Correction: The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0015730.].

Diffuse Correlation Spectroscopy: A Review of Recent Advances in Parallelisation and Depth Discrimination Techniques.

Diffuse correlation spectroscopy is a non-invasive optical modality used to measure cerebral blood flow in real time, and it has important potential applications in clinical monitoring and neuroscience. As such, many research groups have recently been investigating methods to improve the signal-to-noise ratio, imaging depth, and spatial resolution of diffuse correlation spectroscopy. Such methods have included multispeckle, long wavelength, interferometric, depth discrimination, time-of-flight resolution, and acousto-optic detection strategies. In this review, we exhaustively appraise this plethora of recent advances, which can be used to assess limitations and guide innovation for future implementations of diffuse correlation spectroscopy that will harness technological improvements in the years to come.

Increasing the Q-factor of Fabry-Perot etalons using focused Bessel beam illumination.

Sensing and filtering applications often require Fabry-Perot (FP) etalons with an Interferometer Transfer Function (ITF) having high visibility, narrow Full Width at Half Maximum (FWHM), and high sensitivity. For the ITF to have these characteristics, the illumination beam must be matched to the modes of the FP cavity. This is challenging when a small illumination element size is needed, as typical focused beams are not matched to the FP cavity modes. Bessel beams are a potential alternative as their structure resembles the FP cavity modes while possessing a focused core. To study the feasibility of using Bessel beam illumination, in this Letter, ITFs of an FP etalon were measured using Bessel and Gaussian illumination beams. A Bessel beam with core size of 28 µm provided an ITF with visibility 3.0 times higher, a FWHM 0.3 times narrower, and a sensitivity 2.2 times higher than a Gaussian beam with waist 32 µm. The results show that Bessel beam illumination can provide ITFs similar to that of collimated beam illumination while also having with a focused core.

On the inverse problem in optical coherence tomography.

We examine the inverse problem of retrieving sample refractive index information in the context of optical coherence tomography. Using two separate approaches, we discuss the limitations of the inverse problem which lead to it being ill-posed, primarily as a consequence of the limited viewing angles available in the reflection geometry. This is first considered from the theoretical point of view of diffraction tomography under a weak scattering approximation. We then investigate the full non-linear inverse problem using a variational approach. This presents another illustration of the non-uniqueness of the solution, and shows that even the non-linear (strongly scattering) scenario suffers a similar fate as the linear problem, with the observable spatial Fourier components of the sample occupying a limited support. Through examples we demonstrate how the solutions to the inverse problem compare when using the variational and diffraction-tomography approaches.

Numerical model of light propagation through Fabry-Perot etalons composed of interfaces with non-planar surface topography.

We present a model that calculates optical fields reflected and transmitted by a Fabry-Perot (FP) etalon composed of interfaces with non-planar surface topography. The model uses the Rayleigh-Rice theory, which predicts the fields reflected and transmitted by a single interface, to account for the non-planar surface topography of each interface. The Rayleigh-Rice theory is evaluated iteratively to account for all round trips that light can take within the FP etalon. The model predictions can then be used to compute Interferometer transfer function (ITF)s, by performing wavelength or angle resolved simulations enabling predictions of the bandwidth, peak transmissivity, and sensitivity of FP etalons. The model was validated against the Pseudospectral time-domain (PSTD) method, which resulted in good agreement. Since the model accuracy is expected to reduce as the Root mean square (RMS) of the topographic map increases, the error in the model's predictions was studied as a function of topographic map RMS. Finally, application of the model was exemplified by predicting the impact of roughness on ITFs and computing the changes in FP etalon transmissivity as cavity thickness is modulated by an ultrasonic wave.

ABCD transfer matrix model of Gaussian beam propagation in Fabry-Perot etalons.

A numerical model of Gaussian beam propagation in planar Fabry-Perot (FP) etalons is presented. The model is based on the ABCD transfer matrix method. This method is easy to use and interpret, and readily connects models of lenses, mirrors, fibres and other optics to aid simulating complex multi-component etalon systems. To validate the etalon model, its predictions were verified using a previously validated model based on Fourier optics. To demonstrate its utility, three different etalon systems were simulated. The results suggest the model is valid and versatile and could aid in designing and understanding a range of systems containing planar FP etalons. The method could be extended to model higher order beams, other FP type devices such as plano-concave resonators, and more complex etalon systems such as those involving tilted components.

Application of a Taylor series approximation to the Debye-Wolf integral in time-domain numerical electromagnetic simulations.

Finite-difference time-domain (FDTD) and pseudospectral time-domain (PSTD) methods are numerical electromagnetic simulation techniques that have been employed to perform rigorous simulations of broadband illuminations in several contexts. However, the computational cost of calculating the incident source fields introduced into the FDTD/PSTD grid can be considerable. In some cases, this can exceed the computational cost of what might be considered the principal part of the FDTD/PSTD algorithm, which calculates the spatial derivative of fields throughout the computational grid. In this paper, we analyze an existing method that has been used to approximate broadband illumination, which uses knowledge of the field only at a central frequency of the spectrum. We then present a new, to the best of our knowledge, approximation of the broadband illumination, which is more accurate, while remaining computationally tractable. Finally, we present some examples to verify the accuracy and efficiency of the new method and compare these results with the existing method.

Performance optimisation of a holographic Fourier domain diffuse correlation spectroscopy instrument.

We have previously demonstrated a novel interferometric multispeckle Fourier domain diffuse correlation spectroscopy system that makes use of holographic camera-based detection, and which is capable of making in vivo pulsatile flow measurements. In this work, we report on a systematic characterisation of the signal-to-noise ratio performance of our system. This includes demonstration and elimination of laser mode hopping, and correction for the instrument's modulation transfer function to ensure faithful reconstruction of measured intensity profiles. We also demonstrate a singular value decomposition approach to ensure that spatiotemporally correlated experimental noise sources do not limit optimal signal-to-noise ratio performance. Finally, we present a novel multispeckle denoising algorithm that allows our instrument to achieve a signal-to-noise ratio gain that is equal to the square root of the number of detected speckles, whilst detecting up to ∼1290 speckles in parallel. The signal-to-noise ratio gain of 36 that we report is a significant step toward mitigating the trade-off that exists between signal-to-noise ratio and imaging depth in diffuse correlation spectroscopy.

Volumetric High-Resolution X-Ray Phase-Contrast Virtual Histology of Breast Specimens With a Compact Laboratory System.

The assessment of margin involvement is a fundamental task in breast conserving surgery to prevent recurrences and reoperations. It is usually performed through histology, which makes the process time consuming and can prevent the complete volumetric analysis of large specimens. X-ray phase contrast tomography combines high resolution, sufficient penetration depth and high soft tissue contrast, and can therefore provide a potential solution to this problem. In this work, we used a high-resolution implementation of the edge illumination X-ray phase contrast tomography based on "pixel-skipping" X-ray masks and sample dithering, to provide high definition virtual slices of breast specimens. The scanner was originally designed for intra-operative applications in which short scanning times were prioritised over spatial resolution; however, thanks to the versatility of edge illumination, high-resolution capabilities can be obtained with the same system simply by swapping x-ray masks without this imposing a reduction in the available field of view. This makes possible an improved visibility of fine tissue strands, enabling a direct comparison of selected CT slices with histology, and providing a tool to identify suspect features in large specimens before slicing. Combined with our previous results on fast specimen scanning, this works paves the way for the design of a multi-resolution EI scanner providing intra-operative capabilities as well as serving as a digital pathology system.

Angular Airy function: a model of Fabry-Perot etalons illuminated by arbitrary beams.

Fabry-Perot (FP) etalons are used as filters and sensors in a range of optical systems. The reflected and transmitted fields associated with an FP etalon have traditionally been predicted by the Airy function, which assumes a plane wave illumination. FP etalons are, however, often illuminated by non-collimated beams, rendering the Airy function invalid. To address this limitation, we describe the angular Airy function which calculates the reflected and transmitted fields for arbitrary illumination beams, using angular spectrum decomposition. Combined with realistic models of the experimental illumination beams and detection optics, we show that the angular Airy function can accurately predict experimental wavelength resolved intensity measurements. Based on the angular Airy function, we show that the fundamental operating principle of an FP etalon is as an angular-spectral filter. Based on this interpretation we explain the asymmetry, broadening and visibility reduction seen on wavelength resolved intensity measurements from high Q-factor FP etalons illuminated with focused Gaussian beams.

Simulation of statistically accurate time-integrated dynamic speckle patterns in biomedical optics.

The simulation of statistically accurate time-integrated dynamic speckle patterns using a physics-based model that accounts for spatially varying sample properties is yet to be presented in biomedical optics. In this Letter, we propose a solution to this important problem based on the Karhunen-Loève expansion of the electric field and apply our method to the formalisms of both laser speckle contrast imaging and diffuse correlation spectroscopy. We validate our technique against solutions for speckle contrast for different forms of homogeneous field and also show that our method can readily be extended to cases with spatially varying sample properties.

Reducing residual-motion artifacts in iterative 3D CBCT reconstruction in image-guided radiation therapy.

PURPOSE: Recent evaluations of a 3D iterative cone-beam computed tomography (iCBCT) reconstruction method available on Varian radiation treatment devices demonstrated that iCBCT provides superior image quality when compared to analytical Feldkamp-Davis-Kress (FDK) method. However, iCBCT employs statistical penalized likelihood (PL) that is known to be highly sensitive to inconsistencies due to physiological motion occurring during the acquisition. We propose a computationally inexpensive extension of iCBCT addressing this deficiency. METHODS: During the iterative process, the gradients of PL are modified to avoid the generation of motion-related artifacts. To assess the impact of this modification, we propose a motion simulation generating CBCT projections of a moving anatomy together with artifact-free images used as ground truth. Contrast-to-noise ratio and power spectra of difference images are computed to quantify the impact of the motion on reconstructed CBCT volumes as well as the effect of the proposed modification. RESULTS: Using both simulated and clinical data, it is shown that the motion of patient's abdominal wall during the acquisition results in artifacts that can be quantified as low-frequency components in volumes reconstructed with iCBCT. Further, a quantitative evaluation demonstrates that the proposed modification of PL reduces these low-frequency components. While preserving the advantages of PL, it effectively suppresses the propagation of motion-related artifacts into clinically important regions, thus increasing the motion resiliency of iCBCT. CONCLUSIONS: The proposed modified iterative reconstruction method significantly improves the quality of CBCT images of anatomies suffering from residual motion.

Optimization of multipoint phase retrieval in edge illumination X-ray imaging: A theoretical and experimental analysis.

PURPOSE: In this work, an analytical model describing the noise in the retrieved three contrast channels, transmission, refraction, and ultra small-angle scattering, obtained with edge illumination X-ray phase-based imaging system is presented and compared to experimental data. METHODS: In EI, images acquired at different displacements of the presample mask (i.e., different illumination levels referred to as points on the "illumination curve"), followed by pixel-wise curve fitting, are exploited to quantitatively retrieve the three contrast channels. Therefore, the noise in the final image will depend on the error associated with the fitting process. We use a model based on the derivation of the standard error on fitted parameters, which relies on the calculation of the covariance matrix, to estimate the noise and the cross-channel correlation as a function of the position of the sampling points. In particular, we investigated the most common cases of 3 and 5 sampling points. In addition, simulations have been used to better understand the role of the integration time for each sampling point. Finally, the model is validated by comparison with the experimental data acquired with an edge illumination setup based on a tungsten rotating anode X-ray source and a photon counting detector. RESULTS: We found a good match between the predictions of the model and the experimental data. In particular, for the investigated cases, an arrangement of the sampling points leading to minimum noise and cross-channel correlation can be found. Simulations revealed that, given a fixed overall scanning time, its distribution into the smallest possible number of sampling points needed for phase retrieval leads to minimum noise thanks to higher statistics per point. CONCLUSIONS: This work presents an analytical model describing the noise in the various contrast channels retrieved in edge illumination as a function of the illumination curve sampling. In particular, an optimal sampling scheme leading to minimum noise has been determined for the case where 3 or 5 sampling points are used, which represent two of the most common acquisition schemes. In addition, the correlation between noise in the different channels and the role of the number of points and exposure time have been also investigated. In general, our results suggest a series of procedures that should be followed in order to optimize the experimental acquisitions.

Approximate image synthesis in optical coherence tomography.

Full-wave models of OCT image formation, which are based on Maxwell's equations, are highly realistic. However, such models incur a high computational cost, particularly when modelling sample volumes consistent with those encountered in practice. Here, we present an approximate means of synthesizing volumetric image formation to reduce this computational burden. Instead of performing a full-wave scattered light calculation for each A-scan, we perform a full-wave scattered light calculation for a normally incident plane wave only. We use the angular spectrum field representation to implement beam focussing and scanning, making use of an assumption similar to the tilt optical memory effect, to approximately synthesize volumetric data sets. Our approach leads to an order of magnitude reduction in the computation time required to simulate typical B-scans. We evaluate this method by comparing rigorously and approximately evaluated point spread functions and images of highly scattering structured samples for a typical OCT system. Our approach also reveals new insights into image formation in OCT.

Analysing the impact of non-parallelism in Fabry-Perot etalons through optical modelling.

Fabry-Perot (FP) etalons, composed of two parallel mirrors, are used widely as optical filters and sensors. In certain applications, however, such as when FP etalons with polymer cavities are used to detect ultrasound, the mirrors may not be perfectly parallel due to manufacturing limitations. As little is known about how the mirrors being non-parallel impacts upon FP etalon performance, it is challenging to optimize the design of such devices. To address this challenge, we developed a model of light propagation in non-parallel FP etalons. The model is valid for arbitrary monochromatic beams and calculates both the reflected and transmitted beams, assuming full-wave description of light. Wavelength resolved transmissivity simulations were computed to predict the effect that non-parallel mirrors have on the sensitivity, spectral bandwidth and peak transmissivity of FP etalons. Theoretical predictions show that the impact of the non-parallel mirrors increases with both mirror reflectivity and incident Gaussian beam waist. Guidelines regarding the maximum angle allowed between FP mirrors whilst maintaining the sensitivity and peak transmissivity of a parallel mirror FP etalon are provided as a function of mirror reflectivity, cavity thickness and Gaussian beam waist. This information, and the model, could be useful for guiding the design of FP etalons suffering a known degree of non-parallelism, for example, to optimize the sensitivity of polymer based FP ultrasound sensors.

Speckle-dependent accuracy in phase-sensitive optical coherence tomography.

Phase-sensitive optical coherence tomography (OCT) is used to measure motion in a range of techniques, such as Doppler OCT and optical coherence elastography (OCE). In phase-sensitive OCT, motion is typically estimated using a model of the OCT signal derived from a single reflector. However, this approach is not representative of turbid samples, such as tissue, which exhibit speckle. In this study, for the first time, we demonstrate, through theory and experiment that speckle significantly lowers the accuracy of phase-sensitive OCT in a manner not accounted for by the OCT signal-to-noise ratio (SNR). We describe how the inaccuracy in speckle reduces phase difference sensitivity and introduce a new metric, speckle brightness, to quantify the amount of constructive interference at a given location in an OCT image. Experimental measurements show an almost three-fold degradation in sensitivity between regions of high and low speckle brightness at a constant OCT SNR. Finally, we apply these new results in compression OCE to demonstrate a ten-fold improvement in strain sensitivity, and a five-fold improvement in contrast-to-noise by incorporating independent speckle realizations. Our results show that speckle introduces a limit to the accuracy of phase-sensitive OCT and that speckle brightness should be considered to avoid erroneous interpretation of experimental data.

Detection of involved margins in breast specimens with X-ray phase-contrast computed tomography.

Margins of wide local excisions in breast conserving surgery are tested through histology, which can delay results by days and lead to second operations. Detection of margin involvement intraoperatively would allow the removal of additional tissue during the same intervention. X-ray phase contrast imaging (XPCI) provides soft tissue sensitivity superior to conventional X-rays: we propose its use to detect margin involvement intraoperatively. We have developed a system that can perform phase-based computed tomography (CT) scans in minutes, used it to image 101 specimens approximately half of which contained neoplastic lesions, and compared results against those of a commercial system. Histological analysis was carried out on all specimens and used as the gold standard. XPCI-CT showed higher sensitivity (83%, 95% CI 69-92%) than conventional specimen imaging (32%, 95% CI 20-49%) for detection of lesions at margin, and comparable specificity (83%, 95% CI 70-92% vs 86%, 95% CI 73-93%). Within the limits of this study, in particular that specimens obtained from surplus tissue typically contain small lesions which makes detection more difficult for both methods, we believe it likely that the observed increase in sensitivity will lead to a comparable reduction in the number of re-operations.

A Na,K-ATPase-Fodrin-Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis.

Fenestrae are transcellular plasma membrane pores that mediate blood-tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin-fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex.

Modelling Fabry-Pérot etalons illuminated by focussed beams.

Fabry-Pérot (FP) etalons are used as filters and sensors in a range of optical systems. Often FP etalons are illuminated by collimated laser beams, in which case the transmitted and reflected light fields can be calculated analytically using well established models. However, FP etalons are sometimes illuminated by more complex beams such as focussed Gaussian beams, which may also be aberrated. Modelling the response of FP etalons to these beams requires a more sophisticated model. To address this need, we present a model that can describe the response of an FP etalon that is illuminated by an arbitrary beam. The model uses an electromagnetic wave description of light and can therefore compute the amplitude, phase and polarization of the optical field at any position in the system. It can also account for common light delivery and detection components such as lenses, optical fibres and photo-detectors, allowing practical systems to be simulated. The model was validated against wavelength resolved measurements of transmittance and reflectance obtained using a system consisting of an FP etalon illuminated by a focussed Gaussian beam. Experiments with focal spot sizes ranging from 30 µm to 250 µm and FP etalon mirror reflectivities in the range 97.2 % to 99.2 % yielded excellent visual agreement between simulated and experimental data and an average error below 10% for a range of quantitative comparative metrics. We expect the model to be a useful tool for designing, understanding and optimising systems that use FP etalons.