An intravital window to image the colon in real time

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.


Statistics
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

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Bradley B. Barth and Xiling Shen
Oct 20, 2019 No software was utilized during data collection.
Data analysis was completed using a combination of commercially available software packages (JMP, Imaris, ImageJ) and custom code. Each program and application of it is clearly cited in the descriptions of data processing and analysis. Custom code is stored in an internal repository, and will be made available on request, as will any more specific details regarding parameters used by commercial software. We include the following code accessibility statement in our manuscript: The authors declare that the custom code used for data processing and analysis in this study are available from the corresponding author upon reasonable request. The data in Fig. 4 have associated custom code.
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files or available from the corresponding author upon reasonable request. The data in Fig. 1c,d,e,f; Fig. 2; Fig. 3; and Fig. 4 have associated raw data.

nature research | reporting summary
October 2018 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Note that full information on the approval of the study protocol must also be provided in the manuscript.
Sample size was not pre-determined. As the primary goal of this publication is to describe a new tool rather than to explore novel biological phenomena, pilot studies to demonstrate the ability of the window to observe previously described phenomena typically involved 3-5 animals.
Outlier data were only excluded when they could be linked to probable underlying problems in data collection, such as premature death due to extreme response to anesthetic, dislocation of stimulation leads, or gross internal physical abnormalities discovered during necropsy.
All data presented in this manuscript were successfully replicated internally and/or were consistent with previously published results. Individual data points representing unique animals are presented wherever data is quantified to support this statement.
In most of our studies, animals served as their own controls. One of the primary values of the colon window is that animals can be observed at baseline during the preliminary portion of the study, and then re-measured throughout the course of treatment. Animals were randomly allocated into experiment groups where relevant to validate findings in traditional control/treatment experiments.
Image processing and analysis was preformed blinded.
This study did not utilize wild animals.
This study did not utilize field-collected samples.
All animal procedures were reviewed and approved by the Duke University Institutional Care and Use Committee (protocol A139-18-05 and 195-15-05) and Cornell University Institutional Care and Use Committee (protocol 2015-0029). They were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals84 and all relevant regulations for animal testing and research.