Background: Mitochondria are highly dynamic organelles that constantly undergo processes of fission and fusion. The changes in mitochondrial dynamics shape the organellar morphology and influence cellular activity regulation. Soft X-ray tomography (SXT) allows for three-dimensional imaging of cellular structures while they remain in their natural, hydrated state, which omits the need for cell fixation and sectioning. Synchrotron facilities globally primarily use flat grids as sample carriers for SXT analysis, focusing on adherent cells. To investigate mitochondrial morphology and structure in hydrated yeast cells using SXT, it is necessary to establish a method that employs the flat grid system for examining cells in suspension.

Results: We developed a procedure to adhere suspended yeast cells to a flat grid for SXT analysis. Using this protocol, we obtained images of wild-type yeast cells, strains with mitochondrial dynamics defects, and mutant cells possessing distinctive mitochondria. The SXT images align well with the results from fluorescent microscopy. Optimized organellar visualization was achieved by constructing three-dimensional models of entire yeast cells.

Conclusions: In this study, we characterized the mitochondrial network in yeast cells using SXT. The optimized sample preparation procedure was effective for suspended cells like yeast, utilizing a flat grid system to analyze mitochondrial structure through SXT. The findings corresponded with the mitochondrial morphology observed under fluorescence microscopy, both in regular and disrupted dynamic equilibrium. With the acquired image of unique mitochondria in Δhap2 cells, our results revealed that intricate details of organelles, such as mitochondria and vacuoles in yeast cells, can be characterized using SXT. Therefore, this optimized system supports the expanded application of SXT for studying organellar structure and morphology in suspended cells.

Author: Chang CR

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