Atrial fibrillation is the most common cardiac arrhythmia and is a significant cause of morbidity and mortality. A clinical study by Haissaguerre et al (1998), demonstrated that in 94% of cases of atrial fibrillation, electrical activity originated at ectopic foci in the pulmonary vein It is believed the external sleeve of cardiomyocytes along the pulmonary vein is responsible for its arrhythmogenic propensity, with the veins complex structural orientation providing a substrate for the arrhythmia. This study aimed to characterise the cellular organisation of the rat pulmonary vein using histological techniques and state of the art wide field confocal microscopy. Male Sprague-Dawley rats (250-400g) were euthanised by cervical dislocation, the heart and lungs removed en bloc and sections of the extra- and intrapulmonary veins microdissected. Tissue structure was initially assessed in formalin fixed sections of the pulmonary vein using Masson’s Trichrome staining. Transverse sections revealed a thin smooth muscle layer close to the lumen surrounded by the cardiomyocyte sleeve. Longitudinal sections demonstrated a non uniform and complex arrangement of cardiomyocytes that frequently showed abrupt changes in direction and with the cardiomyocytes extending into the intrapulmonary sections. The Confocal Optical Mesolens is the only device of its kind in the world that is capable of producing detailed, high-definition, three dimensional images of sections of living tissue. Using the dye Di4-ANNEPS (1µM), individual cardiomyocytes were visible in transverse and longitudinal directions, consistent with observations from histological studies. The distribution of mitochondria within the pulmonary vein cardiomyocytes was determined with mitotracker green (1µM) or TMRE (1µM) and both indicated that the mitochondria have a regular arrangement within these cells. Nerves were also clearly visible when the pulmonary vein was labelled with these mitochondrial dyes. The pulmonary vein appeared to be highly innervated further supporting the notion that the autonomic nervous system plays an important role in determining its electrical activity. These techniques demonstrated the variable arrangement of cardiomyocytes in the myocardial sleeve of the rat pulmonary vein. Although the histological and epifluorescence techniques created a clear image of cardiomyocytes arrangement on the pulmonary vein, the Confocal Optical Mesolens provides an opportunity to obtain high resolution three dimensional images, without the need for formalin fixation and serial sectioning of tissues, over a larger field of view with the use of fluorescent dyes to label different structures within the cardiomyocytes. An enhanced insight into the cellular organisation of the pulmonary vein with the use of modern technologies will allow a better understanding of its arrhymogenic properties.