Microtome-Free 3-Dimensional Confocal Imaging Method for Visualization of Mouse Intestine With Subcellular-Level Resolution

Background & Aims

The intrinsic opacity of mouse intestinal tissue prevents its evaluation by high-resolution, in-depth optical microscopy. Instead, intestinal tissue is usually sectioned to expose the interior domains of the mucosa and submucosa for microscopic examination. However, microtome sectioning can cause distortions and artifacts that prevent acquisition of an accurate view of the sample. We therefore attempted to develop a microtome-free 3-dimensional (3D) confocal imaging method for characterization of mouse intestine.

Methods

We applied an optical-clearing solution, FocusClear, to permeate and reduce the opacity of mouse colon and ileum. Tissues were labeled with fluorescent probes and examined by confocal microscopy with efficient fluorescence excitation and emission in the FocusClear solution. The voxel-based confocal micrographs were processed with Amira software for 3D visualization and analysis.

Results

Treatment of tissues with the optical-clearing solution improved photon penetration, resulting in the acquisition of images with subcellular-level resolution across the mucosa, submucosa, and muscle layers. Collectively, the acquired image stacks were processed by projection algorithms for 3D analysis of the spatial relations in villi, crypts, and connective tissues. These imaging technologies allowed for identification of spatiotemporal changes in crypt morphology of colon tissues from mice with dextran sulfate sodium–induced colitis as well as detection of transgenic fluorescent proteins expressed in the colon and ileum.

Conclusions

This new optical method for penetrative imaging of mouse intestine does not require tissue sectioning and provides a useful tool for 3D presentation and analysis of diseased and transgenic intestine in an integrated fashion.

Abbreviations used in this paper: 2D, 2-dimensional, 3D, 3-dimensional, DiD, 4-chlorobenzene sulfonate salt, DSS, dextran sulfate sodium, GFP, green fluorescent protein, PBS, phosphate-buffered saline, PI, propidium iodide