Imaging of cortical structures and microvasculature using extended-focus optical coherence tomography at 1.3 µm.

Extended-focus optical coherence tomography (xf-OCT) is a variant of optical coherence tomography (OCT) wherein the illumination and/or detection modes are engineered to provide a constant diffractionless lateral resolution over an extended depth of field (typically 3 to 10× the Rayleigh range). xf-OCT systems operating at 800 nm have been devised and used in the past to image brain structures at high-resolution in vivo, but are limited to ∼500 µm in penetration depth due to their short illumination wavelength. Here we present an xf-OCT system optimized to an image deeper within the cortex by using a longer illumination central wavelength of 1310 nm. The system offers a lateral resolution of 3 and 6.5 µm, over a depth of 900 µm and >1.5 mm using a 10× and 5× objective, respectively, in air. We characterize the system's resolution using microbeads embedded in PDMS and demonstrate its capabilities by imaging the cortical structure and microvasculature in anesthetized mice to a depth of ∼0.8 mm. Finally, we illustrate the difference in penetration depths obtainable with the new system and an xf-OCT system operating at 800 nm.

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.

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.

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.

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.

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 challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a 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 fluorophore densities and photoactivation or photoswitching kinetics.

Extended-focus optical coherence microscopy for high-resolution imaging of the murine brain.

We propose a new method and optical instrumentation for mouse brain imaging based on extended-focus optical coherence microscopy. This in vivo imaging technique allows the evaluation of the cytoarchitecture at cellular level and the circulation system dynamics in three dimensions. This minimally invasive and non-contact approach is performed without the application of contrasting agents. The optical design achieved a resolution of 2.2 µm over a distance of 800 µm, which was sufficient to obtain a detailed three-dimensional image of a wild-type mouse's brain down to the layer III of the cortex. Intrinsically contrasted microvessels and structures similar to the bodies of neurons were distinguishable.

Label-free fast 3D coherent imaging reveals pancreatic islet micro-vascularization and dynamic blood flow.

In diabetes, pancreatic ß-cells play a key role. These cells are clustered within structures called islets of Langerhans inside the pancreas and produce insulin, which is directly secreted into the blood stream. The dense vascularization of islets of Langerhans is critical for maintaining a proper regulation of blood glucose homeostasis and is known to be affected from the early stage of diabetes. The deep localization of these islets inside the pancreas in the abdominal cavity renders their in vivo visualization a challenging task. A fast label-free imaging method with high spatial resolution is required to study the vascular network of islets of Langerhans. Based on these requirements, we developed a label-free and three-dimensional imaging method for observing islets of Langerhans using extended-focus Fourier domain Optical Coherence Microscopy (xfOCM). In addition to structural imaging, this system provides three-dimensional vascular network imaging and dynamic blood flow information within islets of Langerhans. We propose our method to deepen the understanding of the interconnection between diabetes and the evolution of the islet vascular network.

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

AIMS/HYPOTHESIS: It 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. METHODS: NOD 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. RESULTS: We 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/INTERPRETATION: FOCI 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.

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.

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.

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 vector's magnitude in the adult murine brain with xfOCM.

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.

Multi-parametric imaging of murine brain using spectral and time domain optical coherence tomography.

Examination of brain functions in small animal models may help improve the diagnosis and treatment of neurological conditions. Transcranial imaging of small rodents' brains poses a major challenge for optical microscopy. Another challenge is to reduce the measurement time. We describe methods and algorithms for three-dimensional assessment of blood flow in the brains of small animals, through the intact skull, using spectral and time domain optical coherence tomography. By introducing a resonant scanner to the optical setup of the optical coherence tomography (OCT) system, we have developed and applied a high-speed spectral OCT technique that allows us to vary the imaging range of flow and to shorten measurement time. Multi-parameter signal analysis enables us to obtain both qualitative and quantitative information about flow velocity from the same set of data.

Four-dimensional structural and Doppler optical coherence tomography imaging on graphics processing units.

The authors present the application of graphics processing unit (GPU) programming for real-time three-dimensional (3-D) Fourier domain optical coherence tomography (FdOCT) imaging with implementation of flow visualization algorithms. One of the limitations of FdOCT is data processing time, which is generally longer than data acquisition time. Utilizing additional algorithms, such as Doppler analysis, further increases computation time. The general purpose computing on GPU (GPGPU) has been used successfully for structural OCT imaging, but real-time 3-D imaging of flows has so far not been presented. We have developed software for structural and Doppler OCT processing capable of visualization of two-dimensional (2-D) data (2000 A-scans, 2048 pixels per spectrum) with an image refresh rate higher than 120 Hz. The 3-D imaging of 100×100 A-scans data is performed at a rate of about 9 volumes per second. We describe the software architecture, organization of threads, and optimization. Screen shots recorded during real-time imaging of a flow phantom and the human eye are presented.

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.

Flow velocity estimation by complex ambiguity free joint Spectral and Time domain Optical Coherence Tomography.

We show that recently introduced joint Spectral and Time domain Optical Coherence Tomography (STdOCT) can be used for simultaneous complex ambiguity removal and functional Spectral OCT images. This permits to take advantage of higher sensitivity achievable near the zero-path delay. The technique can be used with all Spectral OCT systems that are equipped with an optical delay line (ODL) and provide oversampled scanning patterns. High sensitivity provided by STdOCT allows this technique to be used in Spectral OCT setups with acquisition speed of 100,000 lines/s. We show that different imaging ranges and velocity ranges can be achieved by switching on/off the ODL and a small modification in the processing algorithm. Additionally, the relatively small computational burden of the technique allows for fast computations in the range of less than 5 minutes for 3D data set. We present application of proposed technique to full-range two- and three-dimensional imaging. Morphological and Doppler tomograms of human retina in-vivo are shown. Finally, we identify and discuss artifacts of the technique.

Three-dimensional quantitative imaging of retinal and choroidal blood flow velocity using joint Spectral and Time domain Optical Coherence Tomography.

Recently, joint Spectral and Time domain Optical Coherence Tomography (joint STdOCT) has been proposed to measure ocular blood flow velocity. Limitations of CCD technology allowed only for two-dimensional imaging at that time. In this paper we demonstrate fast three-dimensional STdOCT based on ultrahigh speed CMOS camera. Proposed method is straightforward, fully automatic and does not require any advanced image processing techniques. Three-dimensional distributions of axial velocity components of the blood in human eye vasculature are presented: in retinal and, for the first time, in choroidal layer. Different factors that affect quality of velocity images are discussed. Additionally, the quantitative measurement allows to observe a new interesting optical phenomenon - random Doppler shift in OCT signals that forms a vascular pattern at the depth of sclera.

Anterior segment imaging with Spectral OCT system using a high-speed CMOS camera.

We describe a new ultrahigh speed Spectral OCT instrument making use of a CMOS camera and demonstrate high quality in vivo imaging of the anterior segment of the human eye. The high flexibility of the designed imaging system allows a wide range of imaging protocols. Two- and three-dimensional high quality OCT images of the cornea, the anterior chamber and the crystalline lens are presented. A high acquisition rate, up to 135,000 A-scans/second enables three-dimensional reconstruction of the anterior segment during lenticular accommodation, blinking and pupillary reaction to light stimulus. We demonstrate OCT tomographic real time imaging of the lens dynamics during accommodation and high quality OCT cross-sectional images of the entire anterior segment of the eye from the cornea up to posterior part of the crystalline lens.

Scanning protocols dedicated to smart velocity ranging in spectral OCT.

We introduce a new type of scanning protocols, called segmented protocols, which enable extracting multi-range flow velocity information from a single Spectral OCT data set. The protocols are evaluated using a well defined flow in a glass capillary. As an example of in vivo studies, we demonstrate two- and three-dimensional imaging of the retinal vascular system in the eyes of healthy volunteers. The flow velocity detection is performed using a method of Joint Spectral and Time domain OCT. Velocity ranging is demonstrated in imaging of retinal vasculature in the macular region and in the optic disk area characterized by different flow velocity values. Additionally, an enhanced visualization of retinal capillary network is presented in the close proximity to macula.