The epithelium lining the intestinal tract mediates the selective absorption of desirable substances from the gut lumen while acting as a remarkably effective barrier against undesirable materials (toxic substances, bacteria, etc). The basis for this barrier is a series of defense arranged serially with a set of pre-epithelial defenses (juxtamucosal pH control, mucus layer), epithelial defense (tight junctions, apical membrane impermeability), and subepithelial defenses (mucosal blood flow, neural regulation). Our laboratory has been developing and applying methods to explore the pre-epithelial and epithelial defenses used in the stomach and intestine. We have had some success using confocal and two-photon microscopy of these organs to explore tissue function with subcellular resolution in living (anesthetized) animals having their mucosal surfaces of the tissue surgically exposed to the exterior. In the first part of the presentation I will describe our work asking how the epithelial barrier is sustained during the rapid renewal of the small intestinal epithelium, where it is estimated that approximately 1 cell is lost per villus per minute. We have observed that the living epithelium appears discontinuous, with gaps in 3% of cell positions. These gaps do not take up vital stains that successfully mark neighbouring cell cytoplasm, nuclei, and apical membranes. When we track cell shedding in real time, we observe creation of gaps that are not rapidly filled by other cells after one cell departs. Importantly, when we add a fluorescent dye to the luminal fluid (Lucifer Yellow) it is not able to permeate into gaps. By this marker, and the presence of a substance in the gaps that reflects laser light, we conclude that an unknown substance plugs gaps. In the second half of the presentation, I will discuss our work in the stomach. Here we have created the first model in which rapid perturbation of the epithelium is restricted to microscopic regions, hopefully modeling the type of injuries that would serve as nucleating events in ulcer formation. We use two-photon absorpton to create micro-lesions that specifically kill 2-3 cells in the epithelium. In response to this insult, there is a spreading of damage to an annulus of innocent bystander cells (noted as increased permeability to a luminal dye, and loss of NAD(P)H fluorescence), and then all damaged cells are shed, apparently thru the force of the healthy epithelium migrating in to close off the damaged area. Coincident with the spreading damage is a large pulse of alkali from the tissue that provides a protective alkaline layer over the damaged tissue during subsequent events. The repair is regulated in part by COX-1, as COX-1 null mice have a smaller alkali pulse and a slower repair. In both the intestinal and gastric work, quantitative light microscopy provides windows into processes that are difficult if not impossible to detect by more classic methods.