Diabetes is a rapidly growing disease with 415 million affected adults worldwide. The pancreatic endocrine cells, most importantly the insulin producing β-cells, play an important role in regulating blood glucose homeostasis. Type 1 diabetes (T1D) is characterized by the inability of the pancreas to secrete sufficient amounts of insulin due to autoimmune destruction of insulin producing β-cells. Type 2 diabetes (T2D) on the other hand is characterized by defects in insulin secretion and insulin sensitivity. Alterations in the β-cell mass (BCM) and/or function play a major role in the development and progression of the disease. Understanding BCM dynamics in disease models is therefore a key aspect for better interpretation of research results. In this thesis, we have used optical projection tomography (OPT) as a tool to evaluate a non-invasive imaging modality for β-cell scoring and to study disease dynamics in frequently used animal models for T1D and T2D. The possibility to monitor BCM in vivo would radically improve our competence in studying the pathogenesis of diabetes and in therapeutic interventions. Single photon emission computed tomography (SPECT) is a widely used technique that has become a promising approach to monitor changes in BCM in vivo. A key issue for using this approach is to evaluate the β-cell specificity and read out of the utilized radiotracers. This is most commonly performed by conventional stereological approaches, which rely on the extrapolation of 2D data. We developed a protocol for SPECT-OPT multimodal imaging that enables rapid and accurate cross evaluation of SPECT based assessments of BCM. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of the radiotracer 111In-exendin-3 and insulin positive β-cell volumes respectively between different pancreatic lobes, both visually and quantitatively. We propose SPECT-OPT multimodal imaging as an accurate and better approach for validating the performance of β-cell radiotracers. The leptin deficient ob/ob mouse is a widely used model for studies of metabolic disturbances leading to T2D, including obesity and insulin resistance. By OPT imaging we created the first 3D-spatial and quantitative account of BCM distribution in this model. We observed a previously unreported degree of cystic lesions in hypertrophic islets, that were occupied by red blood cells (RBCs) and/or fibrin mesh. We propose that these lesions are formed by a mechanism involving the extravasation of RBCs/plasma due to increased blood flow and islet vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of these characteristics of β-cell expansion in ob/ob mice presented in this study may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function. The tomographic data, on which this study was based, will be made publically available as a resource to the research community for the planning and interpretation of research involving this model. There are limited studies on early metabolic and functional changes of BCM in the settings of T1D. In order to assess initial metabolic alterations in BCM before the onset of diabetes, we characterized congenic diabetes prone Bio-breeding (BB) DR.lyp/lyp rats, a widely used model for T1D diabetes. We observed lower acute insulin response, reduced islet blood flow and a significant reduction in the BCM of small and medium sized islets at a very early stage (40 days), i.e. before insulitis and development of diabetes. Underlying changes in islet function may be a previously unrecognized factor of importance in the development of T1D.

A major goal in biological imaging is to visualize interactions of different tissues, often fluorescently labeled, during dynamic processes. Only a few of these labels fit into the available spectral range without overlap, but can be separated computationally if the full spectrum of every single pixel is known. In medical imaging, hyperspectral techniques show promise to identify different tissue types without any staining. Yet, microscopists still commonly acquire spectral information either with filters, thus integrating over a few broad bands only, or point-wise, dispersing the spectra onto a multichannel detector, which is inherently slow. Light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) are two techniques to acquire 3D microscopic data fast, photon-efficiently and gently on the specimen. LSFM works in fluorescence mode and OPT in transmission. Both are based on a fast widefield detection scheme where a 2D detector records the spatial information but leaves no room to acquire dispersed spectra. Hyperspectral imaging had not yet been demonstrated for either technique. In this work, I developed a line-scanning hyperspectral LSFM and an excitation scanning OPT to acquire 5D data (3D spatial, 1D temporal, 1D spectral) and optimized the performance of both setups to minimize acquisition times without sacrificing image contrast, spatial or spectral information. I implemented and assessed different evaluation pipelines to classify and unmix relevant features. I demonstrate the efficiency of my workflow by acquiring up to five fluorescent markers and the autofluorescence in \\zf and fruit fly embryos on my hyperspectral LSFM. I extracted both concentration maps and spectra for each of these fluorophores from the multidimensional data. The same methods were applied to investigate the transmission data from my spectral OPT, where I found evidence that OPT image formation is governed by refraction, whereas scattering and absorption only play a minor role. Furthermore, I have implemented a robust, educational LSFM on which laymen have explored the working principles of modern microscopies. This eduSPIM has been on display in the Technische Sammlungen Dresden for one year during the UNESCO international year of light. / Ein wichtiges Ziel biologischer Bildgebung ist die Visualisierung des Zusammenspiels von verschiedenen, meist fluoreszent markierten, Geweben bei dynamischen Prozessen. Nur wenige dieser Farbstoffe passen ohne Überlapp in das zur Verfügung stehende Spektrum. Sie können jedoch rechnerisch getrennt werden, wenn das gesamte Spektrum jedes Pixels bekannt ist. In medizinischen Anwendungen versprechen hyperspektrale Techniken, verschiedene Gewebetypen markierungsfrei zu identifizieren. Dennoch ist es in der Mikroskopie noch immer üblich, spektrale Information entweder mit Filtern über breiten Bändern zu integrieren, oder Punktspektren mithilfe von Dispersion zu trennen und auf einem Multikanaldetektor aufzunehmen, was inhärent langsam ist. Light Sheet Fluorescence Microscopy (LSFM) und Optical Projection Tomography (OPT) nehmen 3D Mikroskopiedaten schnell, photoneneffizient und sanft für die Probe auf. LSFM arbeitet mit Fluoreszenz, OPT in Transmission. Beide basieren auf schneller Weitfelddetektion, wobei die räumliche Information mit einem 2D Detektor aufgenommen wird, der keinen Raum lässt, um die getrennten Spektren zu messen. Hyperspektrale Bildgebung wurde bis jetzt für keine der zwei Techniken gezeigt. Ich habe ein hyperspektrales LSFM mit Linienabtastung und ein OPT mit Wellenlängenabtastung entwickelt, um 5D Daten (3D räumlich, 1D zeitlich, 1D spektral) aufzunehmen. Beide Aufbauten wurden hinsichtlich minimaler Aufnahmezeit optimiert, ohne dabei Kontrast, räumliche oder spektrale Auflösung zu opfern. Ich habe verschiedene Abläufe zum Klassifizieren und Trennen der Hauptkomponenten implementiert. Ich nehme bis zu fünf Fluorophore und Autofluoreszenz in Zebrafisch- und Fruchtfliegenembryos mit dem hyperspektralen LSFM auf und zeige die Effizienz des gesamten Ablaufes, indem ich Spektren und räumliche Verteilung aller Marker extrahiere. Die Transmissionsdaten des spektralen OPT werden mit denselben Methoden untersucht. Ich konnte belegen, dass die Bildformation im OPT massgeblich von Brechung bestimmt ist, und Streuung und Absorption nur einen geringen Beitrag leisten. Außerdem habe ich ein robustes, didaktisches LSFM gebaut, damit Laien die Funktionsweise moderner Mikroskopie erkunden können. Dieses eduSPIM war ein Jahr lang in den Technischen Sammlungen Dresden ausgestellt.

The gastrointestinal tract comprises a number of digestive organs including the stomach and pancreas. The stomach is involved in the digestion and short term storage of food while the pancreas is a mixed endocrine and exocrine gland which provides the body with hormones and enzymes essential for nutritional utilisation. The pancreas consists of three different cell lineages, acinar, ductal and endocrine cells. The endocrine cells, organised in the islets of Langerhans, are scattered throughout the exocrine parenchyma and regulate blood glucose levels by production of hormones such as glucagon and insulin. The Nkx family of homeodomain proteins controls numerous processes during development. Previous studies have identified two members belonging to the Nkx6 subfamily of Nkx proteins, Nkx6.1 and Nkx6.2. We have described the cloning and embryonic expression pattern of Nkx6.3. All three members of the Nkx6 gene family were shown to be expressed in partially overlapping domains during the development of the gastrointestinal tract and the central nervous system. Nkx6.2 was also identified as a transient marker for pancreatic exocrine cells. Analysing gene expression patterns and morphological features in tissues and organs is often performed by stereologic sampling which is a labour-intensive two dimensional approach that rely on certain assumptions when calculating e.g. β-cell mass and islet number in the pancreas. By combined improvements in immunohistochemical protocols, computational processing and tomographic scanning, we have developed a methodology based on optical projection tomography (OPT) allowing for 3D visualisation and quantification of specifically labelled objects within intact adult mouse organs. In the pancreas, this technique allows for spatial and quantitative measurements of total islet number and β-cell mass. We have further developed a protocol allowing for high resolution regional analyses based on global OPT assessments of the pancreatic constitution. This methodology is likely to facilitate detailed cellular and molecular analysis of user defined regions of interest in the pancreas, at the same time providing information on the overall disease state of the gland. Type 1 diabetes mellitus (T1D) can occur at any age and is characterized by the marked inability of the pancreas to secrete insulin due to an autoimmune destruction of the insulin producing β-cells. Information on the key cellular and molecular events underlying the recruitment of lymphocytes, their infiltration of the islets of Langerhans and consequent β-cell destruction is essential for understanding the pathogenesis of T1D. Using the developed methodology we have recorded the spatial and quantitative distribution of islet β-cells and infiltrating lymphocytes in the non obese diabetic (NOD) mouse model for T1D. This study shows that the smaller islets, which are predominantly organised in the periphery of the organ, are the first to disappear during the progression of T1D. The larger islets appear more resistant and our data suggest that a compensatory proliferative process is going on side by side with the autoimmune-induced β-cell destruction. Further, the formation of structures resembling tertiary lymphoid organs (TLOs) in areas apparently unaffected by insulitis suggests that local factors may provide cues for the homing of these lymphocytes back to the pancreas.

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.

A major goal in biological imaging is to visualize interactions of different tissues, often fluorescently labeled, during dynamic processes. Only a few of these labels fit into the available spectral range without overlap, but can be separated computationally if the full spectrum of every single pixel is known. In medical imaging, hyperspectral techniques show promise to identify different tissue types without any staining. Yet, microscopists still commonly acquire spectral information either with filters, thus integrating over a few broad bands only, or point-wise, dispersing the spectra onto a multichannel detector, which is inherently slow. Light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) are two techniques to acquire 3D microscopic data fast, photon-efficiently and gently on the specimen. LSFM works in fluorescence mode and OPT in transmission. Both are based on a fast widefield detection scheme where a 2D detector records the spatial information but leaves no room to acquire dispersed spectra. Hyperspectral imaging had not yet been demonstrated for either technique. In this work, I developed a line-scanning hyperspectral LSFM and an excitation scanning OPT to acquire 5D data (3D spatial, 1D temporal, 1D spectral) and optimized the performance of both setups to minimize acquisition times without sacrificing image contrast, spatial or spectral information. I implemented and assessed different evaluation pipelines to classify and unmix relevant features. I demonstrate the efficiency of my workflow by acquiring up to five fluorescent markers and the autofluorescence in \\zf and fruit fly embryos on my hyperspectral LSFM. I extracted both concentration maps and spectra for each of these fluorophores from the multidimensional data. The same methods were applied to investigate the transmission data from my spectral OPT, where I found evidence that OPT image formation is governed by refraction, whereas scattering and absorption only play a minor role. Furthermore, I have implemented a robust, educational LSFM on which laymen have explored the working principles of modern microscopies. This eduSPIM has been on display in the Technische Sammlungen Dresden for one year during the UNESCO international year of light. / Ein wichtiges Ziel biologischer Bildgebung ist die Visualisierung des Zusammenspiels von verschiedenen, meist fluoreszent markierten, Geweben bei dynamischen Prozessen. Nur wenige dieser Farbstoffe passen ohne Überlapp in das zur Verfügung stehende Spektrum. Sie können jedoch rechnerisch getrennt werden, wenn das gesamte Spektrum jedes Pixels bekannt ist. In medizinischen Anwendungen versprechen hyperspektrale Techniken, verschiedene Gewebetypen markierungsfrei zu identifizieren. Dennoch ist es in der Mikroskopie noch immer üblich, spektrale Information entweder mit Filtern über breiten Bändern zu integrieren, oder Punktspektren mithilfe von Dispersion zu trennen und auf einem Multikanaldetektor aufzunehmen, was inhärent langsam ist. Light Sheet Fluorescence Microscopy (LSFM) und Optical Projection Tomography (OPT) nehmen 3D Mikroskopiedaten schnell, photoneneffizient und sanft für die Probe auf. LSFM arbeitet mit Fluoreszenz, OPT in Transmission. Beide basieren auf schneller Weitfelddetektion, wobei die räumliche Information mit einem 2D Detektor aufgenommen wird, der keinen Raum lässt, um die getrennten Spektren zu messen. Hyperspektrale Bildgebung wurde bis jetzt für keine der zwei Techniken gezeigt. Ich habe ein hyperspektrales LSFM mit Linienabtastung und ein OPT mit Wellenlängenabtastung entwickelt, um 5D Daten (3D räumlich, 1D zeitlich, 1D spektral) aufzunehmen. Beide Aufbauten wurden hinsichtlich minimaler Aufnahmezeit optimiert, ohne dabei Kontrast, räumliche oder spektrale Auflösung zu opfern. Ich habe verschiedene Abläufe zum Klassifizieren und Trennen der Hauptkomponenten implementiert. Ich nehme bis zu fünf Fluorophore und Autofluoreszenz in Zebrafisch- und Fruchtfliegenembryos mit dem hyperspektralen LSFM auf und zeige die Effizienz des gesamten Ablaufes, indem ich Spektren und räumliche Verteilung aller Marker extrahiere. Die Transmissionsdaten des spektralen OPT werden mit denselben Methoden untersucht. Ich konnte belegen, dass die Bildformation im OPT massgeblich von Brechung bestimmt ist, und Streuung und Absorption nur einen geringen Beitrag leisten. Außerdem habe ich ein robustes, didaktisches LSFM gebaut, damit Laien die Funktionsweise moderner Mikroskopie erkunden können. Dieses eduSPIM war ein Jahr lang in den Technischen Sammlungen Dresden ausgestellt.

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.