This PDF file contains the front matter associated with SPIE Proceedings Volume 9334, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Selective plane illumination microscopy (SPIM) is a 3D imaging technique that uses a sheet of light to optically section a sample in vivo. A cylindrical lens focuses collimated light in one dimension, producing a sheet that is formed in the sample via an objective lens. Any optical power within the sample will additionally refract the light sheet passing through it. We exploit this effect to track the development of the optical power of the zebrafish lens over the first 4 days post fertilisation (dpf). We show that light is focussed on to the photoreceptor layer of the retina at 4 dpf.

Studying the dynamic events involved in early preimplantation embryo development during their transport from the ovary to the uterus is of great significance to improve the understanding of infertility, and eventually to help reduce the infertility rate. The mouse is a widely used mammalian model in reproductive biology, however, dynamic imaging studies of mouse preimplantation embryos have been very limited due to the lack of proper imaging tools for such analysis. Here, we introduce an innovative approach, which can potentially be used for three-dimensional imaging and tracking of murine oocytes with optical coherence tomography (OCT) as they exit the ovary and migrate through the oviduct to the uterus. The imaging is performed with spectral-domain OCT system operating at 70 kHz A-scan rate. The preimplantation embryos and surrounding cumulus cells can be clearly visualized. Results from our experiments indicate that OCT has great potential for dynamic imaging of the oviduct and oocyte tracking, which provides the foundation for future investigations aimed at understanding dynamic events during preimplantation stages in normal development as well as in mouse models of infertility.

How an entire nervous system develops remains mysterious. We have developed a light-sheet microscope system to examine neurodevelopment in C. elegans embryos. Our system creates overlapping light sheets from two orthogonally positioned objectives, enabling imaging from the first cell division to hatching (~14 hours) with 350 nm isotropic resolution. We have also developed computer algorithms to computationally straighten nematode embryos, facilitating data comparison and combination from multiple animals. We plan to use these tools to create an atlas showing the position and morphology of all neurons in the developing embryo.

In recent years we have witnessed a shift from qualitative image analysis towards higher resolution, quantitative analyses of imaging data in developmental biology. This shift has been fueled by technological advances in both imaging and analysis software. We have recently developed a tool for accurate, semi-automated nuclear segmentation of imaging data from early mouse embryos and embryonic stem cells. We have applied this software to the study of the first lineage decisions that take place during mouse development and established analysis pipelines for both static and time-lapse imaging experiments. In this paper we summarize the conclusions from these studies to illustrate how quantitative, single-cell level analysis of imaging data can unveil biological processes that cannot be revealed by traditional qualitative studies.

The murine model is a common model for studying developmental diseases. In this study, we compare the performance of the relatively new method of Optical Projection Tomography (OPT) to the well-established technique of Optical Coherence Tomography (OCT) to assess murine embryonic development at three stages, 9.5, 11.5, and 13.5 days post conception. While both methods can provide spatial resolution at the micrometer scale, OPT can provide superior imaging depth compared to OCT. However, OPT requires samples to be fixed, placed in an immobilization media such as agar, and cleared before imaging. Because OCT does not require fixing, it can be used to image embryos in vivo and in utero. In this study, we compare the efficacy of OPT and OCT for imaging murine embryonic development. The data demonstrate the superior capability of OPT for imaging fine structures with high resolution in optically-cleared embryos while only OCT can provide structural and functional imaging of live embryos ex vivo and in utero with micrometer scale resolution.

Understanding mouse embryonic development is an invaluable resource for our interpretation of normal human embryology and congenital defects. Our research focuses on developing methods for live imaging and dynamic characterization of early embryonic development in mouse models of human diseases. Using multidisciplinary methods: optical coherence tomography (OCT), live mouse embryo manipulations and static embryo culture, molecular biology, advanced image processing and computational modeling we aim to understand developmental processes. We have developed an OCT based approach to image live early mouse embryos (E8.5 – E9.5) cultured on an imaging stage and visualize developmental events with a spatial resolution of a few micrometers (less than the size of an individual cell) and a frame rate of up to hundreds of frames per second and reconstruct cardiodynamics in 4D (3D+time). We are now using these methods to study how specific embryonic lethal mutations affect cardiac morphology and function during early development.

The heart undergoes remarkable changes during embryonic development due to genetic programming and epigenetic influences, in which mechanical loads is a key factor. As embryonic research development, an important goal is to develop mathematical models that describe the influence of mechanics on embryonic heart development. However, basic parameters for the modeling are difficult to acquire since the embryonic heart is tiny and beating fast in the early stages. Optical coherence tomography (OCT) technique provides depth-resolved image with high resolution and high acquisition speed in a noninvasive manner. In this paper, we performed 4D[(x,y,z) + t] scan on the outflow tract (OFT) of the chick embryonic heart at stage of HH18(~ 3 days of incubation) in vivo using spectral domain OCT (SDOCT). Parameters such as displacement and geometrical size of the OFT were extracted from the structural images of the SDOCT. Two-dimensional strain vector were solved using strain-displacement relations in curvilinear cylindrical coordinates based on kinetic theory of elasticity. Based on the geometrical size and other initial conditions, two-dimensional elasticity finite element model of the OFT myocardial wall deformation were established and then solved by direct frequency response method. Comparison between experimental data and simulation result shows the utility of the finite element models. Our results demonstrate that mathematical modeling based on parameters provided by SDOCT is a useful approach for studying cardiac development in early stage.

The cardiac development is a complicated process affected by genetic and environmental factors. Wall shear stress (WSS) is one of the components which have been proved to influence the morphogenesis during early stages of cardiac development. To study the mechanism, WSS measurement is a step with significant importance. WSS is caused by blood flow imposed on the inner surface of the heart wall and it can be determined by calculating velocity gradients of blood flow in a direction perpendicular to the wall. However, the WSS of the early stage embryonic heart is difficult to measure since the embryonic heart is tiny and beating fast. Optical coherence tomography (OCT) is a non-invasive imaging modality with high spatial and temporal resolution, which is uniquely suitable for the study of early stage embryonic heart development. In this paper, we introduce a method to measure the WSS of early stage chick embryonic heart based on high speed spectral domain optical coherence tomography (SDOCT). 4D (x,y,z,t) scan was performed on the outflow tract (OFT) of HH18 (~3 days of incubation) chick embryonic heart. After phase synchronization, OFT boundary segmentation, and OFT center line calculation, Doppler angle of the blood flow in the OFT can be achieved (This method has been described in previous publications). Combining with the Doppler OCT results, we calculate absolute blood flow velocity distribution in the OFT. The boundary of the OFT was segmented at each cross-sectional structural image, then geometrical center of the OFT can be calculated. Thus, the gradients of blood flow in radial direction can be calculated. This velocity gradient near the wall is termed wall shear rate and the WSS value is proportional to the wall shear rate. Based on this method, the WSS at different heart beating phase are compare. The result demonstrates that OCT is capable of early stage chicken embryonic heart WSS study.

Multi-frame superresolution technique has been used to improve the lateral resolution of spectral domain optical coherence tomography (SD-OCT) for imaging of 3D microstructures. By adjusting the voltages applied to 𝑥 and 𝑦 galvanometer scanners in the measurement arm, small lateral imaging positional shifts have been introduced among different C-scans. Utilizing the extracted 𝑥-𝑦 plane en face image frames from these specially offset C-scan image sets at the same axial position, we have reconstructed the lateral high resolution image by the efficient multi-frame superresolution technique. To further improve the image quality, we applied the latest K-SVD and bilateral total variation denoising algorithms to the raw SD-OCT lateral images before and along with the superresolution processing, respectively. The performance of the SD-OCT of improved lateral resolution is demonstrated by 3D imaging a microstructure fabricated by photolithography and a double-layer microfluidic device.