In vivo imaging of pancreatic endocrine islets

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.

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

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

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.

Springer Verlag2009

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

Coherent transfer functions and extended depth of field

Theo Lasser, Rainer Leitgeb, Christophe Pache, Martin Villiger

To preserve the speed advantage of Fourier Domain detection in Optical Coherence Microscopy (OCM), extended depth of field is needed. With a narrow probing volume that extends over a long axial range, tissue could be measured in vivo and at cellular resolution. To assess and improve the DOF and the lateral resolution, we analyzed the coherent transfer function (CTF) of OCM. Both the illumination and detection optics contribute equally to the overall imaging performance. In the Fourier domain detection, each pixel of the spectrometer has its specific CTF, sampling a different region of the object’s spatial frequency spectrum. For classical optics and increasing numerical apertures these regions start to overlap and bend, which limits the depth of field. Annular apertures, created with Bessel-like beams produced by axicon lenses or phase filters, circumvent these detrimental effects, but introduce strong side lobes. Decoupling the detection and the illumination apertures is needed to provide the flexibility in engineering a CTF that optimizes the lateral resolution and the DOF at the same time all while reducing these side lobes. We evaluated different combinations of Gaussian and Bessel-like illumination and detection optics, both theoretically and experimentally. Using Bessel-like beams as well in the illumination as in the detection paths, but with annular apertures of different lobe radii, we obtained a lateral resolution of 1.3 μm and an extended depth of field of more than 300 μm, which was completely decoupled from the numerical aperture and scalable to high lateral resolution.

The International Society for Optical Engineering Spie-Int Soc Optical Engineering2010