Microbial Ecology in States of Health and Disease Workshop Summary (2014) / Chapter Skim
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A12 Investigating bacterial-animal symbioses with light sheet microscopy--Michael J. Taormina, Matthew Jemielita, W. Zac Stephens, Adam R. Burns, Joshua V. Troll, Raghuveer Parthasarathy, and Karen Guillemin
A12 Investigating bacterial-animal symbioses with light sheet microscopy--Michael J. Taormina, Matthew Jemielita, W. Zac Stephens, Adam R. Burns, Joshua V. Troll, Raghuveer Parthasarathy, and Karen Guillemin
Pages 323-346
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From page 323... ...
Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and ynamics d of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high-resolution imaging of bacterial colonization of the intestine of Danio rerio, the zebrafish.
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From page 324... ...
Here we describe the application of light sheet microscopy to the field of symbiosis. In particular, we describe the utility of this imaging technique for exploring unanswered questions about the microbial colonization of the vertebrate intestine by using the model organism Danio rerio, the zebrafish.
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One instructive example of insights into host colonization by bacteria comes from live imaging of the partnership between the bobtail squid Euprymna scolopes and the luminescent marine bacterium Vibrio fischerii, which colonizes the light organ of its host and provides light to erase the squid's shadow when the squid is foraging at night in shallow seawater (Nyholm and McFall-Ngai, 2004)
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Zebrafish, like all vertebrates, are colonized by complex microbial communities, with the most numerically abundant communities being found in their digestive tracts. As in humans, the gut microbiota of zebrafish are dominated by a small number of bacterial phyla, with a high diversity of species and strains within these phyla (Rawls et al., 2006)
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Four-Dimensional Microscopy and Light Sheet Imaging Three-dimensional fluorescence imaging of live specimens over time, often denoted four-dimensional microscopy, provides molecule-specific, nondestructive information about the spatial structure and temporal dynamics of biological systems. Four-dimensional imaging of host and microbial cells in a developing intestine presents particular technical challenges, as it simultaneously demands fine spatial resolution to visualize individual cells; fine temporal resolution to track individual motions and cellular shape changes; large fields of view spanning, for example, a larval zebrafish intestine that is several hundred microns long; and low levels of photodamage to enable nondestructive imaging over hours or days.
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Optical sectioning in light sheet microscopy works by creating a plane of excitation light that is coincident with the focal plane of an imaging objective. Since the sectioning occurs during excitation, the emitted light from the whole field of view can be gathered with a standard camera as a two-dimensional image.
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For the purposes of this discussion, we use the term light sheet microscopy to refer to this technology collectively. Materials and Methods Light Sheet Microscope Design Light sheet microscopes are to date commercially unavailable, necessitating custom constructions.
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Abbre viations: AOTF, acousto-optic tunable filter; MG, mirror galvanometer; SL, scan lens; TL, tube lens; EO, excitation objective lens; DO, detection objective lens.
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From page 331... ...
Because of its wide-field method of image collection, the limiting factor for imaging speed in light sheet microscopy is the readout of large-resolution sensors; in order to maximize image acquisition speed, we employ a recently developed CMOS camera that has high readout speeds, resolution, sensitivity, and dynamic range. For some data sets in this paper (Figure A12-5H–M)
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From page 332... ...
The studies illustrated below exemplify the importance of imaging speed -- for example, to discern the motility of individual bacteria -- and large fields of view and low levels of photodamage -- for example, to image an entire organ for long times. Zebrafish Husbandry and Bacterial Colonization All experiments with zebrafish were performed using protocols approved by the University of Oregon Institutional Animal Care and Use Committee and following standard protocols (Westerfield, 2000)
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veronii strains within the zebrafish intestine (Figure A12-4) , ruling out the first model of colonization by a single cell.
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at various times after inoculation, with t = 6 h corresponding to panel B The contrast of each panel was independently adjusted so that the bacterial populations are clearly visible despite fluctuations in overall bacterial abundance.
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It is clear that the simple model of a chemostat with evenly distributed bacterial cells does not accurately describe the real behavior of bacterial populations in the zebrafish intestine. The high speed of light sheet fluorescence imaging also enables observations of the motility of individual bacterial cells.
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. The high speed of light sheet microscopy provides an opportunity to explore host–microbe interactions at much higher resolution and to ask how the microbiota affect the dynamics of individual zebrafish cells, and reciprocally, how host cells affect the microbiota.
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From page 337... ...
. Visualizing their dynamics in i nternal tissues such as the intestine, however, poses technical challenges that we were able to overcome using light sheet microscopy.
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. Simultaneously imaging fluorescently labeled bacteria and neutrophils with light sheet microscopy will enable visualization of neutrophil recruitment to the intestine upon bacterial colonization and will address whether they exhibit different behaviors, such as increased filopodia activity, in the presence of commensal microbes.
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To illustrate this, we first consider the schematic, computer-generated i mage in Figure A12-7A, in which green spots are distributed randomly in two
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(A) A simulated two-dimensional image with randomly positioned green spots and 1.5× larger red spots each placed about 20 pixels from a green spot, randomly oriented.
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Particular coordinate systems can help characterize particular spatial distributions of bacterial populations. For example, by determining the gut center line ( ither manually or by computational analysis of the bounded space)
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From page 342... ...
, but the application of these ideas to naturally occurring H icrobial communities has been hindered by the technical challenges of observm ing population dynamics on the spatial and temporal scales of microbes. Here we present the methodology of light sheet microscopy and its application to visualizing the dynamics of zebrafish intestinal microbiota and associated host cells.
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From page 343... ...
Light sheet imaging could be employed to image microbial colonization of the internal organs of transparent animals, including many marine organisms and insect larvae, and to record bacterial associations of surface tissues of any animal or plant. We speculate that it will be especially powerful when applied to model symbiosis systems of bacterial associations with the squid light organ, the leech crop, the fruit fly and nematode digestive tracts, and fungal and bacterial inter actions with plant roots and rodent skin.
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2011. Epithelial cell pro liferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88.
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2008. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.
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2011. Deep and fast live imaging with two-photon scanned lightsheet microscopy.
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