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Am J Physiol Gastrointest Liver Physiol. 2015 May 1;308(9):G721-35. doi: 10.1152/ajpgi.00310.2014. Epub 2015 Feb 26.

Optical reconstruction of murine colorectal mucosa at cellular resolution.

Author information

1
The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California; Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, California; and.
2
The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California; Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, California; and Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, California dbpolk@chla.usc.edu.

Abstract

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

KEYWORDS:

clearing; imaging; intestine

PMID:
25721303
PMCID:
PMC4421015
DOI:
10.1152/ajpgi.00310.2014
[Indexed for MEDLINE]
Free PMC Article

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