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

P. L. Appleton, A. J. Quyn, S. Swift, Inke Nathke

    Research output: Contribution to journalArticle

    34 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)196-204
    Number of pages9
    JournalJournal of Microscopy
    Volume234
    Issue number2
    DOIs
    Publication statusPublished - May 2009

    Keywords

    • 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

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