In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

Aims/hypothesis Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. Methods Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. Results xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. Conclusions/interpretation Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.

In vivo imaging of pancreatic endocrine islets

Michael Friedrich, Joan Goulley, Anne Grapin-Botton, Theo Lasser, Rainer Leitgeb, Christophe Pache, Martin Villiger

Extended focus optical coherence microscope (xfOCM) circumvents the compromise between lateral resolution and depth of field by us of a Bessel-like illumination beam. The high sensitivity and parallel depth profiling of Fourier domain optical coherence tomography are preserved, and combined with high isotropic resolution of 1.5 - 2 μm. To comply with the requirements for in vivo measurements, beam scanning had to be implemented. We then performed measurements, first of excised pancreas, validated by standard immunohistochemistry, to investigate the structures that can be observed. For a quantitative analysis, a semi-automatic islet detection algorithm evaluated the islet size, position, contrast and homogeneity. The influence of streptozotocin on the signature of the islets was investigated in a next step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo, visualizing pancreatic lobules, ducts, blood vessels and individual islets of Langerhans.

SPIE2009

Towards high resolution optical imaging of beta cells in vivo

Joan Goulley, Anne Grapin-Botton, Theo Lasser, Erica Martin-Williams, Martin Villiger

Endocrine beta cells produce and release insulin in order to tightly regulate glucose homeostasis and prevent metabolic pathologies such as Diabetes Mellitus. Optical imaging has contributed greatly to our current understanding of beta cell structure and function. In vitro microscopy of beta cell lines has revealed the localization of molecular components in the cell and more recently their dynamic behavior. In cultured islets, interactions of beta cells with other islet cells and the matrix as well as paracrine and autocrine signaling or reaction to nutrients have been studied. Lastly, microscopy has been performed on tissue sections, visualizing the islets in an environment closer to their natural surroundings. In most efforts to date, the samples have been isolated for investigation and hence have by definition been divorced from their natural environments and deprived of vascularization and innervations. In such a setting the beta cells lack the metabolic information that is primordial to their basic function of maintaining glucose homeostasis. We review optical microscopy; its general principles, its impact in decoding beta cell function and its recent developments towards the more physiologically relevant assessment of beta cell function within the environment of the whole organism. This requires both large imaging depth and fast acquisition times. Only few methods can achieve an adequate compromise. We present extended focus Optical Coherence Microscopy (xfOCM) as a valuable alternative to both confocal microscopy and two photon microscopy (2PM), and discuss its potential in interpreting the mechanisms underlying glucose homeostasis and monitoring impaired islet function.

Bentham Science Publishers2010

In Vivo extended focus Optical Coherence Microscopy of endocrine islets of Langerhans in mice

Michael Friedrich, Joan Goulley, Anne Grapin-Botton, Theo Lasser, Rainer Leitgeb, Martin Villiger

Rapid acquisition of volumetric data is a mandatory prerequisite for small animal in vivo imaging. Due to its high sensitivity and capacity to extract the sample depth profile in parallel Fourier Domain Optical Coherence Tomography (FDOCT) complies with this requirement. The absence of labeling further simplifies its use for in vivo application. In order to provide the high spatial resolution necessary to gain biologically relevant insights focusing optics from microscopy are usually employed. The resulting focal spot however has a limited depth of field and compromises with imaging speed and parallel depth extraction. Using a Bessel‐like illumination beam, obtained by use of a conical lens, and detection optics with a low numerical aperture, we achieve a compromise between high lateral resolution and extended depth of field and obtain near isotropic resolution. Together with a scanning system deflecting the beam rapidly in the lateral directions and a fast signal acquisition and processing routine this extended focus Optical Coherence Microscope (xfOCM) was used to measure murine islets of Langerhans. These endocrine islets are assemblies of insulin secreting beta cells contained within the exocrine tissue of the pancreas. Assessing the structure and function of individual islets is important to further the understanding of diabetes’ mechanisms. In this work we used xfOCM to measure islets, pancreatic lobules, ducts and blood vessels, first in excised tissue, and then we demonstrate the potential of this method with volumetric measurements of the pancreatic islets in vivo and in situ.

2009