Improved Visualization of Oral Microbial Consortia.

S T Ramirez-Puebla, J L Mark Welch, G G Borisy
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Abstract

Bacteria on the tongue dorsum (TD) form consortia tens to hundreds of microns in diameter organized around a core of epithelial cells. Whole-mount preparations have been instrumental in revealing their organization and specific microbial associations. However, their thickness and intricate 3-dimensional complexity present challenges for a comprehensive spatial analysis. To overcome these challenges, we employed a complementary approach: embedding in hydrophilic plastic followed by sectioning and postsectioning labeling. Samples were labeled by hybridization with multiplexed fluorescent oligonucleotide probes and visualized by spectral imaging and linear unmixing. Application of this strategy to TD biofilms improved the visualization of bacteria that were difficult to resolve in whole-mount imaging. Actinomyces, previously detected as patches, became resolved at the single-cell level. The filamentous taxa Leptotrichia and Lachnospiraceae, located at the core of the consortium, were regularly visualized whereas previously they were rarely detected when using whole mounts. Streptococcus salivarius, heterogeneously detected in whole mounts, were regularly and homogenously observed. Two-dimensional images provide valuable information about the organization of bacterial biofilms. However, they offer only a single plane of view for objects that can extend to hundreds of microns in thickness, and information obtained from such images may not always reflect the complexity of a 3-dimensional object. We combined serial physical sectioning with optical sectioning to facilitate the 3-dimensional reconstruction of consortia, spanning over 100 µm in thickness. Our work showcases the use of hydrophilic plastic embedding and sectioning for examining the structure of TD biofilms through spectral imaging fluorescence in situ hybridization. The result was improved visualization of important members of the human oral microbiome. This technique serves as a complementary method to the previously employed whole-mount analysis, offering its own set of advantages and limitations. Addressing the spatial complexity of bacterial consortia demands a multifaceted approach for a comprehensive and effective analysis.

改进口腔微生物群的可视化。
舌背(TD)上的细菌围绕上皮细胞核心形成直径数十到数百微米的联合体。整片制备有助于揭示它们的组织和特定的微生物关联。然而,它们的厚度和错综复杂的三维复杂性给全面的空间分析带来了挑战。为了克服这些挑战,我们采用了一种补充方法:将样本包埋在亲水性塑料中,然后进行切片和切片后标记。样品通过与多重荧光寡核苷酸探针杂交进行标记,并通过光谱成像和线性非混合成像进行可视化。将这一策略应用于 TD 生物膜,可改善整片成像中难以分辨的细菌的可视化。以前检测到的片状放线菌,现在可以在单细胞水平上分辨出来。位于菌群核心的丝状类群 Leptotrichia 和 Lachnospiraceae 也能经常被观察到,而以前在使用全装片时很少能检测到它们。唾液链球菌(Streptococcus salivarius)在整片装片中检测不均一,而在二维图像中则可以定期观察到。二维图像提供了有关细菌生物膜组织的宝贵信息。然而,对于厚度可达数百微米的物体,二维图像只能提供单一视角,而且从二维图像中获得的信息并不总能反映三维物体的复杂性。我们将序列物理切片与光学切片相结合,促进了厚度超过 100 微米的联合体的三维重建。我们的工作展示了如何利用亲水性塑料包埋和切片技术,通过光谱成像荧光原位杂交技术检查 TD 生物膜的结构。结果改善了人类口腔微生物群重要成员的可视化。这项技术是对之前采用的整片分析方法的补充,有其自身的优势和局限性。要解决细菌群的空间复杂性问题,需要采用多方面的方法进行全面有效的分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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