Superresolution imaging of Drosophila tissues using expansion microscopy.
Superresolution imaging of Drosophila tissues using expansion microscopy.
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DOI：
10.1091/mbc.e17-10-0583
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发表时间：
2018-06-15
影响因子：
3.3
通讯作者：
Parrish JZ
中科院分区：
文献类型：
--
作者：
Jiang N;Kim HJ;Chozinski TJ;Azpurua JE;Eaton BA;Vaughan JC;Parrish JZ
传统衍射限制显微镜的有限分辨能力阻碍了对细胞中小的、密集排列的结构元素的分析。膨胀显微镜（ExM）为这个问题提供了一个优雅的解决方案，通过在可膨胀的聚合物水凝胶中对样品进行物理膨胀，可以提高标准显微镜的分辨率。在这里，我们应用、验证和优化ExM方案，使果蝇胚胎、幼虫大脑、幼虫和成虫体壁的研究成为可能。我们使用标准共聚焦显微镜在果蝇组织中实现了~ 70 nm的横向分辨率，我们使用ExM来分析精细的细胞内结构和细胞间相互作用。首先，我们发现ExM揭示了突触前活跃区（AZ）结构的特征，这些特征是其他超分辨率成像技术可以观察到的，而不是标准共聚焦显微镜。我们进一步表明，已知突触表现出年龄依赖性的活动变化，也表现出AZ结构的年龄依赖性变化。最后，我们使用显著提高的ExM轴向分辨率来显示，体感觉神经元的树突插入上皮细胞的频率比以前共聚焦显微镜研究中报道的要高。总之，我们的研究为ExM在果蝇组织中的应用提供了基础，并强调了ExM程序的组织特异性优化的重要性。
The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.