Catheter-based confocal imaging systems capable of endoscopic tissue characterization at the cellular level are emerging as a clinical diagnostic tool. The application of these systems to diagnose cardiac diseases is limited by the fact that a fluorescent dye must be available in sufficient concentration in the imaged region to obtain high quality images. We addressed this limitation by developing a novel method for the local delivery of dyes to living tissue. We hypothesized that this method can be applied to study changes in cellular structure that have been demonstrated in cardiac disease. Our dye delivery method, consisting of a hydrogel loaded with dye, was applied to study the three-dimensional microstructure of epi-, endo- and myocardial tissue in excised living tissue and beating rabbit heart preparations using an inverted confocal microscope with a 40x objective lens. Imaging occurred through the hydrogel by pressing the hydrogel directly against the tissue with the objective lens. Dextran-conjugated, lysine-fixable Texas Red with a molecular weight of 3 kDa and an excitation wavelength of 595 nm was used. The image stacks were processed to remove background signals and correct for depth-dependent attenuation and shift. Furthermore, point spread functions were measured and used with the Richardson-Lucy algorithm to deconvolve the image stacks. We demonstrated that dye diffused into the myocardium through the endo- and epicardium and was available in sufficient concentration for imaging. Image stacks of atrial and ventricular tissue were obtained with a field of view of 204.8×153.6 µm2 and up to 80 µm into the myocardium and an isometric resolution of 0.20 µm. The image stacks showed details of cardiac microstructure including the clefts between cells (interstitial space), collagen fibers, transverse tubules and capillary beds. Individual myocytes from the image stacks were segmented to characterize cell morphology. In conclusion, we developed a method of applying dyes directly to living tissue using hydrogel carriers and imaging through the carriers. This will lead to further adaptation of these methods for catheter-based confocal systems. In addition, we describe an approach to create models of the tissue and to quantify cell geometry and volume, which are known to be altered in cardiac diseases.