Nanoscale detection of insulin granule sub-structures using dSTORM imaging

Sammanfattning: Diabetes mellitus is a global disease, mainly caused by insufficient insulin secre- tion from pancreatic islet β-cells. Insulin is stored in the granules of islet β-cells and released in response to extracellular stimuli e.g. glucose. The state of insulin structure and maturation determines the quality of the released insulin, However, the insulin state in the granules is rarely investigated due to lack of proper methods. Here, we hypothesize that high-resolution microscopy and appropriate image analysis methods could be used to detect the insulin state in the granule. We used direct stochastic optical reconstruction microscopy (dSTORM), which is a super- resolution imaging method, to capture cellular insulin granules images and obtain the location of insulin molecules labelled by an insulin single-domain fluorescent antibody. The images were processed by the optimized algorithm of layering and filtering until the clusters of fluorescent dots in the images were distinguished. Then the hypothetical structures were used to scan the images to find the complete hexamer. In summary, this study provides novel method to study mature insulin crystals in the granular of islet β-cells.