The Application of 3D Cryo-Volume Electron Microscopy to Visualize and Quantify the Photosynthetic Filamentous Cyanobacterium Anabaena

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Abstract
Cryogenic volume electron microscopy (cryo-vEM) is a rapidly developing nanoscale imaging approach that enables three-dimensional datasets of vitrified cells and tissues at intermediate resolutions without treatment with chemical fixatives or metal contrasting agents. Our goal was to evaluate the suitability of cryo-vEM for whole cells of photosynthetic organisms such as cyanobacteria. Two species of Anabaena , a filamentous cyanobacterium, were captured on poly-L-lysine-coated grids, vitrified by plunge-freezing into liquid ethane, and mapped by secondary electron and integrated fluorescence light microscopy to identify target cells. Cells of interest were coated with platinum, trenched with xenon, and 3D volumes collected by alternating argon milling and through-lens detector (TLD) imaging on a Thermo Scientific Hydra plasma focused ion beam scanning electron microscope. Volumes were segmented via 2.5D U-Net models, then quantified in Dragonfly 3D World. Block-face images revealed the cell wall, thylakoids, phycobilisomes, carboxysomes, cyanophycin and polyphosphate granules. Whole-cell 3D rendering allowed quantification of carboxysomes, cyanophycin granules, and measurement of thylakoid thickness and spacing in A. inaequalis and Anabaena sp. UTEX 2576. Thus, cryo-vEM provided interpretable whole-cell ultrastructure of cyanobacteria without chemical pre-treatment, and its compatibility with cryo-fluorescence and in situ cryo-ET enables contextual multi-scale perspectives from whole cells to molecular structures.

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