In type 1 diabetes (T1D), destruction of beta cells located in the islets of Langerhans throughout the pancreas is extremely difficult to study owing to the organ's inaccessible location, diffuse tissue architecture and abundance of potentially harmful digestive enzymes that make it difficult to obtain biopsy tissue samples
. Despite some differences compared to the human pathophysiology, our knowledge of how T1D develops has benefited significantly from studies in rodent models such as the non-obese diabetic mouse (NOD)
. In mouse and man, documentation of autoimmune events in the pancreatic islets (a process termed ‘insulitis’) has been traditionally achieved by histological techniques in cross-sectional studies
. While such data provide a one-time ‘snapshot’ of islet destruction, there is no clear knowledge of the precise cellular dynamics involved in this process.
Since first reported by Denk and coworkers
, two-photon microscopy has been applied extensively to image immune cells in intact lymphoid organs
. The major advancement associated with the technique is the use of a pulsed infrared laser for fluorescent dye excitation
. This high excitation wavelength allows for deep tissue imaging and its low energy constrained to the focal plane limits phototoxicity. As a consequence, two-photon microscopy has become the technique of choice to assess the dynamic behavior of immune cells in vivo
. To date, however, the handful of studies that applied two-photon microscopy in the context of autoimmune diabetes were limited to the pancreatic draining lymph nodes
, whereas the situation in the pancreas remains uncharted territory. One group performed 2-photon microscopy on islets transferred into the anterior chamber of the eye, but the immune privileged nature of the site precludes inclusion of the hallmark autoimmune component of T1D
. Another recent study by Nyman et al introduced the use of intravital, high-speed confocal scanning in order to determine blood flow dynamics in islets
. The latter approach, however, poses considerable constraints in terms of imaging depth and laser cytotoxicity and thus is unsuitable for tracking immune cells around islets over longer time spans.
The dynamic behavior of immune cells is profoundly dependent on physiological conditions
, and it is particularly questionable whether circulatory deprivation in explanted pancreas preparations would leave the true in situ
parameters of diabetogenic immune responses unaltered. We report here a novel approach to visualize the kinetic properties of immune cells during the development of diabetes in the intact pancreas and islets of living animals. As such, we provide the first real-time visualization of leukocyte-beta cell interactions and dendritic cell recruitment to the islets.