Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis.

Structural and Functional Imaging of Pancreatic Islets using Optical Coherence Microscopy

Corinne Berclaz

In diabetes, pancreatic beta-cells play a key role. These cells are organized within structures called islets of Langerhans inside the pancreas and produce insulin. Insulin is one of the main hormones contributing to glucose homeostasis, which is the hallmark of diabetes. In vivo imaging of beta-cells and their function are required for diagnosis purposes and assessment of new treatments. However, this task is challenging due to the localization of the pancreas deep inside the abdominal cavity and the small size and heterogeneous distribution of islets of Langerhans throughout the organ. Indeed, islets of Langerhans represent only 1-2% of the pancreas' total volume. All state-of-the-art medical imaging techniques such as MRI, SPECT or PET cannot resolve individual islets. Furthermore, they require the use of tracers to detect the beta-cells. Hence, the research for imaging islets of Langerhans, including the identification of specific beta-cell tracers is a highly active field of research. Indeed, no marker is currently available for clinical studies and diagnosis. For animal models, optical techniques offer a sufficient spatial resolution to image individual islets with a high specificity provided by fluorescent markers. Optical Coherence Microscopy (OCM) is a novel interferometric imaging technique measuring the back-scattered light from a sample to reveal its structure in depth. OCM provides an intrinsic contrast depending on the spatial variation of the index of refraction. A Fourier transform of the acquired spectrum extracts the whole depth structure, thereby requiring only scans in two dimensions to obtain a three-dimensional image with a fast acquisition time. In this thesis, we exploited the label-free capabilities and fast acquisition rate of OCM to study islets of Langerhans. We demonstrated that OCM signal is specific to the beta-cell volume due to the dominant scattering of the zinc-insulin crystalline structures inside the secretory granules. Besides structural information, OCM reveals the vascularization of pancreatic islets in situ. These advantages were exploited to image the progression of autoimmune diabetes and for the characterization of beta-cell tracers. The intrinsic contrast of OCM does not require genetically modified mice, nor the use of exogenous agents to image islets and their vascularization. OCM enhanced with a confocal fluorescence channel can assess beta-cell tracers labeled with a fluorophore in vitro and in vivo in wild type mice. As a proof of principle, we assessed the specificity of Cy5.5-exendin-3, an analogue of the glucagon-like peptide-1 receptor (GLP1R) for beta-cells. Our results confirmed the co-localization of the fluorescence-tagged tracer with the OCM islet signal. The high resolution of OCM serves as a pre-clinical optical platform to facilitate the initial tests aiming to determine the specificity of beta-cell tracers in vivo in mice. Finally, in order to perform non-invasive longitudinal studies, islets of Langerhans were transplanted into the anterior chamber of the eye. This transplantation model allowed us to follow individual islets over time in a spontaneous mouse model of type I diabetes. We demonstrated that alterations of the islet microvasculature accompany the progression of diabetes with a strong correlation between the degree of insulitis and the density of the vascular network.

EPFL2015

Structural and Functional Imaging of Pancreatic Islets using Optical Coherence Microscopy

Corinne Berclaz

EPFL2015