Microscopic visualization of intestine under normal and diseased conditions is essential for understanding the physiology and developing screening and diagnostic tools for intestinal diseases.1
Structurally, the intestine is comprised of the mucosa, submucosa, and muscle layers: a proliferating stem cell population located at/near the crypt base in the mucosa and undergoes terminal differentiation as cells migrate from the base to the luminal surface.2
Because standard two-dimensional (2D) tissue analysis confines visualization of the intestinal architecture at a specific cut plane, three-dimensional (3D) representation of image data over an area of interest is preferable for in situ
visualization and assessment of the epithelium.3
Among the available imaging technologies, confocal microscopy generates a sharp 2D image at the plane of focus; incrementing the plane of focus creates a series of optical sections at different depths in the specimen, which allows for construction of a 3D image.4
When a tissue specimen is sufficiently transparent so that light can pass through with minimal scattering (such as the Zebrafish embryo), confocal microscopy provides a useful tool to study the 3D configuration of molecules of interest in the sample. Unfortunately, tissues such as colons and small intestines are non-transparent, which seriously limits their optical accessibility for confocal microscopy or light microscopy in general.
To date, preparation of tissue sections has been the standard laboratory practice used to expose the interior domain of a thick tissue for microscopic examination. There are, however, practical difficulties in using thin layers of tissue sections to acquire an integral view of the sample: artifacts and distortions introduced by microtome slicing are inevitable, let alone the challenge of aligning serial sample sections with precision. Recently, significant progress has been made to develop the confocal laser endoscopy for in vivo
diagnosis of gastrointestinal disorders.5–9
However, the extension of confocal microscopy from the bench to the bedside still leaves unresolved the problem of light scattering as photons encounter the gut wall.
Previously, we have developed a set of bioimaging technologies in sample preparation, confocal microscopy, and post-recording image processing for visualization of neural circuits in the brain of fruitfly Drosophila melanogaster
(~130 μm in thickness) at the subcellular level without employing tissue dehydration, embedding, and sectioning.10–13
In the present study, we extend the developed technologies for characterization of mouse intestine. We appreciate that the intrinsic opacity of the layers of mucosa, submucosa, and muscles can prevent efficient light penetration for high-resolution imaging. We therefore applied an optical-clearing solution (FocusClear™, with a refractive index at 1.46, US Patent 6472216-B1) to permeate and reduce the opacity of the mouse intestine to improve photon penetration during optical microscopy.14
Unlike common optical-clearing procedures involving treatment with xylene, mineral oil, methyl salicylate, or glycerol, which often dissolve labeled fluorescent probes and result in weak signals and blurring images from the sample, the FocusClear™-mediated opacity clearing is fully compatible with fluorochrome staining and fluorescent protein detection in the intestine. In this research, we show that the improved photon penetration led to a clear visualization of the mouse colon and ileum. To our knowledge, this is the first microscopic imaging method to achieve subcellular-level resolution (resolving adjacent nuclei) of the full depth of intestinal mucosa and submucosa without using microtome sectioning.
To demonstrate applications of this microtome-free confocal imaging method, we examined spatio-temporal changes in colonic crypt morphology following dextran sulfate sodium-induced ulcerative colitis in BALB/c mice.15,16
We also inspected nestin-GFP (green fluorescent protein expression driven by the nestin promoter)17
transgenic mice to reveal the 3D expression pattern of nestin in the colon and ileum. Results using optical clearing combined with fluorescence labeling and confocal microscopy for 3D visualization of mouse intestine are presented and discussed in this report.