In vivo wide-area cellular imaging by side-view endomicroscopy

Abstract

In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.

Figure 1. In vivo side-view endomicroscopy

(a) Schematic of a laser-scanning side-viewing endoscope. The raster-scanned beam in x and y is relayed by grade-index lenses in the probe and directed by a 90° prism to a side-view window. ζ and ϕ represent the axial and circumferential coordinates, respectively, in the imaging plane. θ denotes the rotation angle of the probe, or the angle between x and ϕ axes. (b) Coordinate transform between the proximal (x-y) and distal (ζ-ϕ) imaging planes. The trace of raster-scanned beam is depicted in blue solid lines. (c) Imaging set up. The laser beam is projected to an animal stage. Dotted lines depict the outline of the beam diverging after going through the imaging plane. (d) 3-dimensional rendered fluorescence image of the vasculature in the descending colon of a normal C57B6/L mouse. (e) A fly-through rendered image of d. Fluorescence images of the microvasculature in the esophagus (f), vasculature of villi in the small intestine (g) and MHC-II-GFP expression in dendritic cells in the trachea (h). Blood vessels are visualized by intravenously injected FITC-dextran conjugates. In f-h, the horizontal axis represents the circumferential angle ϕ; scale bars, 200 μm.

Figure 2. Visualization of FoxP3⁺ T_reg cells in a DSS-induced colitis model

(a) Large-area map of FoxP3:GFP⁺ T_reg cells taken before DSS treatment (day −1). (b) 2.5x image of the area (red square) marked in a. Inset is 10x image showing T_reg cells (green) and blood vasculature (red). (c) A typical image of T_reg cells at day 7 in the recovery phase of colitis. Blood vessels are visualized by intravenously injected Evans Blue. Scale bars, 200 μm; 20 μm in the inset.

Figure 3. Longitudinal imaging of colorectal tumorigenesis

(a-c) Fluorescence image of colorectal vasculature in a floxed Apc mouse at 11 weeks (a) and 13 weeks (b) after adeno-cre administration, and in a large lesion at week 17 (c) in another Cre-treated mouse. (d) Apc-knockout GFP⁺ cells (green) and blood vessels (red) at the same site in the descending colon, observed at days 10, 12, 14, and 28. Each image is a projection view of 50-μm z-stack. The serial images reveal a GFP⁺ lesion that grows in size over time (*). Other GFP⁺ nodules shrink (▲) or vanish (circle) at day 28. Blood vessels are visualized by intravenously injected FITC-dextran in a-c and by TAMRA-dextran in d. Scale bars, 200 μm.