Super-resolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Super-resolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photo-convertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and re-activated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

超分辨率显微镜以高精度确定荧光蛋白的定位，超出了光的衍射极限。超分辨率显微技术包括光激活定位显微镜 (PALM)，它可以通过随机激活其荧光来定位单个蛋白质。在通过 PALM 确定单分子定位时，每张图像可以分析的分子数量是有限的。因此，需要许多图像来重建细胞中众多分子的定位。然而，大多数荧光蛋白在固定后会失去荧光。在这里，我们结合了两种 Eos 蛋白衍生物 Skylan-S 和 mEos4b 的氨基酸取代，固定抗性Skylan -S ( frSkylan -S)，一种绿色 RSFP。frSkylan-S 蛋白在激发光下失活，并通过紫光照射重新激活，并且在醛固定后比 Skylan-S 保留更多的荧光。在 PALM 观察中，frSkylan-S 融合蛋白的质量足够高，正如使用 α-微管蛋白和网格蛋白轻链所检查的那样。此外，frSkylan-S 可以与抗体染色结合用于多色成像。因此，frSkylan-S 是一种绿色荧光蛋白，适用于醛固定条件下的 PALM 成像。