In vivo identification of the retinal layer containing photopigments in OCT images through correlation with two-photon psychophysics.

Two-photon vision enables near-infrared light perception in humans. We investigate the possibility to utilize this phenomenon as an indicator of the location of the outer segments of photoreceptor cells in the OCT images. Since two-photon vision is independent on OCT imaging, it could provide external to OCT reference relative to which positions of retinal layers visible in OCT imaging could be measured. We show coincidence between OCT imaging of outer retinal layers and two-photon light perception. The experiment utilizes an intrinsic nonlinear process in the retina, two-photon absorption of light by visual photopigments, which triggers perception of near-infrared light. By shifting the focus of the imaging/stimulus beam, we link the peak efficiency of two-photon vision with the visibility of outer segments of photoreceptor cells, which can be seen as in vivo identification of a retinal layer containing visual photopigments in OCT images. Determination of the in-focus retinal layer is achieved by analysis of en face OCT image contrast. We discuss experimental methods and experimental factors that may influence two-photon light perception and the accuracy of the results. The limits of resolution are discussed in analysis of the one-photon and two-photon point spread functions.

Spindle shape and volume differ in high- and low-quality metaphase II oocytes.

In brief: Optical coherence microscopy non-invasively visualizes metaphase II spindles allowing for quantitative analysis of their volume and shape, which may prove useful in the assessment of the oocyte quality. Using a mouse model, we showed also that analysis of spindle length combined with morphokinetics improves the evaluation of the resulting embryos. Abstract: The proper development of embryos strongly depends on the quality of oocytes, so the evaluation of oocytes may be a useful initial step in IVF procedures. Additionally, it enables embryologists to make more informed decisions regarding the treatments chosen for the patients and better manage patients' expectations. Optical coherence microscopy (OCM) allows for non-invasive 3D visualization of intracellular structures, such as spindles or nuclei, which have been linked to the success of embryonic development. Here, we applied a mouse model to examine whether OCM imaging could be used in the quality assessment of metaphase II (MII) oocytes. We showed that quantitative parameters describing the shape and volume of the MII spindle were associated with the quality of the resulting embryos, including the likelihood of blastocyst formation and the embryos' ability to differentiate the trophectoderm and primitive endoderm, but not the epiblast. We also created a multivariate linear regression model, combining OCM-based quantification of MII spindles with morphokinetic analysis of the embryos, that allowed for improved evaluation of the embryo quality. Finally, we proved that OCM does not interfere with the viability of the scanned cells, at least during the preimplantation development. Therefore, we believe that OCM-based quantitative assessment of MII spindles can improve the oocyte and embryo selection in IVF procedures.

The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

Customizable tubular model for n-furcating blood vessels and its application to 3D reconstruction of the cerebrovascular system.

Understanding the 3D cerebral vascular network is one of the pressing issues impacting the diagnostics of various systemic disorders and is helpful in clinical therapeutic strategies. Unfortunately, the existing software in the radiological workstation does not meet the expectations of radiologists who require a computerized system for detailed, quantitative analysis of the human cerebrovascular system in 3D and a standardized geometric description of its components. In this study, we show a method that uses 3D image data from magnetic resonance imaging with contrast to create a geometrical reconstruction of the vessels and a parametric description of the reconstructed segments of the vessels. First, the method isolates the vascular system using controlled morphological growing and performs skeleton extraction and optimization. Then, around the optimized skeleton branches, it creates tubular objects optimized for quality and accuracy of matching with the originally isolated vascular data. Finally, it optimizes the joints on n-furcating vessel segments. As a result, the algorithm gives a complete description of shape, position in space, position relative to other segments, and other anatomical structures of each cerebrovascular system segment. Our method is highly customizable and in principle allows reconstructing vascular structures from any 2D or 3D data. The algorithm solves shortcomings of currently available methods including failures to reconstruct the vessel mesh in the proximity of junctions and is free of mesh collisions in high curvature vessels. It also introduces a number of optimizations in the vessel skeletonization leading to a more smooth and more accurate model of the vessel network. We have tested the method on 20 datasets from the public magnetic resonance angiography image database and show that the method allows for repeatable and robust segmentation of the vessel network and allows to compute vascular lateralization indices.

Optical coherence microscopy allows for quality assessment of immature mouse oocytes.

In brief: Optical coherence microscopy is a label-free and non-invasive imaging technique capable of 3D subcellular structure visualization. Here we show that this method allows for quality assessment of immature mouse oocytes based on their chromatin conformation and can be a valuable addition to the toolkit used in assisted reproduction procedures. Abstract: The success of assisted reproductive technologies, and particularly in vitro maturation, is tightly linked to the quality of oocytes. Therefore, there is a need for robust, reliable, and easy-to-assess biomarkers of oocyte developmental competence. Microscopy techniques visualizing oocyte intracellular structure could provide such biomarkers. However, fluorescence imaging methods, applied frequently in biology and allowing for detailed structural and dynamic studies of single cells, require fluorescent tags to visualize cellular architecture and may cause short- and long-term photo-damage. On the other hand, traditional light microscopy, although relatively non-invasive, does not provide detailed structural information. Optical coherence microscopy (OCM) is a promising alternative, as it does not require sample pre-processing or labelling and can provide 3D images of intracellular structures. Here we applied OCM to assess the chromatin conformation of immature mouse oocytes, a feature that corresponds with their transcriptional status and developmental competence and cannot be examined by traditional light microscopy. We showed that OCM distinguished oocytes with so-called non-surrounded nucleoli (NSN) and surrounded nucleoli (SN) chromatin conformation with very high sensitivity and specificity and that OCM scanning did not decrease the quality of oocytes. Finally, we cross-referenced OCM data with the oocyte ability to undergo normal nuclear and cytoplasmic maturation and proven that indeed oocytes scored with OCM as NSN mature less effectively than oocytes scored as SN. Our results suggest that OCM may be a valuable addition to the imaging toolkit used in assisted reproduction procedures.

Analysis of strain estimation methods in phase-sensitive compression optical coherence elastography.

In compression optical coherence elastography (OCE), deformation is quantified as the local strain at each pixel in the OCT field-of-view. A range of strain estimation methods have been demonstrated, yet it is unclear which method provides the best performance. Here, we analyze the two most prevalent strain estimation methods used in phase-sensitive compression OCE, i.e., weighted least squares (WLS) and the vector method. We introduce a framework to compare strain imaging metrics, incorporating strain sensitivity, strain signal-to-noise ratio (SNR), strain resolution, and strain accuracy. In addition, we propose a new phase unwrapping algorithm in OCE, fast phase unwrapping (FPU), and combine it with WLS, termed WLSFPU. Using the framework, we compare this new strain estimation method with both a current implementation of WLS that incorporates weighted phase unwrapping (WPU), termed WLSWPU, and the vector method. Our analysis reveals that the three methods provide similar strain sensitivity, strain SNR, and strain resolution, but that WLSFPU extends the dynamic range of accurate, measurable local strain, e.g., measuring a strain of 2.5 mɛ with ∼4% error, that is ×11 and ×15 smaller than the error measured using WLSWPU and the vector method, respectively. We also demonstrate, for the first time, the capability to detect sub-resolution contrast in compression OCE, i.e., changes in strain occurring within the strain axial resolution, and how this contrast varies between the different strain estimation methods. Lastly, we compare the performance of the three strain estimation methods on mouse skeletal muscle and human breast tissue and demonstrate that WLSFPU avoids strain imaging artifacts resulting from phase unwrapping errors in WLSWPU and provides improved contrast over the other two methods.

Near-infrared, wavelength, and illumination scanning holographic tomography.

We present a holographic tomography technique in which the projections are acquired using both wavelength and illumination scanning in the near-infrared region. We show how to process the acquired data to obtain correct values of three-dimensional refractive index distributions in both single-wavelength and multi-wavelength data acquisition schemes and how to properly account for the dispersion of the sample. We perform numerical and experimental comparisons of different illumination scenarios to determine the most efficient measurement protocol. We show that the multi-wavelength protocol is advantageous in terms of signal-to-noise ratio and contrast-to-noise ratio over single-wavelength protocols, even for the same number of projections used for reconstructions. Finally, we show that this approach is suitable for providing high-quality refractive index distributions of relatively thick colon cancer samples.

Pupillary Light Reflex Induced by Two-Photon Vision.

Purpose: Two-photon vision relies on the perception of pulsed infrared light due to two-photon absorption in visual pigments. This study aimed to measure human pupil reaction caused by a two-photon 1040-nm stimulus and compare it with pupil responses elicited by 520-nm stimuli of similar color. Methods: Pupillary light reflex (PLR) was induced on 14 dark-adapted healthy subjects. Three types of fovea-centered stimuli of 3.5° diameter were tested: spirals formed by fast scanning 1040-nm (infrared [IR] laser) or 520-nm (visible [VIS] laser) laser beams and uniformly filled circle created by 520-nm LED (VIS light-emitting diode [LED]). The power of visible stimuli was determined with a dedicated procedure to obtain the same perceived brightness equivalent as for 800 µW used for two-photon stimulation. Results: The minimum pupil diameter for IR laser was 88% ± 10% of baseline, significantly larger than for both VIS stimuli: 74% ± 10% (laser) and 69% ± 9% (LED). Mean constriction velocity and time to maximum constriction had significantly smaller values for IR than for both VIS stimuli. Latency times were similar for IR and VIS lasers and slightly smaller for VIS LED. Conclusions: The two-photon stimulus caused a considerably weaker pupil reaction than one-photon stimuli of the same shape, brightness, and similar color. The smaller pupil response may be due to weaker two-photon stimulation of rods relative to cones as previously observed for two-photon vision. Contrary to normal vision, in a two-photon process the stray light is not perceived, which might reduce the number of stimulated photoreceptors and further weaken the PLR.

Accurate calibration of beam trajectories in scanning optical imaging systems.

We present a calibration method for finding the coordinates of points in the trajectory of the scanning beam in flying-spot imaging devices. Our method is based on laterally translating the field of view on the imaging object plane by introducing additional beam deflections. We show that laterally translating the field of view provides a series of images whose relative translations are equal to the distances between the points in the scanning pattern to be calibrated. We show how these distances are mapped to the coordinates of the trajectory points. As an example, we demonstrate the calibration of the scanning patterns in an optical system with two independent microelectromechanical system based scanners. Our method profits from a large collection of distance measurements to find the trajectory coordinates, thereby minimizing the effect of random sources of uncertainty in the positions of points in the scanning pattern. We have found that we are capable of finding the coordinates of points in the scanning patterns with accuracy greater than the optical resolution of the imaging system.

Computational Optimization of the Size of Gold Nanorods for Single-Molecule Plasmonic Biosensors Operating in Scattering and Absorption Modes.

We present a comprehensive computational study on the optimization of the size of gold nanorods for single-molecule plasmonic sensing in terms of optical refractive index sensitivity. We construct an experimentally relevant model of single-molecule-single-nanoparticle sensor based on spherically capped gold nanorods, tip-specific functionalization and passivation layers, and biotin-streptavidin affinity system. We introduce a universal figure of merit for the sensitivity, termed contrast-to-noise ratio (CNR), which relates the change of measurable signal caused by the discrete molecule binding events to the inherent measurement noise. We investigate three distinct sensing modalities relying on direct spectral measurements, monitoring of scattering intensity at fixed wavelength and photothermal effect. By considering a shot-noise-limited performance of an experimental setup, we demonstrate the existence of an optimum nanorod size providing the highest sensitivity for each sensing technique. The optimization at constant illumination intensity (i.e., low-power applications) yields similar values of approximately 20 × 80 nm2 for each considered sensing technique. Second, we investigate the impact of geometrical and material parameters of the molecule and the functionalization layer on the sensitivity. Finally, we discuss the variable illumination intensity for each nanorod size with the steady-state temperature increase as its limiting factor (i.e., high-power applications).

Artefact-removal algorithms for Fourier domain quantum optical coherence tomography.

Quantum Optical Coherence Tomography (Q-OCT) is a non-classical equivalent of Optical Coherence Tomography and is able to provide a twofold axial resolution increase and immunity to resolution-degrading dispersion. The main drawback of Q-OCT are artefacts which are additional elements that clutter an A-scan and lead to a complete loss of structural information for multilayered objects. Whereas there are very practical and successful methods for artefact removal in Time-domain Q-OCT, no such scheme has been devised for Fourier-domain Q-OCT (Fd-Q-OCT), although the latter modality-through joint spectrum detection-outputs a lot of useful information on both the system and the imaged object. Here, we propose two algorithms which process a Fd-Q-OCT joint spectrum into an artefact-free A-scan. We present the theoretical background of these algorithms and show their performance on computer-generated data. The limitations of both algorithms with regards to the experimental system and the imaged object are discussed.

Extraction of phase-based optoretinograms (ORG) from serial B-scans acquired over tens of seconds by mouse retinal raster scanning OCT system.

Several specialized retinal optical coherence tomography (OCT) acquisition and processing methods have been recently developed to allow in vivo probing of light-evoked photoreceptors function, focusing on measurements in individual photoreceptors (rods and cones). Recent OCT investigations in humans and experimental animals have shown that the outer segments in dark-adapted rods and cones elongate in response to the visible optical stimuli that bleach fractions of their visual photopigment. We have previously successfully contributed to these developments by implementing OCT intensity-based "optoretinograms" (ORG), the paradigm of using near-infrared OCT (NIR OCT) to measure bleaching-induced back-scattering and/or elongation changes of photoreceptors in the eye in vivo. In parallel, several groups have successfully implemented phase-based ORGs, mainly in human studies, exploiting changes in the phases of back-scattered light. This allowed more sensitive observations of tiny alterations of photoreceptors structures. Applications of the phase-based ORG have been implemented primarily in high speed and cellular resolution AO-OCT systems that can visualize photoreceptor mosaic, allowing phase measurements of path length changes in outer segments of individual photoreceptors. The phase-based ORG in standard resolution OCT systems is much more demanding to implement and has not been explored extensively. This manuscript describes our efforts to implement a phase analysis framework to retinal images acquired with a standard resolution and raster scanning OCT system, which offers much lower phase stability than line-field or full-field OCT detection schemes due to the relatively slower acquisition speed. Our initial results showcase the successful extraction of phase-based ORG signal from the B-scans acquired at ∼100 Hz rate and its favorable comparison with intensity-based ORG signal extracted from the same data sets. We implemented the calculation of phase-based ORG signals using Knox-Thompson paths and modified signal recovery by adding decorrelation weights. The phase-sensitive ORG signal analysis developed here for mouse retinal raster scanning OCT systems could be in principle extended to clinical retinal raster scanning OCT systems, potentially opening doors for clinically friendly ORG probing.

High-resolution, ultrafast, wide-field retinal eye-tracking for enhanced quantification of fixational and saccadic motion.

We introduce a novel, noninvasive retinal eye-tracking system capable of detecting eye displacements with an angular resolution of 0.039 arcmin and a maximum velocity of 300°/s across an 8° span. Our system is designed based on a confocal retinal imaging module similar to a scanning laser ophthalmoscope. It utilizes a 2D MEMS scanner ensuring high image frame acquisition frequencies up to 1.24 kHz. In contrast with leading eye-tracking technology, we measure the eye displacements via the collection of the observed spatial excursions for all the times corresponding a full acquisition cycle, thus obviating the need for both a baseline reference frame and absolute spatial calibration. Using this approach, we demonstrate the precise measurement of eye movements with magnitudes exceeding the spatial extent of a single frame, which is not possible using existing image-based retinal trackers. We describe our retinal tracker, tracking algorithms and assess the performance of our system by using programmed artificial eye movements. We also demonstrate the clinical capabilities of our system with in vivo subjects by detecting microsaccades with angular extents as small as 0.028°. The rich kinematic ocular data provided by our system with its exquisite degree of accuracy and extended dynamic range opens new and exciting avenues in retinal imaging and clinical neuroscience. Several subtle features of ocular motion such as saccadic dysfunction, fixation instability and abnormal smooth pursuit can be readily extracted and inferred from the measured retinal trajectories thus offering a promising tool for identifying biomarkers of neurodegenerative diseases associated with these ocular symptoms.

Blood flow rate estimation in optic disc capillaries and vessels using Doppler optical coherence tomography with 3D fast phase unwrapping.

The retinal volumetric flow rate contains useful information not only for ophthalmology but also for the diagnosis of common civilization diseases such as diabetes, Alzheimer's disease, or cerebrovascular diseases. Non-invasive optical methods for quantitative flow assessment, such as Doppler optical coherence tomography (OCT), have certain limitations. One is the phase wrapping that makes simultaneous calculations of the flow in all human retinal vessels impossible due to a very large span of flow velocities. We demonstrate that three-dimensional Doppler OCT combined with three-dimensional four Fourier transform fast phase unwrapping (3D 4FT FPU) allows for the calculation of the volumetric blood flow rate in real-time by the implementation of the algorithms in a graphics processing unit (GPU). The additive character of the flow at the furcations is proven using a microfluidic device with controlled flow rates as well as in the retinal veins bifurcations imaged in the optic disc area of five healthy volunteers. We show values of blood flow rates calculated for retinal capillaries and vessels with diameters in the range of 12-150 µm. The potential of quantitative measurement of retinal blood flow volume includes noninvasive detection of carotid artery stenosis or occlusion, measuring vascular reactivity and evaluation of vessel wall stiffness.

In vivo brain imaging with multimodal optical coherence microscopy in a mouse model of thromboembolic photochemical stroke.

We used a new multimodal imaging system that combines optical coherence microscopy and brightfield microscopy. Using this in vivo brain monitoring approach and cranial window implantation, we three-dimensionally visualized the vascular network during thrombosis, with high temporal (18 s) and spatial (axial, 2.5 µ m ; lateral, 2.2 µ m ) resolution. We used a modified mouse model of photochemical thromboembolic stroke in order to more accurately parallel human stroke. Specifically, we applied green laser illumination to focally occlude a branch of the middle cerebral artery. Despite the recanalization of the superficial arteries at 24 h after stroke, no blood flow was detected in the small vessels within deeper regions. Moreover, after 24 h of stroke progression, scattering signal enhancement was observed within the stroke region. We also evaluated the infarct extent and shape histologically. In summary, we present a novel approach for real-time mouse brain monitoring and ischemic variability analysis. This multimodal imaging method permits the analysis of thrombosis progression and reperfusion. Additionally and importantly, the system could be used to study the effect of poststroke drug treatments on blood flow in small arteries and capillaries of the brain.

Spatiotemporal optical coherence (STOC) manipulation suppresses coherent cross-talk in full-field swept-source optical coherence tomography.

Full-field swept-source optical coherence tomography (FF-SS-OCT) provides high-resolution depth-resolved images of the sample by parallel Fourier-domain interferometric detection. Although FF-SS-OCT implements high-speed volumetric imaging, it suffers from the cross-talk-generated noise from spatially coherent lasers. This noise reduces the transversal image resolution, which in turn, limits the wide adaptation of FF-SS-OCT for practical and clinical applications. Here, we introduce the novel spatiotemporal optical coherence (STOC) manipulation. In STOC the time-varying inhomogeneous phase masks are used to modulate the light incident on the sample. By properly adjusting these phase masks, the spatial coherence can be reduced. Consequently, the cross-talk-generated noise is suppressed, the transversal image resolution is improved by the factor of 2 , and sample features become visible. STOC approach is validated by imaging 1951 USAF resolution test chart covered by the diffuser, scattering phantom and the rat skin ex vivo. In all these cases STOC suppresses the cross-talk-generated noise, and importantly, do not compromise the transversal resolution. Thus, our method provides an enhancement of FF-SS-OCT that can be beneficial for imaging biological samples.

Light microscopy of mammalian gametes and embryos: methods and applications.

In recent years, we have witnessed an unprecedented advancement of light microscopy techniques which has allowed us to better understand biological processes occurring during oogenesis and early embryonic development in mammals. In short, two modes of cellular imaging are now available: those involving fluorescent labels and those which are fluorophore-free. Fluorescence microscopy, in its various forms, is used predominantly in research, as it provides detailed information about cellular processes; however, it can involove an increased risk of photodamage. Fluorophore-free techniques provide, on the other hand, a smaller amount of biological data but they are safer for cells and therefore can be potentially used in a clinical setting. Here, we review various fluorescence and fluorophore-free visualisation approaches and discuss their applicability in developmental biology and reproductive medicine.

Computationally effective 2D and 3D fast phase unwrapping algorithms and their applications to Doppler optical coherence tomography.

We propose a simplification for a robust and easy to implement fast phase unwrapping (FPU) algorithm that is used to solve the phase wrapping problem encountered in various fields of optical imaging and metrology. We show that the number of necessary computations using the algorithm can be reduced compared to its original version. FPU can be easily extended from two to three spatial dimensions. We demonstrate the applicability of the two- and three-dimensional FPU algorithm for Doppler optical coherence tomography (DOCT) in numerical simulations, and in the imaging of a flow phantom and blood flow in the human retina in vivo. We introduce an FPU applicability plot for use as a guide in the selection of the most suitable version of the algorithm depending on the phase noise in the acquired data. This plot uses the circular standard deviation of the wrapped phase distribution as a measure of noise and relates it to the root-mean-square error of the recovered, unwrapped phase.

Quality improvement of OCT angiograms with elliptical directional filtering.

We present a method of OCT angiography (OCTA) data filtering for noise suppression and improved visualization of the retinal vascular networks in en face projection images. In our approach, we use a set of filters applied in three orthogonal axes in the three-dimensional (3-D) data sets. Minimization of artifacts generated in B-scan-wise data processing is accomplished by filtering the cross-sections along the slow scanning axis. A-scans are de-noised by axial filtering. The core of the method is the application of directional filtering to the C-scans, i.e. one-pixel thick sections of the 3-D data set, perpendicular to the direction of the scanning OCT beam. The method uses a concept of structuring, directional kernels of shapes matching the geometry of the image features. We use rotating ellipses to find the most likely local orientation of the vessels and use the best matching ellipses for median filtering of the C-scans. We demonstrate our approach in the imaging of a normal human eye with laboratory-grade spectral-domain OCT setup. The "field performance" is demonstrated in imaging of diabetic retinopathy cases with a commercial OCT device. The absolute complex differences method is used for the generation of OCTA images from the data collected in the most noise-wise unfavorable OCTA scanning regime-two frame scanning.