Arno Bouwens

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 β-amyloidosis with extended-focus optical coherence microscopy

We demonstrate label-free imaging of cerebral β-amyloidosis ex vivo and in a living mouse model of Alzheimers disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual β-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 β-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.

Quantitative lateral and axial flow imaging with optical coherence microscopy and tomography

Optical coherence tomography (OCT) and optical coherence microscopy (OCM) allow the acquisition of quantitative three-dimensional axial flow by estimating the Doppler shift caused by moving scatterers. Measuring the velocity of red blood cells is currently the principal application of these methods. In many biological tissues, blood flow is often perpendicular to the optical axis, creating the need for a quantitative measurement of lateral flow. Previous work has shown that lateral flow can be measured from the Doppler bandwidth, albeit only for simplified optical systems. In this work, we present a generalized model to analyze the influence of relevant OCT/OCM system parameters such as light source spectrum, numerical aperture and beam geometry on the Doppler spectrum. Our analysis results in a general framework relating the mean and variance of the Doppler frequency to the axial and lateral flow velocity components. Based on this model, we present an optimized acquisition protocol and algorithm to reconstruct quantitative measurements of lateral and axial flow from the Doppler spectrum for any given OCT/OCM system. To validate this approach, Doppler spectrum analysis is employed to quantitatively measure flow in a capillary with both extended focus OCM and OCT.

Quantitative cerebral blood flow imaging with extended-focus optical coherence microscopy

Quantitative three-dimensional blood flow imaging is a valuable technique to investigate the physiology of the brain. Two-photon microscopy (2PM) allows quantification of the local blood flow velocity with micrometric resolution by performing repeated line scans, but prohibitively long measurement times would be required to apply this technique to full three-dimensional volumes. By multiplexing the image acquisition over depth, Fourier domain optical coherence tomography (FDOCT) enables quantification of blood flow velocities with a high volume acquisition rate, albeit at a relatively low spatial resolution. Extended-focus optical coherence microscopy (xfOCM) increases the lateral resolution without sacrificing depth of field and therefore combines the high volume acquisition rate of FDOCT with a resolution comparable to 2PM. Here, we demonstrate high-resolution quantitative imaging of the blood flow velocity vectors magnitude in the adult murine brain with xfOCM.

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 β-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 μm3). Although this category of the smallest islets represents only a negligible volume compared to the total β-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 β-cell volume only.

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.

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging.

Aims/hypothesisIt is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation.MethodsNOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation.ResultsWe demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin–zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet.Conclusions/interpretationFOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.

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.

Visible light optical coherence correlation spectroscopy.

Optical coherence correlation spectroscopy (OCCS) allows studying kinetic processes at the single particle level using the backscattered light of nanoparticles. We extend the possibilities of this technique by increasing its signal-to-noise ratio by a factor of more than 25 and by generalizing the method to solutions containing multiple nanoparticle species. We applied these improvements by measuring protein adsorption and formation of a protein monolayer on superparamagnetic iron oxide nanoparticles under physiological conditions.

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 μm axial and 0.4 μm lateral resolution maintained over a depth of 40 μm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.