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.

Optical coherence correlation spectroscopy (OCCS).

We present a new method called optical coherence correlation spectroscopy (OCCS) using nanoparticles as reporters of kinetic processes at the single particle level. OCCS is a spectral interferometry based method, thus giving simultaneous access to several sampling volumes along the optical axis. Based on an auto-correlation analysis, we extract the diffusion coefficients and concentrations of nanoparticles over a large concentration range. The cross-correlation analysis between adjacent sampling volumes allows to measure flow parameters. This shows the potential of OCCS for spatially resolved diffusion and flow measurements.

Fast three-dimensional imaging of gold nanoparticles in living cells with photothermal optical lock-in Optical Coherence Microscopy.

We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 µm lateral and 2 µm axial resolution over a 50 µm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.

Label-free imaging of cerebral ß-amyloidosis with extended-focus optical coherence microscopy.

We demonstrate label-free imaging of cerebral ß-amyloidosis ex vivo and in a living mouse model of Alzheimer's disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual ß-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of ß-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.

Diabetes imaging-quantitative assessment of islets of Langerhans distribution in murine pancreas using extended-focus optical coherence microscopy.

Diabetes is characterized by hyperglycemia that can result from the loss of pancreatic insulin secreting ß-cells in the islets of Langerhans. We analyzed ex vivo the entire gastric and duodenal lobes of a murine pancreas using extended-focus Optical Coherence Microscopy (xfOCM). To identify and quantify the islets of Langerhans observed in xfOCM tomograms we implemented an active contour algorithm based on the level set method. We show that xfOCM reveals a three-dimensional islet distribution consistent with Optical Projection Tomography, albeit with a higher resolution that also enables the detection of the smallest islets (≤ 8000 µm(3)). Although this category of the smallest islets represents only a negligible volume compared to the total ß-cell volume, a recent study suggests that these islets, located at the periphery, are the first to be destroyed when type I diabetes develops. Our results underline the capability of xfOCM to contribute to the understanding of the development of diabetes, especially when considering islet volume distribution instead of the total ß-cell volume only.

Dark-field optical coherence microscopy.

Dark-field illumination is known to enhance scattering contrast in optical microscopy. We combined this concept with Fourier domain optical coherence microscopy (OCM). The detection and illumination paths are decoupled, and only the scattered light originating from the sample generates the tomogram signal, whereas any specular reflection is highly suppressed. We analyze and discuss this dark-field OCM concept and present its superior imaging quality on live cell samples.

Digital holographic microscopy real-time monitoring of cytoarchitectural alterations during simulated microgravity.

Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.