Non-Invasive Single-Cell Morphometry and Tracking in Living Bacterial Biofilms

项目摘要/摘要 生物膜是具有凝聚力的多细胞微生物群落，能够附着在生物或非生物表面。 人类微生物群包含许多生物膜形成细菌种类，有助于维持正常的人类 生理学。另一方面，在美国170万医院获得性感染中，超过一半是由 通过形成生物膜的细菌病原体。生物膜的生活方式是有利的，因为表型多样性和 生物膜内细胞行为的协调为细菌种群提供了超越 单个细胞的基因。例如，生物膜对物理、化学、 和生物应激源，最显著的是使用抗生素药物的长期治疗或由 免疫系统。然而，人们在很大程度上仍然不清楚，这种非凡的能力是如何从 单个细胞的行为以及它们之间的相互作用。快速发展的一个关键障碍是无法 传统显微镜在厚(10微米)生物膜中分辨微米大小的细菌细胞 非侵入性的方式。拟议的研究通过以下方式解决这一挑战：(I)开发综合实验 和计算技术，使致病生物膜的非侵入性、3D荧光成像成为可能 通过晶格光片显微镜观察生理相关环境，(Ii)准确的单细胞分割和 基于获取的图像的3D形状测量，以及(Iii)同时对数千个细胞进行3D跟踪 在生物膜内部。在密集的微生物种群中进行单细胞测量的能力将使 研究人员将每个细胞的空间轨迹与该细胞的基因表达和行为相关联 表型。这些信息将提供对细菌如何协调基因的完整理解 在三维空间和时间中的表达和社会行为。对生物膜生物学有一个基本的了解将会有所帮助 为人类利用微生物种群的新功能提供新的战略 有益于去除有害环境中的致病生物膜。

Project Summary/Abstract Biofilms are cohesive, multicellular microbial communities that are able to adhere to biotic or abiotic surfaces. The human microbiome contains numerous biofilm-forming bacterial species that help maintain normal human physiology. On the other hand, more than half of the 1.7 million hospital-acquired infections in the US are caused by biofilm-forming bacterial pathogens. The biofilm lifestyle is advantageous, because phenotypic diversity and coordination of cellular behaviors within biofilms provide bacterial populations with emergent capabilities beyond those of individual cells. For example, biofilms are orders of magnitude more tolerant towards physical, chemical, and biological stressors, most notably long-term treatments with antibiotic drugs or clearance attempts by the immune system. However, it remains largely unknown how such remarkable capabilities emerge from the behaviors of individual cells and the interactions between them. A critical barrier to rapid progress is the inability of conventional microscopes to resolve micrometer-sized bacterial cells in thick (>10 micrometers) biofilms in a non-invasive manner. The proposed research addresses this challenge by (i) developing integrated experimental and computational technologies that enable non-invasive, 3D fluorescence imaging of pathogenic biofilms in physiologically relevant environments by lattice-light sheet microscopy, (ii) accurate single-cell segmentation and 3D shape measurements based on the acquired images, and (iii) simultaneous 3D tracking of thousands of cells inside biofilms. The ability to make single-cell measurements in dense microbial populations will enable researchers to correlate the spatial trajectory of each cell with that cells’ gene expression and behavioral phenotype. Such information will provide an integrated understanding of how bacteria coordinate gene expression and social behaviors in 3D space and time. A fundamental understanding of biofilm biology will help inform new strategies for harnessing the emergent functional capabilities of microbial populations for human benefit and for removing pathogenic biofilms from undesired environments.