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.

中文翻译：

---

改进的口腔微生物联盟的可视化。


舌背细菌 （TD） 形成直径数十至数百微米的联盟，围绕上皮细胞的核心组织。整装制剂有助于揭示它们的组织和特异性微生物关联。然而，它们的厚度和错综复杂的 3 维复杂性为全面的空间分析带来了挑战。为了克服这些挑战，我们采用了一种互补方法：包埋在亲水性塑料中，然后进行切片和切片后标记。通过与多重荧光寡核苷酸探针杂交来标记样品，并通过光谱成像和线性解混进行可视化。将这种策略应用于 TD 生物膜改善了在整装成像中难以解决的细菌的可视化。以前检测为斑块的放线菌在单细胞水平上得到分离。位于联盟核心的丝状分类群 Leptotrichia 和 Lachnospiraceae 经常被可视化，而以前在使用整个坐骑时很少检测到它们。唾液链球菌，在整个支架中异质检测到，定期和均匀地观察到。二维图像提供了有关细菌生物膜组织的宝贵信息。但是，它们仅为厚度可延伸到数百微米的物体提供单个视图平面，并且从此类图像中获得的信息可能并不总是反映 3D 物体的复杂性。我们将连续物理切片与光学切片相结合，以促进厚度超过 100 μm 的财团的 3 维重建。 我们的工作展示了使用亲水性塑料包埋和切片通过光谱成像荧光原位杂交来检查 TD 生物膜的结构。结果是改善了人类口腔微生物组重要成员的可视化。该技术作为以前采用的整体安装分析的补充方法，具有自己的优点和局限性。解决细菌群落的空间复杂性需要一种多方面的方法进行全面有效的分析。