Diabetic Retinopathy (DR), a leading cause of adult blindness, is a progressive development of microvascular and neurovascular damage in the retina which leads to vision-threatening complications. The disruption of the neurovascular unit (NVU) is a primary factor in the pathogenesis of DR. The NVU is an interdependent unit made up of many cell types including pericytes, endothelium, glial cells and neurons, which work synergistically to maintain the function of the retina and allows adaptation to change in the physiological environment; for example, by regulating blood flow and by maintaining the blood retinal barrier (BRB). In order to assist with improving our understanding of retinal biology, and developing strategies to correct NVU dysfunction, detailed knowledge of the heterocellular interactions of the NVU is required. Serial block face scanning electron microscopy (SBF-SEM), and computational image reconstruction enables the first three-dimensional ultrastructural analysis of the NVU in retinal capillaries.  Examination of the data in the x-, y- and z-planes is performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities to provide visualisation and analysis of the data to help explore these issues with respect spatial changes across and within the retina, between species and with disease. Heterocellular relationships within the retina have been assessed in mouse and human tissues in health and diabetes. Initial findings have revealed, with the onset on DR, features of the NVU showed areas of detachment from the Basement Membrane (BM), leaving intermittent gaps between the cell plasma membranes and the BM. Pericyte-endothelial interactions via peg and socket formations in non-diabetic capillaries show both cell membranes in direct contact, however, there appears to be space surrounding the peg in the socket area of diabetic capillaries. Similar electron lucent gaps were present in the endothelium of diabetic capillaries. An increase in the number of leukocytes were present in the luminal space of diabetic capillaries, which were found to make direct communication with endothelial projections. This work provides new information on the ultrastructural changes to the murine retinal NVU during the onset and progression DR, which in turn can serve as a platform to inform future studies on how to delay or prevent the progression of the diseased retina.