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.

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.

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.

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.

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.

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.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Spectrometer calibration for spectroscopic Fourier domain optical coherence tomography.

We propose a simple and robust procedure for Fourier domain optical coherence tomography (FdOCT) that allows to linearize the detected FdOCT spectra to wavenumber domain and, at the same time, to determine the wavelength of light for each point of detected spectrum. We show that in this approach it is possible to use any measurable physical quantity that has linear dependency on wavenumber and can be extracted from spectral fringes. The actual values of the measured quantity have no importance for the algorithm and do not need to be known at any stage of the procedure. As example we calibrate a spectral OCT spectrometer using Doppler frequency. The technique of spectral calibration can be in principle adapted to of all kind of Fourier domain OCT devices.

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.

Assessment of the flow velocity of blood cells in a microfluidic device using joint spectral and time domain optical coherence tomography.

Although Doppler optical coherence tomography techniques have enabled the imaging of blood flow in mid-sized vessels in biological tissues, the generation of velocity maps of capillary networks remains a challenge. To better understand the origin and information content of the Doppler signal from small vessels and limitations of such measurements, we used joint spectral and time domain optical coherence tomography to monitor the flow in a model, semitransparent microchannel device. The results obtained for Intralipid, whole blood, as well as separated red blood cells indicate that the technique is suitable to record velocity profiles in vitro, in a range of microchannel configurations.