Fluorescence Microscope for Time-Lapse Imaging of Bacteria

Rudner, David

Abstract

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.