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This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901plusmn1713 and 10299plusmn3598 mum3 (meanplusmnsd), respectively. Atrial and ventricular myocyte volume fractions were 72.4plusmn4.7% and 79.7plusmn2.9% (mean plusmn sd), respectively. Atrial and ventricular myocyte density was 165571plusmn55836 and 86957plusmn32280 cells/mm3 (mean plusmn sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.