simpler optical setup and much lower laser power than has previously been possible. We have now started developing RSFP based Ca+2 indicator using Kohinoor for functional Ca+2 imaging.