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We present a validation of red blood cell flux and speed measurements based on the passage of erythrocytes through the OCT’s focal volume. We compare the performance of the so-called RBC-passage OCT technique to co-localized and simultaneously acquired two-photon excitation fluorescence microscopy (TPEF) measurements. Using concurrent multi-modal imaging, we show that fluctuations in the OCT signal display highly similar features to TPEF time traces. Furthermore, we demonstrate an overall difference in RBC flux and speed of 2.5 ± 3.27 RBC/s and 0.12 ± 0.67 mm/s (mean ± S.D.), compared to TPEF. The analysis also revealed that the OCT RBC flux estimation is most accurate between 20 RBC/s to 60 RBC/s, and is severely underestimated at fluxes beyond 80 RBC/s. Lastly, our analysis shows that the RBC speed estimations increase in accuracy as the speed decreases, reaching a difference of 0.16 ± 0.25 mm/s within the 0–0.5 mm/s speed range.

Recent studies suggested that cerebrovascular micro-occlusions, i.e. microstokes, could lead to ischemic tissue infarctions and cognitive deficits. Due to their small size, identifying measurable biomarkers of these microvascular lesions remains a major challenge. This work aims to simulate potential MRI signatures combining arterial spin labeling (ASL) and multi-directional diffusion-weighted imaging (DWI). Driving our hypothesis are recent observations demonstrating a radial reorientation of microvasculature around the micro-infarction locus during recovery in mice. Synthetic capillary beds, randomly- and radially-oriented, and optical coherence tomography (OCT) angiograms, acquired in the barrel cortex of mice (n = 5) before and after inducing targeted photothrombosis, were analyzed. Computational vascular graphs combined with a 3D Monte-Carlo simulator were used to characterize the magnetic resonance (MR) response, encompassing the effects of magnetic field perturbations caused by deoxyhemoglobin, and the advection and diffusion of the nuclear spins. We quantified the minimal intravoxel signal loss ratio when applying multiple gradient directions, at varying sequence parameters with and without ASL. With ASL, our results demonstrate a significant difference (p < 0.05) between the signal-ratios computed at baseline and 3 weeks after photothrombosis. The statistical power further increased (p < 0.005) using angiograms measured at week 4. Without ASL, no reliable signal change was found. We found that higher ratios, and accordingly improved significance, were achieved at lower magnetic field strengths (e.g., B0 = 3T) and shorter echo time TE (< 16 ms). Our simulations suggest that microstrokes might be characterized through ASL-DWI sequence, providing necessary insights for posterior experimental validations, and ultimately, future translational trials.

In this work, we demonstrate the application of Rose Bengal as a photo-thrombotic agent using 2-photon and 3-photon excitation by measuring the response to multiphotonic excitation. We also demonstrate, in the case of 2-photon excitation, that the mechanism used is different from laser injury alone with a control group of FitC injected mice. Preliminary results show that a capillary photo-thrombosis could be performed up to 200 μm and OCT imaging could confirm blockage.

Recent studies suggested that cerebrovascular micro-occlusions, i.e. microstokes, could lead to ischemic tissue infarctions and cognitive deficits. Due to their small size, identifying measurable biomarkers of these microvascular lesions remains a major challenge. This work aims to simulate potential MRI signatures combining arterial spin labeling (ASL) and multi-directional diffusion-weighted imaging (DWI). Driving our hypothesis are recent observations demonstrating a radial reorientation of microvasculature around the micro-infarction locus during recovery in mice. Synthetic capillary beds, randomly- and radially-oriented, and optical coherence tomography (OCT) angiograms, acquired in the barrel cortex of mice (n=5) before and after inducing targeted photothrombosis, were analyzed. Computational vascular graphs combined with a 3D Monte-Carlo simulator were used to characterize the magnetic resonance (MR) response, encompassing the effects of magnetic field perturbations caused by deoxyhemoglobin, and the advection and diffusion of the nuclear spins. We quantified the minimal intravoxel signal loss ratio when applying multiple gradient directions, at varying sequence parameters with and without ASL. With ASL, our results demonstrate a significant difference (p

We present an application of massively parallel processing of quantitative flow measurements data acquired using spectral optical coherence microscopy (SOCM). The need for massive signal processing of these particular datasets has been a major hurdle for many applications based on SOCM. In view of this difficulty, we implemented and adapted quantitative total flow estimation algorithms on graphics processing units (GPU) and achieved a 150 fold reduction in processing time when compared to a former CPU implementation. As SOCM constitutes the microscopy counterpart to spectral optical coherence tomography (SOCT), the developed processing procedure can be applied to both imaging modalities. We present the developed DLL library integrated in MATLAB (with an example) and have included the source code for adaptations and future improvements. Program summary Program title: CudaOCMproc Catalogue identifier: AFBT_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AFBT_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: GNU GPLv3 No. of lines in distributed program, including test data, etc.: 913552 No. of bytes in distributed program, including test data, etc.: 270876249 Distribution format: tar.gz Programming language: CUDA/C, MATLAB. Computer: Intel x64 CPU, GPU supporting CUDA technology. Operating system: 64-bit Windows 7 Professional. Has the code been vectorized or parallelized?: Yes, CPU code has been vectorized in MATLAB, CUDA code has been parallelized. RAM: Dependent on users parameters, typically between several gigabytes and several tens of gigabytes Classification: 6.5, 18. Nature of problem: Speed up of data processing in optical coherence microscopy Solution method: Utilization of GPU for massively parallel data processing Additional comments: Compiled DLL library with source code and documentation, example of utilization (MATLAB script with raw data) Running time: 1,8 s for one B-scan (150 × faster in comparison to the CPU data processing time)

Over the past decades, optical coherence tomography has emerged as an important imaging technique to study biological processes through its ability to perform three-dimensional imaging at high acquisition rates and non-invasively. Furthermore, OCT has shown a growing interest in brain imaging through its capacity in obtaining functional information such as cellular viability, hematocrit and blood flow velocity. Although OCT can reach image depths spanning a few millimeters, the effective imaging depth is typically dictated by the depth-of-field of the imaging optics. In traditional OCT systems, this depth-of-field is given by the Rayleigh range and is thus coupled to the lateral resolution. As such, increasing the numerical aperture of the system reduces the imaging depth, ultimately hampering the depth-multiplexing advantage of OCT. Wavefront engineering schemes have been devised to overcome this limitation, providing the OCT systems with an extended-focus. We present here two extended-focus OCT systems (xf-OCT) optimized for cerebral imaging. The first system operates in the visible wavelength range and is designed to image the superficial cortex of mice at high contrast and at high resolution. Its high axial and lateral resolution of 0.8 and 1.4 um respectively, maintained over 200 um enable resolving structures such as myelinated axons, neuronal cells and micro-vessels in vivo. The second system is optimized for deep microvascular cortical imaging and operates in the infrared spectral range. Through its extended-focus and increased penetration, the second system can provide maps of cortical microvasculature over 800 um in depth in the cortex in vivo.

Spectral domain optical coherence tomography (OCT) is a widely employed, minimally invasive bio-medical imaging technique, which requires a broadband light source, typically implemented by super-luminescent diodes. Recent advances in soliton based photonic integrated frequency combs (soliton microcombs) have enabled the development of low-noise, broadband chipscale frequency comb sources, whose potential for OCT imaging has not yet been unexplored. Here, we explore the use of dissipative Kerr soliton microcombs in spectral domain OCT and show that, by using photonic chipscale Si3N4 resonators in conjunction with 1300 nm pump lasers, spectral bandwidths exceeding those of commercial OCT sources are possible. We characterized the exceptional noise properties of our source (in comparison to conventional OCT sources) and demonstrate that the soliton states in microresonators exhibit a residual intensity noise floor at high offset frequencies that is ca. 3 dB lower than a traditional OCT source at identical power, and can exhibit significantly lower noise performance for powers at the milli-Watt level. Moreover, we demonstrate that classical amplitude noise of all soliton comb teeth are correlated, i.e., common mode, in contrast to superluminescent diodes or incoherent microcomb states, which opens a new avenue to improve imaging speed and performance beyond the thermal noise limit., Superluminescent diodes, that provide a broadband spectrum are typically used in spectral domain coherence tomography. Here, the authors use chipscale silicon nitride resonators to generate soliton microcombs with a lower noise flor that could substitute the diode sources.

Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here we expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover glass) and compare these results with an objective devoid of collar-correction. Finally, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window, thereby significantly improving the imaging quality. The performance of the device is demonstrated in an in vivo setting and a tilt-correction procedure is described, enabling the accurate alignment (

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.

We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume.

In diabetes, pancreatic beta-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. (C) 2016 Optical Society of America

Magnetic Resonance Imaging has revolutionised our understanding of brain function through its ability to image human cerebral structures non-invasively over the entire brain. By exploiting the different magnetic properties of oxygenated and deoxygenated blood, functional MRI can indirectly map areas undergoing neural activation. Alongside the development of fMRI, powerful statistical tools have been developed in an effort to shed light on the neural pathways involved in processing of sensory and cognitive information. In spite of the major improvements made in fMRI technology, the obtained spatial resolution of hundreds of microns prevents MRI in resolving and monitoring processes occurring at the cellular level. In this regard, Optical Coherence Microscopy is an ideal instrumentation as it can image at high spatio-temporal resolution. Moreover, by measuring the mean and the width of the Doppler spectra of light scattered by moving particles, OCM allows extracting the axial and lateral velocity components of red blood cells. The ability to assess quantitatively total blood velocity, as opposed to classical axial velocity Doppler OCM, is of paramount importance in brain imaging as a large proportion of cortical vascular is oriented perpendicularly to the optical axis. We combine here quantitative blood flow imaging with extended-focus Optical Coherence Microscopy and Statistical Parametric Mapping tools to generate maps of stimuli-evoked cortical hemodynamics at the capillary level.

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. ispartof: PLoS One vol:12 issue:7 ispartof: location:United States status: published

We demonstrate 3D velocimetry of blood flow in the mouse brain with extended-focus optical coherence microscopy (xfOCM). xfOCM combines multiplexed depth acquisition with a resolution and penetration comparable to two-photon microscopy.

We review optical coherence microscopy and present selected structural and functional imaging applications ranging from diabetes to brain imaging, including new concepts for cell imaging with an emphasis on the underlying optical concepts.

Microvascular stalling, the process occurring when a capillary temporarily loses perfusion, has gained increasing interest in recent years through its demonstrated presence in various neuropathologies. Studying the impact of such stalls on the surrounding brain tissue is of paramount importance to understand their role in such diseases. Despite efforts trying to study the stalling events, investigations are hampered by their elusiveness and scarcity. In an attempt to alleviate these hurdles, we present here a novel methodology enabling transient occlusions of targeted microvascular segments through multiphoton excitation of Rose Bengal, an established photothrombotic agent. With n = 7 mice C57BL/6 J (5 males and 2 females) and 95 photothrombosis trials, we demonstrate the ability of triggering reversible blockages by illuminating a capillary segment during ∼300 s at 1000 nm, using a standard Ti:Sapphire femtosecond laser. Furthermore, we performed concurrent Optical Coherence Microscopy (OCM) angiography imaging of the microvascular network to highlight the specificity of the targeted occlusion and its duration. Through comparison with a control group, we conclude that blood flow cessation is indeed created by the photothrombotic agent via multiphoton excitation and is temporary, followed by a flow recovery in less than 24 h. Moreover, Immunohistology points toward a stalling mechanism driven by adherence of the neutrophil in the vascular lumen. This observation seems to be promoted by the inflammation locally created via multiphoton activation of Rose Bengal.

Alzheimer's disease (AD) is characterized by amyloidosis of brain tissues. This phenomenon is studied with genetically-modified mouse models. We propose a method to quantify amyloidosis in whole 5xFAD mouse brains, a model of AD. We use optical projection tomography (OPT) and a random forest voxel classifier to segment and measure amyloid plaques. We validate our method in a preliminary cross-sectional study, where we measure 6136 +/- 1637, 8477 +/- 3438, and 17267 +/- 4241 plaques (AVG +/- SD) at 11, 17, and 31 weeks. Overall, this method can be used in the evaluation of new treatments against AD. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

In recent years, three-dimensional mesoscopic imaging has gained significant importance in life sciences for fundamental studies at the whole-organ level. In this manuscript, we present an optical projection tomography (OPT) method designed for imaging of the intact mouse brain. The system features an isotropic resolution of ~50 µm and an acquisition time of four to eight minutes, using a 3-day optimized clearing protocol. Imaging of the brain autofluorescence in 3D reveals details of the neuroanatomy, while the use of fluorescent labels displays the vascular network and amyloid deposition in 5xFAD mice, an important model of Alzheimer's disease (AD). Finally, the OPT images are compared with histological slices.

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 figures

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.

Optical coherence microscopy (OCM) is an interferometric technique providing 3D images of biological samples with micrometric resolution and penetration depth of several hundreds of micrometers. OCM differs from optical coherence tomography (OCT) in that it uses a high numerical aperture (NA) objective to achieve high lateral resolution. However, the high NA also reduces the depth-of-field (DOF), scaling with 1/NA2. Interferometric synthetic aperture microscopy (ISAM) is a computed imaging technique providing a solution to this trade-off between resolution and DOF. An alternative hardware method to achieve an extended DOF is to use a non-Gaussian illumination. Extended focus OCM (xfOCM) uses a Bessel beam to obtain a narrow and extended illumination volume. xfOCM detects back-scattered light using a Gaussian mode in order to maintain good sensitivity. However, the Gaussian detection mode limits the DOF. In this work, we present extended ISAM (xISAM), a method combining the benefits of both ISAM and xfOCM. xISAM uses the 3D coherent transfer function (CTF) to generalize the ISAM algorithm to different system configurations. We demonstrate xISAM both on simulated and experimental data, showing that xISAM attains a combination of high transverse resolution and extended DOF which has so far been unobtainable through conventional ISAM or xfOCM individually. ispartof: Optics Express vol:25 issue:24 pages:30807-30819 ispartof: location:United States status: published