A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens

Baker, Ryan; Taormina, Michael J.; Jemielita, Matthew; Parthasarathy, Raghuveer

doi:10.1111/jmi.12220

Cited by 19 publications

(27 citation statements)

References 32 publications

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“…We next asked whether the presence of Vibrio leads to changes in the mechanics of intestinal motility. To test this, we imaged intestinal motility in larval zebrafish using differential interference contrast microscopy (DIC) [27] and calculated the dominant period and amplitude of intestinal contractions. Comparing GF fish with Vibrio or Aeromonas mono-associated fish, or fish in which Aeromonas is challenged after 24 hours by Vibrio , there is no notable difference in period or amplitude (S4 Fig).…”

Section: Resultsmentioning

confidence: 99%

“…Larval intestinal motility was assessed from images captured using differential interference contrast (DIC) microscopy, performed as previously described [27]. The displacement field from frame to frame in time-series was determined using particle image velocimetry (PIV) algorithms [60], which calculate the motions necessary for regions in one frame to be mapped onto regions in another.…”

Section: Methodsmentioning

confidence: 99%

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Host Gut Motility and Bacterial Competition Drive Instability in a Model Intestinal Microbiota

Wiles

Jemielita

Baker

et al. 2016

Preprint

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30The gut microbiota is a complex consortium of microorganisms with the ability to 31 influence important aspects of host health and development. Harnessing this 'microbial 32 organ' for biomedical applications requires clarifying the degree to which host and 33 bacterial factors act alone or in combination to govern the stability of specific lineages. 34To address this we combined bacteriological manipulation and light sheet fluorescence 35 microscopy to monitor the dynamics of a defined two-species microbiota within the 36 vertebrate gut. We observed that the interplay between each population and the gut 37 environment produced distinct spatiotemporal patterns. Consequently, one species 38 dominates while the other experiences dramatic collapses that are well fit by a 39 stochastic mathematical model. Modeling revealed that bacterial competition could only 40 partially explain the observed phenomena, suggesting that a host factor is also 41 important in shaping the community. We hypothesized the host determinant to be gut 42 motility, and tested this mechanism by measuring colonization in hosts with enteric 43 nervous system dysfunction due to mutation in the Hirschsprung disease locus ret. In 44 mutant hosts we found reduced gut motility and, confirming our hypothesis, robust 45 coexistence of both bacterial species. This study provides evidence that host-mediated 46 spatial structuring and stochastic perturbation of communities along with bacterial 47Zebrafish larvae are highly amenable to gnotobiotic techniques and can be reared 99 germ-free (GF) in large numbers [19]. At four days post-fertilization (dpf) larvae possess 100 an open and functional digestive tract that is permissive to microbial colonization, the 101 timing of which is controlled by adding bacteria to the water column. Importantly, larval 102 zebrafish share many physiological traits with humans, including aspects of innate 103 immunity, neurological development, and intestinal function [20]. Therefore, interactions 104 between zebrafish and their microbial symbionts are expected to reflect analogous 105 interactions that occur in other vertebrates. LSFM, combined with the optical 106 transparency of larval zebrafish, enables three-dimensional visualization of the entire 107 intestine with single-bacterium resolution, rapid image acquisition to avoid blurring due 108 to intestinal motility, and extended live imaging with low phototoxicity [21, 22]. This 109 experimental setup provides an unprecedented opportunity to investigate ecological 110 interactions within the vertebrate intestine at a range of spatial and temporal scales. 111 With this model system we found that an apparent competitive interaction 112 between two species native to the zebrafish gut, Aeromonas veronii and Vibrio 113 cholerae, is characterized by sudden and catastrophic collapses of the Aeromonas 114 population, which appear to be driven by mechanical forces related to host intestinal 115 motility. The differential behavior of these two species can be explained by their ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Host Gut Motility and Bacterial Competition Drive Instability in a Model Intestinal Microbiota

Wiles

Jemielita

Baker

et al. 2016

Preprint

View full text Add to dashboard Cite

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“…Imaging was performed using a custom-designed and custom-built microscope capable of differential interference contrast microscopy as well as light sheet fluorescence microscopy (Baker et al, 2015). The specimen was illuminated by a polarized 447nm LED (Quadica Developments Luxeon Star Brantford, Ontario, Canada) and imaged using a standard microscope objective (Zeiss Oberkochen, Germany DICMMPlan Apochromat, 40x/1.0).…”

Section: Imaging Experimentsmentioning

confidence: 99%

“…The specimen was illuminated by a polarized 447nm LED (Quadica Developments Luxeon Star Brantford, Ontario, Canada) and imaged using a standard microscope objective (Zeiss Oberkochen, Germany DICMMPlan Apochromat, 40x/1.0). A Nomarski prism and polarizer were oriented in such a way as to provide Differential Interference Contrast (DIC) (Baker et al, 2015). The resulting image was then focused onto a sCMOS Camera (Cooke, Kelheim, Germany, pco.edge).…”

Section: Imaging Experimentsmentioning

confidence: 99%

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Image velocimetry and spectral analysis enable quantitative characterization of larval zebrafish gut motility

Ganz

Baker

Hamilton

et al. 2017

Preprint

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2 Summary StatementWe present a new image analysis technique using image velocimetry and spectral analysis that returns quantitative measures of gut contraction strength, frequency, and wave speed that can be used to study gut motility and other cellular movements. AbstractNormal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Whereas previous image analysis approaches have successfully extracted properties related to temporal frequency of motility modes, robust measures of contraction magnitude remain elusive. We developed a new image analysis method based on image velocimetry and spectral analysis that reveals temporal characteristics such as frequency and wave propagation speed, while also providing quantitative measures of the amplitude of gut motions. We validate this approach using several challenges to larval zebrafish, imaged with differential interference contrast microscopy. Both acetylcholine exposure and feeding increase frequency and amplitude of motility. Larvae lacking enteric nervous system gut innervation show the same average motility frequency, but reduced and less variable amplitude compared to wild-types. Our image analysis approach enables insights into gut dynamics in a wide variety of developmental and physiological contexts and can also be extended to analyze other types of cell movements.

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Light Sheet Microscopy to Measure Protein Dynamics

Rieckher

2016

J. Cell. Physiol

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Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution, and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio-temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole-organism context. J. Cell. Physiol. 232: 27-35, 2017. © 2016 Wiley Periodicals, Inc.

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A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens

Cited by 19 publications

References 32 publications

Host Gut Motility and Bacterial Competition Drive Instability in a Model Intestinal Microbiota

Host Gut Motility and Bacterial Competition Drive Instability in a Model Intestinal Microbiota

Image velocimetry and spectral analysis enable quantitative characterization of larval zebrafish gut motility

Light Sheet Microscopy to Measure Protein Dynamics

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