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Residual β cell function can positively affect diabetes regulation in type 1 diabetes (T1D), but details on residual β cell mass distribution in T1D is largely lacking in a whole organ context. Implementing an optical 3D imaging pipeline, we generated a complete account of the remaining β cells throughout an entire human late onset T1D pancreas at a microscopic resolution. Our data show that most of the residual β cells were present as scattered individual cells or as punctated clusters of individual β cells, spatially separated from each other and all other endocrine cell types. Compared to islet-associated β cells, extra-islet β cells appeared in a substantially higher relative abundance in the head region of the T1D pancreas. This 3D depiction of an entire T1D pancreas shows that individual β cells may be preserved and/or formed in a highly regionalized manner, potentially reflecting key aspects of disease dynamics, advocating for increased focus on extra-islet islet β cells in attempts to develop strategies for pancreatic β cell preservation in T1D.

Although major advances have been made in the field of mesoscopic imaging and associated tissue clearing protocols, these applications are greatly challenged when applied to imaging of pancreatic ductal adenocarcinoma (PDAC) tissue. Most importantly, penetration of labelling agents, typically antibodies, can be drastically reduced from the characteristically dense PDAC stroma. To circumvent this issue, we present a method by which machine learning assisted segmentation is applied to resolve the 3D PDAC microarchitecture from autofluorescence (AF) based light-sheet fluorescence microscopy (LSFM) scans. Hereby, PDAC tissue features could be studied in 3D space without the need for labelling or sectioning. In this proof of principle study, we applied this imaging pipeline on surgical specimens from five PDAC patients and normal pancreatic tissue, generating mosaics of cm3-sized tissue discs at micrometre resolution, each on scanning depths corresponding to thousands of standard pathological 2D tissue sections. Using this method, we generated 3D volumes for quantification of blood vasculature, neoplastic epithelium, islets of Langerhans and stromal components. We further showcase the potential for downstream 2D histochemical and immunohistochemical analysis of scanned specimen. As such, the method may facilitate studies of metastatic routes, vessel microarchitecture, islet phenotypes, and spatial relationships in the PDAC tumour microenvironment.

To facilitate the understanding of how a complex organ such as the pancreas is formed, this chapter illustrates the general anatomical dynamics of pancreas morphogenesis that occur during development in mice (and in humans where relevant). By applying recent advances in optical imaging techniques, including optical projection tomography and light sheet fluorescence microscopy (LSFM), this chapter presents a full image series demonstrating pancreatic bud formation and growth, as well as key morphological events that result in murine and human organs that are anatomically quite different. Further, it is now well established that pancreas development is governed by complex gene regulatory networks, where the timing and duration of gene expression, as well as the degree of molecular interactions are critical. Where appropriate, these key molecular determinants in inductive processes or other events are discussed in relation to pancreas organogenesis. Finally, this chapter describes the spatial and quantitative distribution of insulin as an example of pancreatic endocrine structure-function relationships, where lobular islet heterogeneity in the adult pancreata of mice and humans are evaluated and discussed.

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research.

The possibility to quantitatively study specific molecular/cellular features of complete human organs with preserved spatial 3D context would have widespread implications for pre-clinical and clinical medicine. Whereas optical 3D imaging approaches have experienced a formidable revolution, they have remained limited due to current incapacities in obtaining specific labelling within large tissue volumes. We present a simple approach enabling reconstruction of antibody labeled cells within entire human organs with preserved organ context. We demonstrate the utility of the approach by providing volumetric data and 3D distribution of hundreds of thousands of islets of Langerhans within the human pancreas. By assessments of pancreata from non-diabetic and type 2 diabetic individuals, we display previously unrecognized features of the human islet mass distribution and pathology. As such, this method may contribute not only in unraveling new information of the pancreatic anatomy/pathophysiology, but it may be translated to essentially any antibody marker or organ system.

The exocrine-endocrine multipart organization of the pancreas makes it an exceedingly challenging organ to analyze, quantitatively and spatially. Both in rodents and humans, estimates of the pancreatic cellular composition, including beta-cell mass, has been largely relying on the extrapolation of 2D stereological data originating from limited sample volumes. Alternatively, they have been obtained by low resolution non-invasive imaging techniques providing little detail regarding the anatomical organization of the pancreas and its cellular and/or molecular make up. In this mini-review, the state of the art and the future potential of currently existing and emerging high-resolution optical imaging techniques working in the mm-cm range with μm resolution, here referred to as mesoscopic imaging approaches, will be discussed regarding their contribution toward a better understanding of pancreatic anatomy both in normal conditions and in the diabetic setting. In particular, optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) imaging of the pancreas and their associated tissue processing and computational analysis protocols will be discussed in the light of their current capabilities and future potential to obtain more detailed 3D-spatial, quantitative, and molecular information of the pancreas.

The possibility to assess pancreatic anatomy with microscopic resolution in three dimensions (3D) would significantly add to pathological analyses of disease processes. Pancreatic ductal adenocarcinoma (PDAC) has a bleak prognosis with over 90% of the patients dying within 5 years after diagnosis. Cure can be achieved by surgical resection, but the efficiency remains drearily low. Here we demonstrate a method that without prior immunohistochemical labelling provides insight into the 3D microenvironment and spread of PDAC and premalignant cysts in intact surgical biopsies. The method is based solely on the autofluorescent properties of the investigated tissues using optical projection tomography and/or light-sheet fluorescence microscopy. It does not interfere with subsequent histopathological analysis and may facilitate identification of tumor-free resection margins within hours. We further demonstrate how the developed approach can be used to assess individual volumes and numbers of the islets of Langerhans in unprecedently large biopsies of human pancreatic tissue, thus providing a new means by which remaining islet mass may be assessed in settings of diabetes. Generally, the method may provide a fast approach to provide new anatomical insight into pancreatic pathophysiology.

Somapacitan is a long-acting, once-weekly, albumin-binding growth hormone (GH) derivative. The reversible albumin-binding properties leads to prolonged circulation half-life. Here, we investigated and compared somapacitan with human GH on downstream receptor signaling in primary hepatocytes and hepatocellular models and using isothermal titration calorimetry to characterize receptor binding of somapacitan in the presence or absence of human serum albumin (HSA). With non-invasive fluorescence imaging we quantitatively visualize and compare the temporal distribution and examine the tissue-specific growth hormone receptor (GHR) activation at distribution sites. We found that signaling kinetics were slightly more rapid and intense for GH compared with somapacitan. Receptor binding isotherms were characterized by a high and a low affinity interaction site with or without HSA. Using in vivo optical imaging we found prolonged systemically biodistribution of somapacitan compared with GH, which correlated with plasma pharmacokinetics. Ex vivo mouse organ analysis revealed that the temporal fluorescent intensity in livers dosed with somapacitan was significantly increased compared with GH-dosed livers and correlated with the degree of downstream GHR activation. Finally, we show that fluorescent-labeled analogs distributed to the hypertrophic zone in the epiphysis of proximal tibia of hypophysectomized rats and that somapacitan and GH activate the GHR signaling in epiphyseal tissues.

Mouse models of Streptozotocin (STZ) induced diabetes represent the most widely used preclinical diabetes research systems. We applied state of the art optical imaging schemes, spanning from single islet resolution to the whole organ, providing a first longitudinal, 3D-spatial and quantitative account of β-cell mass (BCM) dynamics and islet longevity in STZ-treated mice. We demonstrate that STZ-induced β-cell destruction predominantly affects large islets in the pancreatic core. Further, we show that hyperglycemic STZ-treated mice still harbor a large pool of remaining β-cells but display pancreas-wide downregulation of glucose transporter type 2 (GLUT2). Islet gene expression studies confirmed this downregulation and revealed impaired β-cell maturity. Reversing hyperglycemia by islet transplantation partially restored the expression of markers for islet function, but not BCM. Jointly our results indicate that STZ-induced hyperglycemia results from β-cell dysfunction rather than β-cell ablation and that hyperglycemia in itself sustains a negative feedback loop restraining islet function recovery.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.