Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy

被引:35
作者
Appleton, P. L. [1 ]
Quyn, A. J. [1 ]
Swift, S. [2 ]
Naethke, I. [1 ]
机构
[1] Univ Dundee, Div Cell & Dev Biol, Dundee DD1 5EH, Scotland
[2] Univ Dundee, Coll Life Sci, Light Microscopy Facil, Dundee DD1 5EH, Scotland
基金
英国惠康基金;
关键词
BABB; colorectal cancer; crypt; deep tissue imaging; mounting media; MPLSM; TDE; 3D gut architecture; two-photon microscopy; TOTAL INTERNAL-REFLECTION; FLUORESCENCE MICROSCOPY; REFRACTIVE-INDEX; CONFOCAL MICROSCOPY; ORIENTATION; CELLS; APC;
D O I
10.1111/j.1365-2818.2009.03163.x
中图分类号
TH742 [显微镜];
学科分类号
080401 [精密仪器及机械];
摘要
Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 mu m within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of <= 10-15 mu m into the sample, further compounding the ability to image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology.
引用
收藏
页码:196 / 204
页数:9
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