High-resolution 3D spatial distribution of complex microbial colonies revealed by mass spectrometry imaging,Journal of Advanced Research

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High-resolution 3D spatial distribution of complex microbial colonies revealed by mass spectrometry imaging
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2024-08-29 , DOI: 10.1016/j.jare.2024.08.031
Yuting Shen ₁ , Yisu Wang ₂ , Jianing Wang ₁ , Peisi Xie ₁ , Chengyi Xie ₁ , Yanyan Chen ₁ , Niaz Banaei ₃ , Kangning Ren ₄ , Zongwei Cai ₁

Affiliation

Introduction

Bacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples.

Objectives

The study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies.

Methods

This article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization.

Results

Combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains.

Conclusion

These results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.

中文翻译：

质谱成像揭示复杂微生物菌落的高分辨率 3D 空间分布

介绍

使用质谱成像（MSI）广泛研究了细菌的生存状态和微生物菌落信号分子的分布。然而，目前的方法通常将 3D 菌落视为平面 2D 圆盘，无意中遗漏了有价值的细节。由于微生物样品的独特特性，在生物膜中实现 3D MSI 的挑战仍然存在。

目标

该研究旨在开发一种新的生物膜样品制备方法，该方法可以实现细菌菌落的高分辨率 3D MSI，以揭示细菌菌落的空间组织。

方法

本文介绍了水分辅助冷冻切片（MACS）方法，可实现平行于生长平面的无包埋切片。MACS 方法通过控制环境湿度和切片厚度来防止分子离域，从而确保切片完整。

结果

结合基质辅助激光解吸电离质谱（MALDI）-MSI，MACS 方法为铜绿假单胞菌（P.铜绿假单胞菌）生物膜，包括异构体对。此外，分析的菌落被重新转化为 3D 模型，生动地描绘了从内层到外层的分子分布。此外，我们研究了多个菌落中的代谢物时空动力学，观察随时间的变化以及单个菌落与合并菌落的不同模式。这些发现揭示了多菌落形成的排斥-合并过程。此外，我们的研究监测了抗生素处理后生物膜内的化学反应，显示随着时间的推移，生物膜外层的抗生素水平增加，而内部区域的抗生素水平保持在较低水平。此外，MACS 方法证明了其对其他细菌菌株的普遍性和适用性。