Graduate Group in Biophysics, University of California, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):E135-43. doi: 10.1073/pnas.1107547108. Epub 2011 Dec 12.
Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.
我们利用非常适合研究生物样本的超低光强，通过非线性结构光照明显微镜实现了全细胞超分辨率成像。结构光照明显微镜可以将宽场显微镜的空间分辨率提高两倍，如果样品的发射率对光强呈非线性响应，则可以进一步提高分辨率。荧光团激发态的饱和就是这样一种非线性响应，而饱和结构光照明显微镜的实现已经在染料填充的聚苯乙烯珠上达到了约 50nm 的分辨率。不幸的是，由于饱和需要极高的光强，这些光强很可能会加速甚至固定组织的光漂白和损伤，因此这种方法在研究生物样本方面的应用有限。在这里，荧光蛋白的可逆光开关提供了所需的非线性，其光强比饱和所需的光强低六个数量级。我们通过用荧光可光开关蛋白 Dronpa 标记的纯化微管进行实验证明了约 40nm 的分辨率，并通过成像哺乳动物核孔和肌动蛋白细胞骨架来可视化细胞结构。因此，非线性结构光照明显微镜现在是一种具有生物相容性的超分辨率成像方法。
