Fluorescence Microscope for Time-Lapse Imaging of Bacteria

描述(由申请人提供)：这项共享仪器的提案是为一个倒置的荧光显微镜配备的，并致力于细菌的延时成像。使用者是一群微生物学家，他们的研究集中在细菌生理学的基础和突出问题上。在过去的十年里，使用荧光显微镜对细菌进行细胞学分析已经成熟，并改变了细菌学领域。我们能够可视化亚细胞结构、细胞骨架元素，甚至染色体的定义区域，这从根本上改变了我们对细菌细胞的看法以及我们对细菌细胞提出的问题的类型和深度。蛋白质的亚细胞定位不仅为我们思考这些分子的功能提供了信息，也为探索细胞分裂、染色体分离和形态发生等基本细胞生物学过程的机制提供了分析方法。此外，对含有启动子和荧光蛋白融合的细胞的分析揭示了种群和群落中单个细菌细胞的惊人变异和专门化。许多使用荧光显微镜的革命性发现都来自于获取单幅图像(称为“快照”显微镜)。越来越清楚的是，这些快照只是讲述了故事的一部分，通过时间推移成像实时研究这些过程还有很多有待发现。分析活细胞内蛋白质的动态行为；正在进行的形态发生过程；以及在复杂群落中生长的细胞是细菌细胞生物学的下一个前沿。拟议的工具将帮助我们探索这一前沿，并刺激下一波突破。我们实验室、系里和校园里现有的大部分显微镜只能用来对细菌细胞进行基本的延时成像。我们机构专门为延时显微镜设计的少数系统严重超额认购，缺乏对细菌大小的细胞进行成像所需的关键功能。拟议中的专门用于延时成像的荧光显微镜将加快当前的研究项目，使发现成为可能，并启动新的项目。拟议中的仪器将被安置在哈佛医学院微生物学和分子遗传学系中心的一个开放式房间里。因此，它还将成为我们整个微生物学家社区的资源和培训场所。

DESCRIPTION (provided by applicant): This shared instrument proposal is for an inverted epifluorescence microscope outfitted for, and dedicated to, time-lapse imaging of bacteria. The users are a group of microbiologists whose research is focused on fundamental and outstanding problems in bacterial physiology. Cytological analysis of bacteria using fluorescence microscopy has come of age over the last decade and has transformed the field of bacteriology. Our ability to visualize subcellular structures, cytoskeletal elements, and even defined regions of the chromosome has fundamentally changed our perception of the bacterial cell and the types and depth of questions we ask about it. Subcellular localization of proteins not only informs our thinking about how these molecules function it also provides assays to probe the mechanisms underlying basic cell biological processes like cell division, chromosome segregation and morphogenesis. In addition, the analysis of cells harboring promoter fusions to fluorescent proteins has revealed surprising variation and specialization of individual bacterial cells within populations and communities. Many of the revolutionary discoveries using fluorescence microscopy have come from the acquisition of single images (called "snap-shot" microscopy). It is becoming increasingly clear that these snap shots are only telling part of the story and that much remains to be discovered by studying these processes in real time by time- lapse imaging. Analysis of the dynamic behavior of proteins inside living cells; of ongoing morphogenetic processes; and of cells growing within complex communities is the next frontier in bacterial cell biology. The proposed instrument will help us explore this frontier and stimulate the next wave of breakthroughs. Most of the existing microscopes in our laboratories, our department, and across our campus can only be used for rudimentary time-lapse imaging of bacterial cells. The few systems at our institution specifically designed for time-lapse microscopy are significantly over-subscribed and lack the critical features necessary for imaging cells the size of bacteria. The proposed fluorescent microscope dedicated to time-lapse imaging would accelerate current research projects, enable discovery, and launch new projects. The proposed instrument will be housed in an open-access room in the heart of the department of Microbiology and Molecular Genetics at Harvard Medical School. Accordingly, it will also serve as a resource and training site for our entire community of microbiologists.