Abstract 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.

Wei-Ling Huang

Chang-Lin Chen

Zi-Jing Lin

Chia-Chun Hsieh

Mo Da-Sang Hua

Chih-Chan Cheng

Tzu-Hao Cheng

Lee-Jene Lai

Chuang-Rung Chang

Biology Direct

BIOLOGY DIRECT 作为开放获取、同行评审的在线期刊服务于生命科学研究社区，为作者和读者提供了传统同行评审模式的替代方案。 BIOLOGY DIRECT 考虑目前被认为最有利于开放审查方法的学科领域的原创研究文章、假设、评论、发现笔记和评论，主要是那些具有重要非实验成分的领域。

Biology Direct is an open-access, peer-reviewed online journal that serves the life sciences research community. It provides authors and readers with an alternative to the traditional peer review model. Biology Direct considers original research articles, hypotheses, reviews, discovery notes, and commentaries, particularly in fields that are currently viewed as most conducive to open review methods.

《Biology Direct》是一份开放获取、同行评审的在线期刊，旨在服务于生命科学研究社区。它为作者和读者提供了一种传统同行评审模式的替代方案。《Biology Direct》接受原创研究文章、假设、评论、发现笔记和评论，特别关注那些具有重要非实验成分的学科领域。

Category	Quartile
生物学	2
生物学, 生物学	2

Soft X-ray tomography analysis of mitochondria dynamics in Saccharomyces cerevisiae

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