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 nutrie nt absorption, protein and fluid secretion, and the regulated symbiosis wit h intraluminal contents. Research in epithelial biology has benefited signi ficantly from the use of cultured monolayer preparations, which closely rep licate the structure and function of normal gastrointestinal mucosa. Given the explicit importance of epithelial architecture to its physiology, inves tigations of epithelial biology should be enhanced by the capacity to track microscopic structures and substances in live cells. In order to achieve t his goal, it is necessary to employ a microscopic technique with the capabi lity 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 absorptio n and scattering coefficients. This allows the efficient collection of ligh t energy from sites hundreds of microns deep, with only minimal tissue dama ge. In this report, we have presented an introduction to the theory and pra ctice of two-photon microscopy for imaging the GI tract epithelium, and hav e presented examples of its utility in discerning three-dimensional structu re and function in CaCo2A epithelial cell monolayers. (C) 2000 Wiley-Liss. Inc.