Diabetes mellitus results from the dysfunction and/or destruction of insulin producing beta cells. Glucose homeostasis is managed by daily insulin injections. However, miscalculations in macronutrient uptakes and ongoing insulin demands result in glycemic variabilities leading to debilitating secondary complications. Restoration of euglycemia and insulin independence in people living with diabetes is an ambitious goal. Stem cells derived-beta cells provide a tangible alternative for cell replacement therapy. However, even with the remarkable progress in creating insulin producing cells, the development of fully functional cells that imitate all aspects of native beta cells has remained out of reach. The molecular cues that progressively direct progenitor cells to insulin secreting mature beta cells are not completely understood. Moreover, in a native islet, beta cells exist in intricate three-dimensional structures in close contact with vascular endothelial and other endocrine cells. The complex islet cytoarchitecture has a profound effect on beta cell survival, insulin synthesis and stimulated secretion in native islets. Yet, the aspect of the beta cell niche is not fully investigated or controlled while generating insulin producing spheroids. This collection aims to highlight evolving techniques in beta cell differentiation, modulation of beta cell microenvironment, 3D printing and microfluidics, characterization of differentiated spheroids using confocal and Stimulated Emission Depletion (STED) microscopy, 2-photon and live-cell calcium imaging, single-cell sequencing, and metabolomics studies. This collection further aims to emphasize the survivability and the behavior of implanted cells in in vivo animal models by highlighting deep tissue imaging of implanted cells using 2-photon, time resolved 3-D live cell imaging, Optical bioluminescent/fluorescence Tomography, Light Sheet Microscopy, or any other innovative optical techniques.