Abstract
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.
Highlights
Fungi play important roles in human nutrition and well-being
We successfully degraded the cell wall in F. oxysporum and A. fumigatus germlings and U. maydis sporidia using a mixture of glucanex, driselase and chitinase
We noted that the time point of cell wall digestion within the protocol workflow significantly affected the morphology obtained after the expansion process
Summary
Fungi play important roles in human nutrition and well-being. These tiny organisms serve as biofactories in biotechnology and food industry, are essential for the biodegradation of complex organic compounds, and act as highly destructive pathogens of plants, animals, and humans (Kendrick, 2011; Lange, 2014; Meyer et al, 2016; Cerimi et al, 2019). Microscopy allows to gain new insights at high spatial and temporal resolution into essential cellular processes such as protein localization, physiological activity, and growth dynamics (Hickey et al, 2004; Knaus et al, 2013; Chapuis et al, 2019). Fluorescence microscopy of fungi is limited by the small size of their organelles, which is below the diffraction-limited resolution provided by conventional fluorescence microscopes. Fungi tend to exhibit strong autofluorescence (Knaus et al, 2013), further complicating high-end fluorescence imaging
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