School of Biomedical Engineering, University of British Columbia; Department of Microbiology and Immunology, University of British Columbia.
School of Biomedical Engineering, University of British Columbia; Department of Microbiology and Immunology, University of British Columbia; Humans and the Microbiome Program, Canadian Institute for Advanced Research (CIFAR);
J Vis Exp. 2021 Jul 9(173). doi: 10.3791/62646.
Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health. This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.
在体内环境中测量微生物的定位是揭示微生物群与脊椎动物肠道之间功能关系的关键步骤。肠道微生物群的空间格局受物理特征（肠道黏液、隐窝和褶皱）的严格控制，并受宿主控制的特性（如 pH 值、氧气供应和免疫因素）的影响。这些特性限制了共生微生物和病原体在肠道中稳定定植的能力。在微米尺度上，微生物组织决定了不同微生物之间的近距离相互作用以及微生物与其宿主之间的相互作用。这些相互作用进而影响器官的整体功能和宿主的健康。本方案能够从细胞间距离到器官范围可视化肠道微生物群的空间组织。该方法基于在保留肠道结构和黏液特性的同时固定肠道组织。然后将固定的样本包埋、切片并染色，通过荧光原位杂交（FISH）突出显示特定的细菌种类。宿主特征，如黏液和宿主细胞成分，通过荧光标记的凝集素进行标记。最后，使用共聚焦显微镜对染色的切片进行成像，利用高倍放大的平铺扫描成像来弥合微米到厘米长度尺度之间的差距。这种成像可以应用于来自动物模型的肠道切片和来自人类组织的活检，以确定健康和疾病状态下肠道微生物群的生物地理学。
