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Expansion Microscopy for Cell Biology Analysis in Fungi.
Frontiers in Microbiology ( IF 5.2 ) Pub Date : 2020-04-03 , DOI: 10.3389/fmicb.2020.00574
Ralph Götz 1 , Sabine Panzer 1 , Nora Trinks 1 , Janna Eilts 1 , Johannes Wagener 2 , David Turrà 3 , Antonio Di Pietro 3 , Markus Sauer 1 , Ulrich Terpitz 1
Affiliation  

Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

中文翻译:

用于真菌细胞生物学分析的扩展显微镜。

超分辨率显微镜已经发展成为一种对细胞和细胞器进行亚衍射分辨率荧光成像的有力方法,但是需要复杂且昂贵的安装。基于感兴趣的细胞结构的物理扩展的扩展显微镜(ExM)提供了一种廉价的替代方法,可绕过衍射极限并实现常规荧光显微镜上的超分辨率成像。尽管ExM在放大显示细胞和组织中的蛋白质和RNA方面显示出令人印象深刻的结果,但由于其复杂的细胞壁,它尚未应用于真菌。在这里,我们开发了一种方法,使细胞壁降解酶处理后,能够可靠地各向同性地扩展子囊菌和担子菌。共聚焦激光扫描显微镜(CLSM)和结构化照明显微镜(SIM)图像,表达表达组蛋白H1-mCherry以及Lifeact-sGFP或mRFP靶向的组蛋白H1-mCherry的尖孢镰刀菌或烟曲霉的荧光真菌视紫红质和烟丝菌的UStilago的4.5倍扩增的孢子虫对线粒体的研究揭示了亚细胞结构的细节,估计其空间分辨率约为30 nm。因此,ExM非常适合常规荧光显微镜下真菌中的细胞生物学研究。揭示了亚细胞结构的细节,估计的空间分辨率约为30 nm。因此,ExM非常适合常规荧光显微镜下真菌中的细胞生物学研究。揭示了亚细胞结构的细节,估计的空间分辨率约为30 nm。因此,ExM非常适合常规荧光显微镜下真菌中的细胞生物学研究。
更新日期:2020-04-03
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