| 2. |
- Hahn, Max, 1993-, et al.
(author)
-
3D optical molecular imaging of the rodent pancreas by OPT and LSFM
- 2023
-
In: Type-1 diabetes. - New York : Humana Press. - 9781071628065 - 9781071628096 - 9781071628072 ; , s. 1-19
-
Book chapter (peer-reviewed)abstract
- The rodent pancreas is the prevalent model system for preclinical diabetes research. However, due to the compound endocrine–exocrine organization of the gland, with the endocrine islets of Langerhans scattered by the thousands throughout the much greater exocrine parenchyma, stereological assessments of endocrine cell mass, commonly insulin-producing ß-cells, are exceedingly challenging. In recent years, optical mesoscopic imaging techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) have seen dramatic developments, enabling 3D visualization of fluorescently labeled cells in mm- to cm-sized tissues with μm resolution. Here we present a protocol for 3D visualization and “absolute” quantitative assessments of, for example, islet mass throughout the volume of rodent pancreata with maintained spatial context.
|
|
| 3. |
- Hahn, Max, 1993-
(author)
-
Characterizing the pancreatic "isletome" : 3D optical imaging to study diabetes
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- The pancreas is a specialised multipurpose organ, that can be separated into two major compartments: endocrine and exocrine. The exocrine part makes up the majority of the organ volume and functions to secrete digestive enzymes into the small intestine. Notably, endocrine islets of Langerhans are embedded and scattered in vast numbers throughout the exocrine space. These miniature functional units are composed of different cell types that secrete hormones into the blood stream. The most abundant islet-cell is the insulin-producing β-cell. Highly coordinated, the endocrine cells are the primary regulators of energy homeostasis in the body. Together, the collective islet volume constitutes the pancreatic “isletome”, a synchronised, complex and size-equilibrated system that is able to respond to various metabolic conditions. Indeed, environmental and/or genetic conditions often lead to impaired islet function and/or β-cell destruction leading to elevated blood glucose levels over time and eventually diabetes. Diabetes mellitus is a disease that currently affects more than 400 million individuals worldwide. As such, understanding pancreatic disease-related mechanisms is pivotal to the development of new and more effective therapeutic, or even curative, regimens. The deep location of the pancreas in the abdomen and the relatively low resolution of current clinical imaging approaches, however, render the pancreatic islets difficult to study when visually assessing endocrine function. Although non-invasive imaging techniques have yet to reach their full potential, post-mortem studies of the pancreas and rodent disease models offer unique insights into the process of diabetes disease dynamics.Diabetes induced by streptozotocin (STZ) is a widely used model system in pre-clinical research, where it is generally believed that the b-cells are depleted upon the administration of the drug. Yet, quantification of β-cell volume dynamics and underlying disease mechanisms have not been extensively described. Using optical projection tomography (OPT), light sheet fluorescence microscopy (LSFM) and advanced protocols for ex vivo whole organ three-dimensional (3D) imaging, this study demonstrated that STZ-induced β-cell depletion is modest, primarily affecting large islets, and is not the primary cause for the development of diabetes in STZ-diabetic mice. Combined with islet gene expression studies, the remaining β-cell volume in STZ-diabetic mice displayed a downregulation of glucose transporter type 2 (GLUT2), a transmembrane carrier vital for sensing blood glucose levels. Islet transplantation into the anterior chamber of the eye (ACE) reversed the STZ-induced hyperglycaemia and partially restored islet function, including GLUT2, but did not restore β-cell volume loss. Extensive 3D image datasets were generated as a resource to the research community. The combined results of this study indicated that STZ-induced hyperglycaemia is not caused by β-cell loss, but rather by dysfunctional β-cells and that recovery of islet function is restrained by continuous hyperglycaemia.3D imaging using OPT has proven to be a reliable technique in quantifying cellular/anatomical features of the mouse pancreas. However, the technique has rarely been applied to patient-derived tissues. Here, a label-free and non-destructive method was developed to assess clinical biopsies within hours of collection. Specifically, this study showed that autofluorescence-based imaging can be used to delineate tumours of the pancreas (pancreatic ductal adenocarcinoma, PDAC) in 3D, which may aid in identifying tumour margins in conjunction with resective surgery. Importantly, the protocol included a reversal pipeline so that other histological workflows could be applied to the same specimen. Furthermore, this study demonstrated that natural fluorescent substances in the endocrine cells provide sufficient contrast when quantifying both the volume and number of islets of Langerhans in the healthy pancreas. Altogether, the developed technique may provide a novel tool for the rapid 3D analysis of pancreatic biopsies that may complement and improve traditional pathological assessments.With the emergence of islet transplantation networks worldwide, access to fixed pancreatic tissues from diseased donors has dramatically improved. Hereby, the near instant autolysis of the pancreas post-mortem can generally be avoided, which provides the opportunity to quantitatively study the entire gland ex vivo within a conserved spatial context. Yet, mesoscopic 3D imaging of the pancreas (by OPT and/or LSFM) has been limited predominantly due to the obstacle of labelling larger tissue volumes. As such, a simple approach to antibody labelling and cellular imaging was developed in cubic centimetre-sized tissue cuboids that were mapped to the whole organ. By stitching the resultant datasets back into 3D space, this approach demonstrated how essentially any human organ may be analysed in full with high resolution. This technique was applied to pancreata from non-diabetic and type 2 diabetic (T2D) donors, analysing over 200 thousand islets, revealing features of the human pancreas that were not analysed in 3D previously, including high islet dense regions and intra-islet haemorrhaging. Crucially, this new technique may contribute to unveil a wealth of new insights into the complex pathophysiology of the “diabetic pancreas”.By applying the above method to the entire volume of the human pancreas, the absolute distribution and volume of insulin-positive cells in a pancreas from a donor with longstanding type 1 diabetes (T1D) was demonstrated for the first time. By dividing the 19 cm long organ into smaller pieces, followed by insulin labelling, OPT imaging and reconstruction in 3D space, approximately 173,000 insulin-positive objects were identified. By utilising tissue autofluorescence, the entire organ was reconstructed in 3D, together with blood vessels and ducts. These data indicated several important regional differences in β-cell mass, such as the uncinate process showing the highest density, which potentially reflects key aspects of disease dynamics. Furthermore, regions with a “punctated distribution” of single β-cells in close proximity to each other were identified. Although the significance of these observations needs to be elucidated, we speculate that these regions could be associated with pancreatic regeneration, which might permit the development of new interventions for clinical regenerative processes in the future. Altogether, this study represents the first whole organ account of β-cell distribution at the current level of resolution in an entire organ. As such, it may serve as an important advancement towards detailed whole organ analyses of endocrine cell identity/function, via a wide range of markers, in the study of normal anatomy and pathophysiology of the human pancreas.
|
|
| 5. |
- Willekens, Stefanie M. A., et al.
(author)
-
An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience
- 2024
-
In: Frontiers in Neuroscience. - : Frontiers Media S.A.. - 1662-4548 .- 1662-453X. ; 18
-
Journal article (peer-reviewed)abstract
- Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited.Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images.Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR.Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.
|
|
