| The advances of super-resolution imaging in recent years expedite the development of life science and serve as an incredible tool for life science communities. Along with it, the eager expectations for suitable probes become more urgent. Reversibly switchable fluorescent proteins (RSFPs) are fluorescent molecules that can reversible switch between fluorescent state and dark state in response to specific light activation, and play vital roles in live-cell super-resolution imaging. Howerver, there are only limited RSFPs with modest property which need to be optimized. Here we performed a series of mutagenesis in the position of mEos 3.1 (His62) and found that some of them yielded high brightness, increased photostability, high on/off contrast ratio. Moreover, they also can be turned off by 488-nm laser light or be reactivated by 405-nm laser, which is similar to Dronpa. We name those novel monomeric green RSFPs as Skylan (Sky lantern) family.In super-resolution optical fluctuation imaging (SOFI), Skylan-S exhibits very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state and hence a 4-fold improvement in the fluctuation ranges of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. With these outstanding photochemical properties of Skylan-S, even better resolution of below 100 nm could be achieved and mature CCPs were resolved as a ring structure within 1.5 s under ultrafast frame rate (333 frames/second) during a 60 s time window (20,000 frames) to reconstruct 40 SOFI images. These results prove that Skylan-S is promising for long-time SOFI imaging with high spatial-temporal resolution.In live-cell patterned activation NL-SIM imaging, remarkably Skylan-NS has higher brightness of the "on" state fluorescence, fluorescence contrast ratio in "on/off" states and photostability, which enables low intensity (-100 W/cm2) live cell SR imaging achieve-60 nm resolution at sub-second acquisition times for tens of time points over broad field-of-view. Also, finer cytoskeleton structures could be observed when labeled with Skylan-NS than those labeled with Dronpa or rsEGFP2. These advantages make it suitable for various live-cell super-resolution imaging applications with enhanced resolution.The application and development of super-resolution imaging enable us to detect the localization of biological molecules with high precision. We are trying to explore more physiological problems by super-resolution imaging with our outstanding novel fluorescent probes. As we know apoptosome refers to the adaptor protein complex that mediates the activation of an initiator caspase at the onset of apoptosis. The assembly of the mammalian apoptosome, which is responsible for the activation of caspase-9, involves Apaf-1 and requires cytochrome c and ATP/dATP binding, but the certain process is unclear in insects. So we used Skylan and other different FPs labeled Lepidoptera apoptosome components respectively in mammalian cells for multicolor 3D-SIM imaging, revealed that cytochrome c is released from mitochondria and binds to the cytosolic protein Apaf-1 to facilitate the formation of apoptosome. The wheel-shape 3D structure of the Lepidoptera apoptosome was first revealed by 3D-SIM imaging. More importantly, it provides the dynamics information and resolution that has not been able to be observed by EM and fluorescent microscopy, respectively. This serves as a showcase for Skylans’ broad applications in studying biological questions. |
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