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. 2013 Sep 4;8(9):e73491.
doi: 10.1371/journal.pone.0073491. eCollection 2013.

Design and implementation of a custom built optical projection tomography system

Michael D Wong  1 , Jun Dazai, Johnathon R Walls, Nicholas W Gale, R Mark Henkelman
Affiliations

Design and implementation of a custom built optical projection tomography system

Michael D Wong et al. PLoS One. .

Abstract

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 cm(3). 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.

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Conflict of interest statement

Competing Interests: Nicholas Gale and Johnathon Walls are employees of Regeneron Pharmaceuticals and helped with the design of the proposed system through personal communication. It was ensured that this manuscript does not conflict with any patents, products in development or marketed products at Regeneron. Their employment at Regeneron Pharmaceuticals does not alter the authors' adherence to PLOS ONE policies of sharing and materials as detailed in http://www.PLOSone.org/static/editorial.action#competing.

Figures

Figure 1
Figure 1. Diagram of custom-built OPT hardware set-up.
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 Table 1.
Figure 2
Figure 2. Alignment of the stage assembly with the OPT microscope carving out an ellipse using a bead phantom.
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).
Figure 3
Figure 3. Flow chart of the Labview OPT software.
Figure 4
Figure 4. 3 dimensional point spread function (PSF) of the described OPT system.
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).
Figure 5
Figure 5. E12.5 mouse embryo autofluorescence image acquired with the presented custom OPT system.
(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.
Figure 6
Figure 6. E12.5 mouse embryo autofluorescence image acquired by the presented custom OPT and the commercial Bioptonics 3001 OPT Scanner.
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.
See this image and copyright information in PMC

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