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

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

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

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

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

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

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

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

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature

<div><p>Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.</p></div