Imaging of three-dimensional epithelial architecture and function in cultured CaCo2a monolayers with two-photon excitation microscopy

The principal functions of the gastrointestinal tract mucosa include nutrient absorption, protein and fluid secretion, and the regulated symbiosis with intraluminal contents. Research in epithelial biology has benefited significantly from the use of cultured monolayer preparations, which closely replicate the structure and function of normal gastrointestinal mucosa. Given the explicit importance of epithelial architecture to its physiology, investigations of epithelial biology should be enhanced by the capacity to track microscopic structures and substances in live cells. In order to achieve this goal, it is necessary to employ a microscopic technique with the capability of imaging deep into the tissue or cell preparation, without adversely affecting its physiology. Two-photon excitation microscopy may constitute such a technique, due to its ability to provide fluorescence excitation of fluorophores using near infrared radiation, that has lower tissue absorption and scattering coefficients. This allows the efficient collection of light energy from sites hundreds of microns deep, with only minimal tissue damage. In this report, we have presented an introduction to the theory and practice of two-photon microscopy for imaging the GI tract epithelium, and have presented examples of its utility in discerning three-dimensional structure and function in CaCo2A epithelial cell monolayers.