The mouse pancreas is a mixed exocrine and endocrine glandconsisting of three lobular compartments: the splenic, duodenal and gastric lobes. During embryogenesis, the pancreas forms from two progenitor populations located on the dorsal and ventral side of the primitive gut tube. These anlagen are brought in close proximity as the gut elongates and rotates, and fuse to form a single organ. The splenic and duodenal lobes develop from the dorsal and ventral anlagen, respectively. In the adult pancreas, exocrine tissue secretes digestive enzymes intothe gut lumen to support nutrient uptake. The endocrine Islets of Langerhans are scattered throughout the exocrine tissue and aid in regulation of energy homeostasis through the secretion of hormones. One of the key players in energy homeostasis is the pancreatic ß-cell, which is the most abundant cell type of the islets. The β-cells regulates blood glucose levels through the action of insulin. Conditions where this regulation does not function properly are gathered under the common name of Diabetes mellitus. Type 1 diabetes (T1D) is characterized by insulin deficiency due to autoimmune destruction of the ß-cells. Using recently developed protocols for optical projection tomography (OPT) whole-organ imaging, we have revealed new spatial and quantitative aspects on ß-cell mass dynamics and immune infiltration during the course of T1D development in the non-obese diabetic (NOD) mouse model. We show that although immune infiltration appears to occur asynchronously throughout the organ, smaller islets, mainly located in the periphery of the organ, preferentially loose their ß-cells during early stages of disease progression. Larger islets appear more resistant to the autoimmune attack and our data indicate the existence of a compensatory proliferative capacity within these islets. We also report the appearance of structures resembling tertiary lymphoid organs (TLOs) in association with the remaining islets during later phases of T1D progression. OPT has already proven to be a useful tool for assessments of ß-cellmass in the adult mouse pancreas. However, as with other techniques, previous protocols have relied on a tedious degree of manual postivacquisition editing. To further refine OPT-based assessment of pancreatic ß-cell mass distribution in the murine pancreas, we implemented a computational statistical approach, Contrast-Limited Adaptive Histogram Normalisation (CLAHE), to the OPT projection data of pancreata from C57Bl/6 mice. This methodology provided increased islet detection sensitivity, improved islet morphology and diminished subjectivity in thresholding for reconstruction and quantification. Using this approach, we could report a substantially higher number of islets than previously described for this strain and provide evidence of significant differences in islet mass distribution between the pancreatic lobes. The gastric lobe stood out in particular and contained a 75% higher islet density as compared to the splenic lobe. Although the development of the early pancreatic buds has been relatively well studied, later morphogenetic events are less clear and information regarding the formation of the gastric lobe has largely been missing. Using OPT we have generated a quantitative three-dimensional road map of pancreatic morphogenesis in the mouse. We show that the gastric lobe forms as a perpendicular outgrowth fromthe stem of the dorsal pancreas at around embryonic day (e) 13.5, which grows into a mesenchymal domain overlaying the pyloric sphincter and proximal part of the glandular stomach. By analyzing mutant mice with aberrant spleen development, we further demonstrate that proper formation of the gastric lobe is dependent on the initial formation of the closely positioned spleen, indicating a close interplay between pancreatic and splenic mesenchyme during development. Additionally, we show that the expression profile of markers for pancreatic multipotent progenitors within the pancreas is heterogenous with regards to lobular origin. Altogether, our studies regarding the morphogenesis and adult constitution of the mouse pancreas recognize lobular heterogeneities that add important information for future interpretations of this organ.

Diabetes is a rapidly increasing health problem. In a global perspective,approximately 415 million people suffered from diabetes in 2015 and this number ispredicted to increase to 640 million by 2040. To tackle this pandemic there is a needfor better analytical tools by which we can increase our understanding of the disease.One discipline that has already provided much needed insight to diabetes etiology isoptical molecular imaging. Using various forms of light it is possible to create animage of the analysed sample that can provide information about molecularmechanistic aspects of the disease and to follow spatial and temporal dynamics. The overall aim of this thesis is to improve and adapt existing andnovel optical imaging approaches for their specific use in diabetes research. Hereby,we have focused on three techniques: (I) Optical projection tomography (OPT),which can be described as the optical equivalent of x-ray computed tomography(CT), and two vibrational microspectroscopic (VMS) techniques, which records theunique vibrational signatures of molecules building up the sample: (II) Fouriertransforminfrared vibrational microspectroscopy (FT-IR) and (III) Ramanvibrational microspectroscopy (Raman). The computational tools and hardware applications presented here generallyimprove OPT data quality, processing speed, sample size and channel capacity.Jointly, these developments enable OPT as a routine tool in diabetes research,facilitating aspects of e.g. pancreatic β-cell generation, proliferation,reprogramming, destruction and preservation to be studied throughout the pancreaticvolume and in large cohorts of experimental animals. Further, a novel application ofmultivariate analysis of VMS data derived from pancreatic tissues is introduced.This approach enables detection of novel biochemical alterations in the pancreasduring diabetes disease progression and can be used to confirm previously reportedbiochemical alterations, but at an earlier stage. Finally, our studies indicate thatRaman imaging is applicable to in vivo studies of grafted islets of Langerhans,allowing for longitudinal studies of pancreatic islet biochemistry.viIn summary, presented here are new and improved methods by which opticalimaging techniques can be utilised to study 3D-spatial, quantitative andmolecular/biochemical alterations of the normal and diseased pancreas.