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 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method 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 mouse brain tissue.Comment: 15 pages, 7 figure

Efficient reduction of speckle noise in Optical Coherence Tomography

Speckle pattern, which is inherent in coherence imaging, influences significantly axial and transversal resolution of Optical Coherence Tomography (OCT) instruments. The well known speckle removal techniques are either sensitive to sample motion, require sophisticated and expensive sample tracking systems, or involve sophisticated numerical procedures. As a result, their applicability to in vivo real-time imaging is limited. In this work, we propose to average multiple A-scans collected in a fully controlled way to reduce the speckle contrast. This procedure involves non-coherent averaging of OCT A-scans acquired from adjacent locations on the sample. The technique exploits scanning protocol with fast beam deflection in the direction perpendicular to lateral dimension of the cross-sectional image. Such scanning protocol reduces the time interval between A-scans to be averaged to the repetition time of the acquisition system. Consequently, the averaging algorithm is immune to bulk motion of an investigated sample, does not require any sophisticated data processing to align cross-sectional images, and allows for precise control of lateral shift of the scanning beam on the object. The technique is tested with standard Spectral OCT system with an extra resonant scanner used for rapid beam deflection in the lateral direction. Ultrahigh speed CMOS camera serves as a detector and acquires 200,000 spectra per second. A dedicated A-scan generation algorithm allows for real-time display of images with reduced speckle contrast at 6 frames/second. This technique is applied to in vivo imaging of anterior and posterior segments of the human eye and human skin

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

Label-free three-dimensional imaging of Caenorhabditis elegans with visible optical coherence microscopy

Fast, label-free, high-resolution, three-dimensional imaging platforms are crucial for high-throughput in vivo time-lapse studies of the anatomy of Caenorhabditis elegans, one of the most commonly used model organisms in biomedical research. Despite the needs, methods combining all these characteristics have been lacking. Here, we present label-free imaging of live Caenorhabditis elegans with three-dimensional sub-micrometer resolution using visible optical coherence microscopy (visOCM). visOCM is a versatile optical imaging method which we introduced recently for tomography of cell cultures and tissue samples. Our method is based on Fourier domain optical coherence tomography, an interferometric technique that provides three-dimensional images with high sensitivity, high acquisition rate and micrometer-scale resolution. By operating in the visible wavelength range and using a high NA objective, visOCM attains lateral and axial resolutions below 1 μm. Additionally, we use a Bessel illumination offering an extended depth of field of approximately 40 μm.We demonstrate that visOCM’s imaging properties allow rapid imaging of full sized living Caenorhabditis elegans down to the sub-cellular level. Our system opens the door to many applications such as the study of phenotypic changes related to developmental or ageing processes

Optical Coherence Correlation Spectroscopy (OCCS)

A classical technique to monitor dynamical processes at the single-molecule level is fluorescence correlation spectroscopy (FCS). However, FCS requires fluorescent labels that are typically limited by photobleaching and saturation. We present a new method, optical coherence correlation spectroscopy (OCCS), based on noble-metal nanoparticles that overcome those photobleaching and saturation limitations. OCCS is a correlation spectroscopy technique based on dark-field optical coherence microscopy (dfOCM), a Fourier domain optical coherence microscopy technique. OCCS is based on the amplified backscattered light caused by diffusing nanoparticles. Due to the interferometric principle of OCCS, several sampling volumes along the optical axis are measured simultaneously with high detection sensitivity. This adds the possibility to assess axial flow, which is similar to a lateral flow measurement in dual-focus fluorescence correlation. Using a mode-locked Ti:Sapphire laser (780nm central wavelength) we performed experiments with nanoparticles down to 30nm in diameter. We present these first experimental results and an associated theoretical fit model allowing the extraction of the particles’ concentrations and diffusion parameters. The experimental determination of the diffusion time and concentration of gold nanoparticles based on this method is presented as a proof of principle and shows the potential of this technique. In the near future, we aim at investigating smaller gold nanoparticles assessing biological phenomena. As a first application we apply this method to membrane receptor interaction using functionalized nanoparticles

Statistical parametric mapping of stimuli evoked changes in total blood flow velocity in the mouse cortex obtained with extended-focus optical coherence microscopy

Functional magnetic resonance (fMRI) imaging is the current gold-standard in neuroimaging. fMRI exploits local changes in blood oxygenation to map neuronal activity over the entire brain. However, its spatial resolution is currently limited to a few hundreds of microns. Here we use extended-focus optical coherence microscopy (xfOCM) to quantitatively measure changes in blood flow velocity during functional hyperaemia at high spatio-temporal resolution in the somatosensory cortex of mice. As optical coherence microscopy acquires hundreds of depth slices simultaneously, blood flow velocity measurements can be performed over several vessels in parallel. We present the proof-of-principle of an optimised statistical parametric mapping framework to analyse quantitative blood flow timetraces acquired with xfOCM using the general linear model. We demonstrate the feasibility of generating maps of cortical hemodynamic reactivity at the capillary level with optical coherence microscopy. To validate our method, we exploited 3 stimulation paradigms, covering different temporal dynamics and stimulated limbs, and demonstrated its repeatability over 2 trials, separated by a week

Statistical parametric mapping of stimuli evoked changes in total blood flow velocity in the mouse cortex obtained with extended-focus optical coherence microscopy

Functional magnetic resonance (fMRI) imaging is the current gold-standard in neuroimaging. fMRI exploits local changes in blood oxygenation to map neuronal activity over the entire brain. However, its spatial resolution is currently limited to a few hundreds of microns. Here we use extended-focus optical coherence microscopy (xfOCM) to quantitatively measure changes in blood flow velocity during functional hyperaemia at high spatio-temporal resolution in the somatosensory cortex of mice. As optical coherence microscopy acquires hundreds of depth slices simultaneously, blood flow velocity measurements can be performed over several vessels in parallel. We present the proof-of-principle of an optimised statistical parametric mapping framework to analyse quantitative blood flow timetraces acquired with xfOCM using the general linear model. We demonstrate the feasibility of generating maps of cortical hemodynamic reactivity at the capillary level with optical coherence microscopy. To validate our method, we exploited 3 stimulation paradigms, covering different temporal dynamics and stimulated limbs, and demonstrated its repeatability over 2 trials, separated by a week

Imaging of the stroke-related changes in the vascular system of the mouse brain with the use of extended focus Optical Coherence Microscopy

We used Optical Coherence Microscopy (OCM) to monitor structural and functional changes due to ischemic stroke in small animals brains in vivo. To obtain lateral resolution of 2.2 μm over the range of 600 μm we used extended focus configuration of OCM instrument involving Bessel beam. It provided access to detailed 3D information about the changes in brain vascular system up to the level of capillaries across I and II/III layers of neocortex. We used photothrombotic stroke model involving photoactive application of rose bengal to assure minimal invasiveness of the procedure and precise localization of the clot distribution center. We present the comparative analysis involving structural and angiographic maps of the stroke-affected brain enabling in-depth insight to the process of development of the disorder

In vivo high-resolution cortical imaging with extended-focus optical coherence microscopy in the visible-NIR wavelength range

Visible light optical coherence tomography has shown great interest in recent years for spectroscopic and high-resolution retinal and cerebral imaging. Here, we present an extended-focus optical coherence microscopy system operating from the visible to the near-infrared wavelength range for high axial and lateral resolution imaging of cortical structures in vivo. The system exploits an ultrabroad illumination spectrum centered in the visible wavelength range (λc  =  650  nm, Δλ  ∼  250  nm) offering a submicron axial resolution (∼0.85  μm in water) and an extended-focus configuration providing a high lateral resolution of ∼1.4  μm maintained over ∼150  μm in depth in water. The system’s axial and lateral resolution are first characterized using phantoms, and its imaging performance is then demonstrated by imaging the vasculature, myelinated axons, and neuronal cells in the first layers of the somatosensory cortex of mice in vivo.status: publishe