Irina V. Larina

Live imaging of blood flow in mammalian embryos using Doppler swept-source optical coherence tomography

Studying hemodynamic changes during early mammalian embryonic development is critical for further advances in prevention, diagnostics, and treatment of congenital cardiovascular (CV) birth defects and diseases. Doppler optical coherence tomography (OCT) has been shown to provide sensitive measurements of blood flow in avian and amphibian embryos. We combined Doppler swept-source optical coherence tomography (DSS-OCT) and live mouse embryo culture to analyze blood flow dynamics in early embryos. SS-OCT structural imaging was used for the reconstruction of embryo morphology and the orientation of blood vessels, which is required for calculating flow velocity from the Doppler measurements. Spatially and temporally resolved blood flow profiles are presented for the dorsal aorta and a yolk sac vessel in a 9.5-day embryo. We demonstrate that DSS-OCT can be successfully used for structural analysis and spatially and temporally resolved hemodynamic measurements in developing early mammalian embryos.

Hemodynamic measurements from individual blood cells in early mammalian embryos with Doppler swept source OCT

The most common and lethal birth defects affect the cardiovascular (CV) system. The mouse is a superior model for identifying and understanding mammalian CV birth defects, but there is a great need for tools that can detect early and subtle deficiencies in cardiac function in mouse embryos. We combined swept source optical coherence tomography (SS-OCT) with live mouse embryo culture protocols to generate structural two-dimensional and three-dimensional imaging and hemodynamic measurements in a live 8.5 day embryo just a few hours after the beginning of a heartbeat. Our data show that individual circulating blood cells can be visualized with structural SS-OCT, and using Doppler SS-OCT the velocity of single moving blood cells were measured during different phases of the heartbeat cycle. These results demonstrate that Doppler SS-OCT is an extremely useful tool for structural and hemodynamic analysis at the earliest stages of mammalian blood circulation.

A membrane associated mCherry fluorescent reporter line for studying vascular remodeling and cardiac function during murine embryonic development

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell–cell and cell‐matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr‐mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr‐mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr‐mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B‐EYFP) line, cell number in addition to cell architecture is revealed, making it possible to determine how individual endothelial cells contribute to the structure of the vessel. Anat Rec, 2008.

Optical coherence tomography for high-resolution imaging of mouse development in utero

Although the mouse is a superior model to study mammalian embryonic development, high-resolution live dynamic visualization of mouse embryos remain a technical challenge. We present optical coherence tomography as a novel methodology for live imaging of mouse embryos through the uterine wall thereby allowing for time lapse analysis of developmental processes and direct phenotypic analysis of developing embryos. We assessed the capability of the proposed methodology to visualize structures of the living embryo from embryonic stages 12.5 to 18.5 days postcoitus. Repetitive in utero embryonic imaging is demonstrated. Our work opens the door for a wide range of live, in utero embryonic studies to screen for mutations and understand the effects of pharmacological and toxicological agents leading to birth defects.

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology.

The Flk1‐myr::mCherry mouse as a useful reporter to characterize multiple aspects of ocular blood vessel development and disease

The highly vascularized mouse eye is an excellent model system in which to elucidate the molecular genetic basis of blood vessel development and disease. However, the analysis of ocular vessel defects has traditionally been derived from fixed tissue, which fails to account for dynamic events such as blood flow and cell migration. To overcome the limitations of static analysis, tremendous advances in imaging technology and fluorescent protein reporter mouse lines now enable the direct visualization of developing cells in vivo. Here, we demonstrate that the Flk1‐myr::mCherry transgenic mouse is an extremely useful live reporter with broad applicability to retinal, hyaloid, and choroid vascular research. Developmental Dynamics 238:2318–2326, 2009.

Sequential Turning Acquisition and Reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures

Optical coherence tomography allows for dynamic, three-dimensional (3D+T) imaging of the heart within animal embryos. However, direct 3D+T imaging frame rates remain insufficient for cardiodynamic analysis. Previously, this limitation has been addressed by reconstructing 3D+T representations of the beating heart based on sets of two-dimensional image sequences (2D+T) acquired sequentially at high frame rate and in fixed (and parallel) planes throughout the heart. These methods either require additional hardware to trigger the acquisition of each 2D+T series to the same phase of the cardiac cycle or accumulate registration errors as the slices are synchronized retrospectively by pairs, without a gating signal. Here, we present a sequential turning acquisition and reconstruction (STAR) method for 3D+T imaging of periodically moving structures, which does not require any additional gating signal and is not prone to registration error accumulation. Similarly to other sequential cardiac imaging methods, multiple fast image series are consecutively acquired for different sections but in between acquisitions, the imaging plane is rotated around the center line instead of shifted along the direction perpendicular to the slices. As the central lines of all image-sequences coincide and represent measurements of the same spatial position, they can be used to accurately synchronize all the slices to a single inherent reference signal. We characterized the accuracy of our method on a simulated dynamic phantom and successfully imaged a beating embryonic rat heart. Potentially, this method can be applied for structural or Doppler imaging approaches with any direct space imaging modality such as computed tomography, ultrasound, or light microscopy.

4D Reconstruction of the Beating Embryonic Heart From Two Orthogonal Sets of Parallel Optical Coherence Tomography Slice-Sequences

Current methods to build dynamic optical coherence tomography (OCT) volumes of the beating embryonic heart involve synchronization of 2D+time slice-sequences acquired over separate heartbeats. Temporal registration of these sequences is performed either through gating or postprocessing. While synchronization algorithms that exclusively rely on image-intrinsic signals allow forgoing external gating hardware, they are prone to error accumulation, require operator-supervised correction, or lead to nonisotropic resolution. Here, we propose an image-based, retrospective reconstruction technique that uses two sets of parallel 2D+T slice-sequences, acquired perpendicularly to each other, to yield accurate and automatic reconstructions with isotropic resolution. The method utilizes the similarity of the data at the slice intersections to spatio-temporally register the two sets of slice sequences and fuse them into a high-resolution 4D volume. We characterize our method by using 1) simulated heart phantom datasets and 2) OCT datasets acquired from the beating heart of live cultured E9.5 mouse and E10.5 rat embryos. We demonstrate that while our method requires greater acquisition and reconstruction time compared to methods that use slices from a single direction, it produces more accurate and self-validating reconstructions since each set of reconstructed slices acts as a reference for the slices in the perpendicular set.

Optical Coherence Tomography for live imaging of mammalian development

Understanding the nature and mechanism of congenital defects of the different organ systems in humans has heavily relied on the analysis of the corresponding mutant phenotypes in rodent models. Optical Coherence Tomography (OCT) has recently emerged as a powerful tool to study early embryonic development. This non-invasive optical methodology does not require labeling and allows visualization of embryonic tissues with single cell resolution. Here, we will discuss how OCT can be applied for structural imaging of early mouse and rat embryos in static culture, cardiodynamic and blood flow analysis, and in utero embryonic imaging at later stages of gestation, demonstrating how OCT can be used to assess structural and functional birth defects in mammalian models.

Optical coherence tomography for live phenotypic analysis of embryonic ocular structures in mouse models

Mouse models of ocular diseases provide a powerful resource for exploration of molecular regulation of eye development and pre-clinical studies. Availability of a live high-resolution imaging method for mouse embryonic eyes would significantly enhance longitudinal analyses and high-throughput morphological screening. We demonstrate that optical coherence tomography (OCT) can be used for live embryonic ocular imaging throughout gestation. At all studied stages, the whole eye is within the imaging distance of the system and there is a good optical contrast between the structures. We also performed OCT eye imaging in the embryonic retinoblastoma mouse model Pax6-SV40 T-antigen, which spontaneously forms lens and retinal lesions, and demonstrate that OCT allows us to clearly differentiate between the mutant and wild type phenotypes. These results demonstrate that OCTin utero imaging is a potentially useful tool to study embryonic ocular diseases in mouse models.