• 3-D Imaging of T-cell Localization in Inflamed Colon Tissues

3-D Imaging of T-cell Localization in Inflamed Colon Tissues

Light sheet fluorescence microscopy (LSFM) provides accurate 3-dimensional images of inflammed intestine of mice with colitis, researchers show in a Gastroenterology in Motion article and video in the October issue of Gastroenterology. The authors demonstrate use of the technology to analyze migration of human T cells in colons of mice with colitis.

Improved techniques are needed to image inflammed tissues in animal models and patients with inflammatory bowel diseases (IBD)—these might be used to better assess disease progression and the effects of therapeutic agents. Conventional imaging techniques are limited in penetration depth, field of view, or resolution resulting in assessment of only small parts of the gut.

Sebastian Zundler et al show that LSFM can overcome these limitations.

Top: Colon tissues of mice injected with human T cells (green) without vedolizumab (top) and with (bottom).

LSFM combines features of wide-field and confocal microscopy. To emit fluorescence signals, samples are illuminated by lasers, which are expanded by optical devices to form a thin sheet of light (5–40 μm) that penetrates the sample from opposite sides and excites a defined plane, rather than a single point. This provides superior optical sectioning, speeds image acquisition, and reduces photobleaching.

Three-dimensional datasets can be acquired by wide-range shifting of the sample through fixed light sheets in z-direction and sequential imaging at every position. Samples of up to 1.5 cm x 1.5 cm, stained with as many as 8 colors, can be analyzed by LSFM.

The video introduces readers to the principles of LSFM and demonstrates image analysis of the tissues.

Zundler et al used LSFM to visualize inflammed intestinal tissues, in their original anatomical configuration, in mice with colitis. Human CD4+ T cells incubated with or without vedolizumab (an antibody against the α4β7 integrin) were injected into mice with colitis and LSFM was used analyze colon tissues.

Using LSFM, the authors demonstrated that vedolizumab reduced the accumulation of T cells in the colon. This shows that blocking the integrin α4β7 impedes migration of human immune cells to the intestinal mucosa

The authors conclude that LSFM allows faster acquisition and better interpretation of optical information. It could be used to visualize angiogenesis, tumor dissemination or infiltration, cell movements, and development of inflammation—especially in mice expressing fluorescent proteins. LSFM can also be used in analysis of whole-mount stained biopsies of patients with IBD or cancer.

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