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。拟议的研究通过开发综合实验来解决这一挑战 和计算技术，通过以下方式实现病原生物膜的非侵入性3D荧光成像 晶格光片显微镜、精确的单细胞分割和基于 获取的图像，以及对生物膜内数千个细胞的同时3D跟踪。有能力让单人- 密集微生物种群中的细胞测量将使研究人员能够关联 每个细胞都有该细胞的基因表达和行为表型。这些信息将提供一个综合的 了解细菌如何在三维空间和时间中协调基因表达和社会行为。一个 对生物膜生物学的基本了解将有助于制定新的战略，以利用新出现的 微生物种群的功能能力和去除有害生物膜的能力 环境。

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 developing integrated experimental and computational technologies that enable non-invasive, 3D fluorescence imaging of pathogenic biofilms by lattice-light sheet microscopy, accurate single-cell segmentation and 3D shape measurements based on the acquired images, and 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 and for removing pathogenic biofilms from undesired environments.