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

由于没有合适的基于荧光蛋白(FP)的探针，在生命系统的纳米水平上进行功能成像是困难的。基于可逆光开关荧光蛋白(RSFP)的探针是最好的选择。然而，由于传统RSFP的性能较差，我们试图开发一种具有快速光开关、高光稳定性和优异亮度的RSFP。经过定点突变和多次随机突变循环，我们最终获得了一株RSFP，Kohinoor，它在其亲本RSFP Padron中包含7个突变。Kohinoor具有最快的开关速度和最高的光稳定性，其开关开关周期是其亲本Padron RSFP的约25倍。Kohinoor的量子产率为0.71，是所有RSFP中最高的。使用Kohinooor，我们用Palm方法成功地观察到了纳米尺度的帕西林在焦点粘连中的动力学。在可逆饱和光学荧光过渡纳米显微镜中，Kohinoor可以使用更简单的光学设置和比以前可能的更低的激光功率获得波形蛋白的超分辨率图像。我们现在已经开始开发基于RSFP的钙指示剂，使用Kohinoor进行功能钙成像。

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.