Christophe Pache

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.

Dark-field optical coherence microscopy

Many solutions have been proposed to produce phase quantitative images of biological cell samples. Among these, Spectral Domain Phase Microscopy combines the fast imaging speed and high sensitivity of Optical Coherence Microscopy (OCM) in the Fourier domain with the high phase stability of common-path interferometry. We report on a new illumination scheme for OCM that enhances the sensitivity for backscattered light and detects the weak sample signal, otherwise buried by the signal from specular reflection. With the use of a Bessel-like beam, a dark-field configuration was realized. Sensitivity measurements for three different illumination configurations were performed to compare our method to standard OCM and extended focus OCM. Using a well-defined scattering and reflecting object, we demonstrated an attenuation of -40 dB of the DC-component and a relative gain of 30 dB for scattered light, compared to standard OCM. In a second step, we applied this technique, referred to as dark-field Optical Coherence Microscopy (dfOCM), to living cells. Chinese hamster ovarian cells were applied in a drop of medium on a coverslide. The cells of ~15 μm in diameter and even internal cell structures were visualized in the acquired tomograms.

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.

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.

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.

Coherent transfer functions and extended depth of field

To preserve the speed advantage of Fourier Domain detection in Optical Coherence Microscopy (OCM), extended depth of field (DOF) is needed. To assess and improve the DOF and the lateral resolution, we analyzed the coherent transfer function (CTF) of OCM. In the spectral domain detection, each wavelength has its own specific CTF, sampling a different part of the objects spatial frequency spectrum. For classical optics and increasing numerical apertures these regions start to overlap and bend, which limits the depth of field. Bessel-like beams produced by axicon lenses circumvent these detrimental effects, but introduce side lobes. Decoupling the detection and the illumination apertures gives more flexibility in engineering a CTF and 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. Using Bessel-like beams as well in the illumination as in the detection paths, but with different side-lobe radii, we obtained a lateral resolution of 2μm invariant over an extended depth of field of more than 300μm, at a signal penalty of only 12dB compared to classical Gaussian optics.

Measurement of retinal physiology using functional Fourier domain OCT concepts

Fourier Domain OCT proved to be an outstanding tool for measuring 3D retinal structures with high sensitivity, resolution, and speed. We extended the FDOCT concept towards functional imaging by analyzing the spectroscopic tissue properties, polarization contrast and Doppler velocity imaging. Differential spectral contrast FDOCT allows optical staining of retinal tomograms and to contrast tissue of high pigmentation such as the retinal pigment epithelium (RPE). The latter shows strong correlation if compared to polarization sensitive OCT images. First implementations of Doppler FDOCT systems demonstrated the capability of measuring in-vivo retinal blood flow profiles and pulsatility. We developed a new concept of Doppler FDOCT that allows measuring also large flow velocities typically close to the optic nerve head. Studies of retinal perfusion based on Laser Doppler Flowmetry (LDF) demonstrated the high sensitivity of blood flow to external stimuli. We performed first experiments of studying retinal perfusion in response to flicker stimulation. An increase in vessel diameter by 11% and of flow velocity by 49% was measured. We believe that a multi-modal functional imaging concept is of high value for an accurate and early diagnosis and understanding of retinal pathologies and pathogenesis.

Dual-mode digital holographic and fluorescence microscopy for the study of morphological changes in cells under simulated microgravity

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.