Vangindertael Jeroen, Beets Isabel, Rocha Susana, Dedecker Peter, Schoofs Liliane, Vanhoorelbeke Karen, Hofkens Johan, Mizuno Hideaki
Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.
Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak. E. Sabbelaan 53, 8500 Kortrijk, Belgium.
Sci Rep. 2015 Sep 1;5:13532. doi: 10.1038/srep13532.
Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals.
光激活定位显微镜（PALM）是一种基于对单个荧光分子进行检测及后续定位的超分辨率成像技术。因此，PALM是在最终分析检测极限下解析生物分子结构和假定相互作用的强大工具。然而，其有限的成像深度使得PALM大多局限于体外应用。考虑到对多细胞生物体成像时对解剖学背景的额外需求，这些限制使得在完整动物中使用PALM变得困难。在此，我们将PALM与共聚焦显微镜相结合，用于秀丽隐杆线虫神经系统的相关成像，我们将这项技术称为共聚焦相关PALM（ccPALM）。位于几层组织下方的神经元在动物体内可达10μm深处仍可被可视化。通过ccPALM，我们以20nm的精度可视化了秀丽隐杆线虫中离子型谷氨酸受体的分布，揭示了我们绘制到动物体内注释神经元上的受体簇的超分辨率结构。我们研究结果的关键在于通过对标记受体表达和定位进行基因调控以有效降低背景荧光，实现了不依赖全内反射荧光（TIRF）的单分子检测。通过将PALM与共聚焦显微镜相关联，该平台能够在完整动物的生理相关背景下以单分子分辨率剖析生物结构。
