Fluorescent two-photon selective-plane illumination microscopy (2P-SPIM) enables deep imaging of cellular information such as proliferation, type identification, and signaling using fluorescence. Optical coherence tomography (OCT) can capture complementary structural information based on intrinsic optical scattering. We developed a specialized multimodal high-resolution embryonic imaging system combining the benefits of OCT with 2P-SPIM. The OCT and 2P-SPIM beams were optically co-aligned and scanned using the same scanners and the same objective lens. The resulting light sheet thickness was ~13 µm with a transverse resolution of ~2.1 µm. The OCT system was based on a 1050 nm centered swept source laser with a bandwidth of ~100 nm and a sweep rate of 100 kHz. The OCT system utilized a Michelson-style interferometer and had a lateral resolution of ~15 µm and an axial resolution of ~7 µm. The capabilities of the multimodal imaging system were demonstrated using images of fluorescent microbeads and a fluorescently tagged mouse embryo at gestational day 9.5. Due to the co-alignment of the OCT and 2P-SPIM systems, image registration was simple and allowed for high-throughput multimodal imaging without the use of sophisticated registration methods.
Optical coherence tomography (OCT) and light sheet fluorescence microscopy (LSFM) are well-established imaging techniques preferred in developmental biology, e.g., embryonic imaging. However, each technique has its own drawbacks, such as resolution and molecular specificity with OCT and field-of-view (FOV) and speed with LSFM. To overcome these limitations for small animal embryo imaging, we have developed a co-aligned multimodal imaging system combining OCT and LSFM. The OCT probe and LSFM excitation beams were combined and scanned with a galvanometer-mounted mirror through the same objective lens. The light sheet thickness was ~13 μm. The LSFM collection arm consisted of a 0.8 numerical aperture water immersion objective, tube lens, and CCD camera, resulting in a transverse resolution of ~2.1 μm. The OCT system was based on a 100 kHz swept-source laser with a central wavelength of 1050 nm and had a lateral resolution of ~15 µm and an axial resolution of ~7 μm. Images of fluorescent microbeads and a fluorescent-tagged mouse embryo at gestational day 9.5 showed the capabilities of the multimodal imaging system. Since the OCT system and LSFM system were co-aligned, image registration was straightforward and enabled high-throughput multimodal imaging without the need for complex registration techniques.
We developed a high-resolution multimodal system for mouse embryonic imaging that combines Optical Coherence Tomography (OCT) and Light Sheet Fluorescence Microscopy (LSFM). LSFM Illumination is restricted to fluorophores in the focal volume, and collecting the light using a microscope objective increases the signal from that plane and reduces the noise coming from outside of the plane. Colinearly aligning this modality with the OCT beam allows one to acquire the structural information from the same plane that is illuminated by the LSFM beam. A 3D image of 9.5 day mouse embryo was captured using this multimodal system.
Hemodynamic force is vital to cardiovascular remodeling in the early post-implantation mouse embryo. Here, we present work using microCT and lightsheet microscopy to establish the critical sequence of developmental events required for forming functional vasculature and circulation in the embryo, yolk sac, and placenta in the context of normal and impaired flow. A flow impaired model, Mlc2a+/- will be used to determine how hemodynamic force affects the specific events during embryonic development and vascular remodeling between the 4 and 29-somite stage using microCT. We have recently established high-resolution methods for the generation of 3D image volumes from the whole embryo within the deciduum (Hsu et al., in revision). This method enables the careful characterization of 3D images of vitelline and umbilical vessel remodeling to define how poor blood flow impacts both vitelline and umbilical vessel remodeling. Novel lightsheet live imaging techniques will be used to determine the consequence of impaired blood flow on yolk sac vasculature remodeling and formation of umbilical vessels using transgenic reporters: Flk-myr::mCherry, Flk1-H2B::YFP, or εGlobin-GFP. High-resolution 3D imaging of fixed and ScaleA2-cleared whole mount embryos labeled with Ki67 and Caspase3 will also be performed using lightsheet microscopy to quantify the proliferation and apoptotic indexes of early post-implanted embryos and yolk sac. This multi-modality approach is aimed at revealing further information about the cellular mechanisms required for proper vessel remodeling and the initial stages in placentation during early post-implantation development.
Both optical coherence tomography (OCT) and selective plane illumination microscopy (SPIM) are frequently used in
mouse embryonic research for high-resolution three-dimensional imaging. However, each of these imaging methods
provide a unique and independent advantage: SPIM provides morpho-functional information through
immunofluorescence and OCT provides a method for whole-embryo 3D imaging. In this study, we have combined
rotational imaging OCT and SPIM into a single, dual-modality device to image E9.5 mouse embryos. The results
demonstrate that the dual-modality setup is able to provide both anatomical and functional information simultaneously
for more comprehensive tissue characterization.
Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development.
The mouse is a common model for studying developmental diseases. Different optical techniques have been developed to investigate mouse embryos, but each has its own set of limitations and restrictions. In this study, we imaged the same E9.5 mouse embryo with rotational imaging Optical Coherence Tomography (RI-OCT) and Selective Plane Illumination Microscopy (SPIM), and compared the two techniques. Results demonstrate that both methods can provide images with micrometer-scale spatial resolution. The RI-OCT technique was developed to increase imaging depth of OCT by performing traditional OCT imaging at multiple sides and co-registering the images. In SPIM, optical sectioning is achieved by illuminating the sample with a sheet of light. In this study, the images acquired from both techniques are compared with each other to evaluate the benefits and drawbacks of each technique for embryonic imaging. Since 3D stacks can be obtained by SPIM from different angles by rotating the sample, it might be possible to build a hybrid setup of two imaging modalities to combine the advantages of each technique.
Optical coherence tomography (OCT) has been widely used to study mammalian embryonic development with the advantages of high spatial and temporal resolutions and without the need for any contrast enhancement probes. However, the limited imaging depth of traditional OCT might prohibit visualization of the full embryonic body. To overcome this limitation, we have developed a new methodology to enhance the imaging range of OCT in embryonic day (E) 9.5 and 10.5 mouse embryos using rotational imaging. Rotational imaging OCT (RI-OCT) enables full-body imaging of mouse embryos by performing multiangle imaging. A series of postprocessing procedures was performed on each cross-section image, resulting in the final composited image. The results demonstrate that RI-OCT is able to improve the visualization of internal mouse embryo structures as compared to conventional OCT.
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.
Optical coherence tomography (OCT) has proved to be an effective tool to study the development of mammalian
embryos due to its high resolution and contrast. However, light attenuation is an important factor which constrains the imaging depth of OCT. Limitation of imaging depth will inhibit us to better study the structural characteristics of
mouse embryos. Here we propose a new method, rotational imaging OCT (riOCT), to improve the imaging depth and provide full-body embryonic imaging. The experimental setup comprises the swept source OCT system and the square glass tube mounted on a rotational stage. The E10.5 mouse embryos are dissected and immersed in the glass
tube using 0.9% saline solution. 3D structural imaging is performed at four different angles with the interval of 90 degrees. The OCT image records the optical distances of different components such as glass, gelatin and tissue. The position of rotation center is determined by the track of the glass tube center at different angles. The final image is acquired by rotating the images at different angles according to the rotation center. Results indicate that this method is able to improve the visualization of structural information of mouse embryo compared to conventional OCT.
Because of the ease in generating transgenic/gene knock out models and accessibility to early stages of embryogenesis, mouse and rat models have become invaluable to studying the mechanisms that underlie human birth defects. To study precisely how structural birth defects arise, Ultrasound, MRI, microCT, Optical Projection Tomography (OPT), Optical Coherence Tomography (OCT) and histological methods have all been used for imaging mouse/rat embryos. However, of these methods, only OCT enables live, functional imaging with high spatial and temporal resolution. However, one of the major limitations of conventional OCT imaging is the light depth penetration, which limits acquisition of structural information from the whole embryo. Here we introduce new imaging scheme by OCT imaging from different sides of the embryos that extend the depth penetration of OCT to permit high-resolution imaging of 3D and 4D volumes.
Optical coherence tomography (OCT) is gaining popularity as live imaging tool for embryonic research in
animal models. Recently we have demonstrated that OCT can be used for live imaging of cultured early mouse
embryos (E7.5-E10) as well as later stage mouse embryos in utero (E12.5 to the end of gestation). Targeted
delivery of signaling molecules, drugs, and cells is a powerful approach to study normal and abnormal
development, and image guidance is highly important for such manipulations. Here we demonstrate that OCT
can be used to guide microinjections of gold nanoshell suspensions in live mouse embryos. This approach can
potentially be used for variety of applications such as guided injections of contrast agents, signaling molecules,
pharmacological agents, cell transplantation and extraction, as well as other image-guided micromanipulations.
Our studies also reveal novel potential for gold nanoshells in embryonic research.
We have used a swept-source optical coherence tomography (OCT) system to study the development of eyes in mice embryo in utero at different development stages from E13.5 - 18.5. Obtained results demonstrate capability of OCT technology for high-resolution imaging of ocular tissues in utero and capability of assessing key developmental characteristics of the eye during embryonic development.
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.
Congenital abnormalities of the limbs are common birth defects. These include missing or extra fingers or toes,
abnormal limb length, and abnormalities in patterning of bones, cartilage or muscles. Optical Coherence
Tomography (OCT) is a 3-D imaging modality, which can produce high-resolution (~8 μm) images of developing
embryos with an imaging depth of a few millimeters. Here we demonstrate the capability of OCT to perform 3D
imaging of limb development in normal embryos and a mouse model with congenital abnormalities. Our results
suggest that OCT is a promising tool to analyze embryonic limb development in mammalian models of congenital
defects.
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.
Studying progression of congenital diseases in animal models can greatly benefit from live embryonic imaging
Mouse have long served as a model of mammalian embryonic developmental processes, however, due to intra-uterine
nature of mammalian development live imaging is challenging. In this report we present results on live mouse
embryonic imaging in utero with Optical Coherence Tomography. Embryos from 12.5 through 17.5 days post-coitus
(dpc) were studied through the uterine wall. In longitudinal studies, same embryos were imaged at developmental stages
13.5, 15.5 and 17.5 dpc. This study suggests that OCT can serve as a powerful tool for live mouse embryo imaging.
Potentially this technique can contribute to our understanding developmental abnormalities associated with mutations,
toxic drugs.
Live imaging of an eye during embryonic development in mammalian model is important for understanding
dynamic aspects of normal and abnormal eye morphogenesis. In this study, we used Swept Source Optical
Coherence Tomography (SS-OCT) for live structural imaging of mouse embryonic eye through the uterine wall. The
eye structure was reconstructed in mouse embryos at 13.5 to 17.5 days post coitus (dpc). Despite the limited imaging
depth of OCT in turbid tissues, we were able to visualize the whole eye globe at these stages. These results suggest
that live in utero OCT imaging is a useful tool to study embryonic eye development in the mouse model.
Congenital abnormalities are often caused by genetic disorders which alter the normal development of the eye.
Embryonic eye imaging in mouse model is important for understanding of normal and abnormal eye development and
can contribute to prevention and treatment of eye defects in humans. In this study, we used Swept Source Optical
Coherence Tomography (SS-OCT) to image eye structure in mouse embryos at 12.5 to 17.5 days post coitus (dpc). The
imaging depth of the OCT allowed us to visualize the whole eye globe at these stages. Different ocular tissues including
lens, cornea, eyelids, and hyaloid vasculature were visualized. These results suggest that OCT imaging is a useful tool to
study embryonic eye development in the mouse model.
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.
Optical coherence tomography (OCT) holds great promise as a routine research tool for 3-D analysis of mammalian embryos. However, despite the depth penetration afforded by this imaging modality, light attenuation in tissues imposes limitations. Here we studied the optical clearing effect of different concentrations of glycerol in mouse embryos. Depth- and time-resolved profiles for OCT signal enhancement are presented. We found that application of 50% glycerol resulted in 51.5±12.5% improvement of the OCT signal, while 25% glycerol enhanced the OCT signal by 25.2±7.3% at the depth of about 200 to 500 µm, and the glycerol permeability rate was estimated as 26.7±6 µm/min. These results demonstrate that embryonic imaging is improved by application of glycerol as optical clearing agent.
Congenital cardiovascular defects are very common, occurring in 1% of live births, and cardiovascular failures are the leading cause of birth defect-related deaths in infants. To improve diagnostics, prevention and treatment of cardiovascular abnormalities, we need to understand not only how cells form the heart and vessels but also how physical factors such as heart contraction and blood flow influence heart development and changes in the circulatory network. Mouse models are an excellent resource for studying cardiovascular development and disease because of the resemblance to humans, rapid generation time, and availability of mutants with cardiovascular defects linked to human diseases. In this work, we present results on development and application of Doppler Swept Source Optical Coherence Tomography (DSS-OCT) for imaging of cardiovascular dynamics and blood flow in the mouse embryonic heart and vessels. Our studies demonstrated that the spatial and temporal resolution of the DSS-OCT makes it possible to perform sensitive measurements of heart and vessel wall movements and to investigate how contractile waves facilitate the movement of blood through the circulatory system.
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.
KEYWORDS: Fluorescence resonance energy transfer, In vivo imaging, Luminescence, Sensors, Heart, Fluorescent proteins, Blood circulation, In vitro testing, Tissues, Biophysics
Endothelial cells that comprise vessels and line the heart are known to respond to mechanical forces imparted
by fluid flow. It is also known that blood flow is required for vascular remodeling and that abnormal heart contractions
lead to the failure of the vasculature to remodel properly. Although there is considerable evidence to indicate that flow is
necessary, little is known about how mechanical signals are transduced in endothelial cells in the embryo. This project is
focused on understanding the role mechanical forces play in the development of the cardiovascular system using recently
generated FRET (Fluorescence Resonance Energy Transfer) reporter that can detect real-time Src-kinase activity in cells
using fluorescence microscopy. Src kinase regulates integrin-cytoskeleton interactions that are essential for
mechanotransduction, and its activity is upregulated in cultured endothelial cells exposed to flow. Experiments reported
here were focused on testing potential feasibility of the proposed technique to sense Src changes in vivo. Successful
implementation of this project will reveal previously unknown signaling events involved in the mechanism of vascular
remodeling and their relation to the blood flow, thus providing a unique tool for in vivo sub-cellular imaging of
mechanotransduction in the vasculature and other organs.
With the availability of new confocal laser scanning microscopes, fast biological processes, such as the blood flow in living organisms at early stages of the embryonic development, can be observed with unprecedented time resolution. When the object under study has a periodic motion, e.g. a beating embryonic heart, the imaging capabilities can be extended to retrieve 4D data. We acquire nongated slice-sequences at increasing depth and retrospectively synchronize them to build dynamic 3D volumes. Here, we present a synchronization procedure based on the temporal correlation of wavelet features. The method is designed to handle large data sets and to minimize the influence of artifacts that are frequent in fluorescence imaging techniques such as bleaching, nonuniform contrast, and photon-related noise.
KEYWORDS: Heart, Wavelets, Cardiac imaging, Point spread functions, Confocal microscopy, Data acquisition, Microscopes, Wavelet transforms, In vivo imaging, 3D image processing
Being able to acquire, visualize, and analyze 3D time series (4D data) from living embryos makes it possible to understand complex dynamic movements at early stages of embryonic development. Despite recent technological breakthroughs in 2D dynamic imaging, confocal microscopes remain quite slow at capturing optical sections at successive depths. However, when the studied motion is periodic—such as for a beating heart—a way to circumvent this problem is to acquire, successively, sets of 2D+time slice sequences at increasing depths over at least one time period and later rearrange them to recover a 3D+time sequence. In other imaging modalities at macroscopic scales, external gating signals, e.g., an electro-cardiogram, have been used to achieve proper synchronization. Since gating signals are either unavailable or cumbersome to acquire in microscopic organisms, we have developed a procedure to reconstruct volumes based solely on the information contained in the image sequences. The central part of the algorithm is a least-squares minimization of an objective criterion that depends on the similarity between the data from neighboring depths. Owing to a wavelet-based multiresolution approach, our method is robust to common confocal microscopy artifacts. We validate the procedure on both simulated data and in vivo measurements from living zebrafish embryos.
Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.
In order to study complex cellular interactions in the developing somite and nervous system, we have been refining techniques for labeling and imaging individual cells within the living vertebrate embryo. Most recently, we have been using MPLSM to analyze cellular behaviors, such as cell migration, filopodial extension, cell process collapse, and neuron pathfinding using time-lapse microscopy in 3-dimensions (3-d). To enhance the efficiency of two-photon excitation in these samples, we have been using a Zeiss LSM 510 NLO fiber delivery system with a Grating Dispersion Compensator (GDC). This system not only offers the convenience of fiber delivery for coupling our Ti:Sapphire laser to the microscope, but also affords us precise control over the pulsewidth of the mode- locked beam. In addition, we have developed a novel peptide/non-cationic lipid gene delivery system to introduce GFP plasmid into somite cells. This approach has allowed us to generate detailed 3-d images of somite cell morphologies at various stages of somite development in a way that best preserves the vitality of the cells being imaged.
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