Imaging Bacterial Colonization of the Zebrafish Gut with Selective Plane Illumination

Abstract

Microbes are a major component of living animals, outnumbering the human cells in humans, for example, by an order of magnitude. The mechanisms by which resident microbial communities assemble, however, are very poorly understood, in large part due to the difficulty of imaging microbes inside animals and to the scarcity of tractable model systems. Selective plane illumination microscopy (SPIM), also known as light sheet fluorescence microscopy, is a recently developed imaging technique that is able to acquire high-speed three-dimensional images over long periods of time with high resolution, a wide field of view, and low levels of phototoxicity. Zebrafish (Danio rerio) are well-studied model vertebrates that are transparent during larval life when their intestines are first colonized by bacteria. We can rear germ-free zebrafish that can then be inoculated with particular bacterial species expressing fluorescent proteins. Using a home-built SPIM microscope, we have begun to investigate the dynamics of bacterial colonization of the larval zebrafish intestine. We provide here the first demonstration that SPIM has the resolution and speed necessary to image individual bacteria as well as larger colonies over periods of several hours. Imaging colonies allows quantification of changes in the spatial distribution of bacterial populations, while imaging individuals makes it possible to explore the motility of these organisms in more natural settings than conventional culture-based assays allow.