Three-Dimensional Analysis of Vascular Development in the Mouse Embryo

Key vasculogenic (de-novo vessel forming) and angiogenic (vessel remodelling) events occur in the mouse embryo between embryonic days (E) 8.0 and 10.0 of gestation, during which time the vasculature develops from a simple circulatory loop into a complex, fine structured, three-dimensional organ. Interpretation of vascular phenotypes exhibited by signalling pathway mutants has historically been hindered by an inability to comprehensively image the normal sequence of events that shape the basic architecture of the early mouse vascular system. We have employed Optical Projection Tomography (OPT) using frequency distance relationship (FDR)-based deconvolution to image embryos immunostained with the endothelial specific marker PECAM-1 to create a high resolution, three-dimensional atlas of mouse vascular development between E8.0 and E10.0 (5 to 30 somites). Analysis of the atlas has provided significant new information regarding normal development of intersomitic vessels, the perineural vascular plexus, the cephalic plexus and vessels connecting the embryonic and extraembryonic circulation. We describe examples of vascular remodelling that provide new insight into the mechanisms of sprouting angiogenesis, vascular guidance cues and artery/vein identity that directly relate to phenotypes observed in mouse mutants affecting vascular development between E8.0 and E10.0. This atlas is freely available a

Design and Implementation of a Custom Built Optical Projection Tomography System

<div><p>Optical projection tomography (OPT) is an imaging modality that has, in the last decade, answered numerous biological questions owing to its ability to view gene expression in 3 dimensions (3D) at high resolution for samples up to several cm³. This has increased demand for a cabinet OPT system, especially for mouse embryo phenotyping, for which OPT was primarily designed for. The Medical Research Council (MRC) Technology group (UK) released a commercial OPT system, constructed by Skyscan, called the Bioptonics OPT 3001 scanner that was installed in a limited number of locations. The Bioptonics system has been discontinued and currently there is no commercial OPT system available. Therefore, a few research institutions have built their own OPT system, choosing parts and a design specific to their biological applications. Some of these custom built OPT systems are preferred over the commercial Bioptonics system, as they provide improved performance based on stable translation and rotation stages and up to date CCD cameras coupled with objective lenses of high numerical aperture, increasing the resolution of the images. Here, we present a detailed description of a custom built OPT system that is robust and easy to build and install. Included is a hardware parts list, instructions for assembly, a description of the acquisition software and a free download site, and methods for calibration. The described OPT system can acquire a full 3D data set in 10 minutes at 6.7 micron isotropic resolution. The presented guide will hopefully increase adoption of OPT throughout the research community, for the OPT system described can be implemented by personnel with minimal expertise in optics or engineering who have access to a machine shop.</p></div

Optical parameters of the described OPT system.

<p>Optical parameters of the described OPT system are listed for both maximum and minimum magnification. MAG = magnification, N.A. = numerical aperture, D.O.F. = depth of field, F.O.V = field of view.</p

3 dimensional point spread function (PSF) of the described OPT system.

<p>Line profiles of the image intensity through the center of a fluorescent bead along the x (blue) and z (red) axes discretized by pixel number. The full width at half maximum of these line profiles is demonstrative of the lateral and axial resolution of the system (6.77 and 6.72 microns respectively).</p

Diagram of custom-built OPT hardware set-up.

<p>OPT system set-up is presented in three different views to identify each hardware component and its relation to all other parts in the OPT system. The hardware parts are labeled in the view in which they are best displayed. The assembly of parts is described in the text and the parts list is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073491#pone-0073491-t001" target="_blank">Table 1</a>.</p

Alignment of the stage assembly with the OPT microscope carving out an ellipse using a bead phantom.

<p>In this example, the stage assembly is rotated in both the XY and YZ planes with respect to the microscope and the center of rotation is currently at pixel 1100. (A) To align the stage assembly with the microscope in the XY plane, the angle (θ) between the x-axis and the major axis of the ellipse should be reduced from 0.1 to less than 0.01. (B) To align the stage assembly with the microscope in the YZ plane, the diameter of the minor axis should be reduced from 4 pixels to less than 1.5 pixels. (C) To move the center or rotation to the center of the CCD, move the stage assembly such that the center of the ellipse is positioned at the center pixel of the field-of-view (i.e. 1024).</p

OPT hardware parts list.

<p>All parts needed for the presented OPT system are listed here, both commercially available and custom-made. Illumination assembly is separated for hardware parts required for white light (WH) and ultraviolent (UV) illumination. A more descriptive parts lists along with optional system additions are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073491#pone.0073491.s001" target="_blank">Table S1</a>.</p

E12.5 mouse embryo autofluorescence image acquired with the presented custom OPT system.

<p>(A) 3D textured rendering of the whole volume of the mouse embryo is illustrated in orange. Digital sections of the same mouse embryo are illustrated in gray-scale demonstrating autofluorescence anatomy data in sagittal (B), coronal (C), and axial planes (D). An equivalent location in anatomy is shown by the location of the red cross-hair. The scale bar is 2 mm.</p

E12.5 mouse embryo autofluorescence image acquired by the presented custom OPT and the commercial Bioptonics 3001 OPT Scanner.

<p>Similarly positioned sagittal sections through an E12.5 mouse embryo acquired by the custom OPT (A) and the Bioptonics system (B). The higher resolution produced by the custom scanner is visually evident through sharper and more defined edges as well as the observation of individual blood pooling in the vasculature. The scale bar is 2 mm.</p

Flow chart of the Labview OPT software.

<p>Flow chart of the Labview OPT software.</p