Abstract
We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 μm). All three methods use the microscope’s existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations’ viability.
Highlights
In biological and biomedical research there is an increasing trend towards 3D cell cultures and in vivo model organisms to provide more physiologically relevant context
In this paper we describe a simple and inexpensive opensource adaptor that sits in a standard microscope stage, providing the necessary alignment and sample rotation required for optical projection tomography (OPT) [1], allowing 3D reconstructions from transmitted light and/or fluorescence acquisitions of ~mm sized samples using the microscope’s incoherent light sources
The plate, which slots into a standard microscope stage, incorporates a stepper motor for sample rotation and a tilt adjustment to ensure that the axis of rotation is perpendicular to the optical axis
Summary
In biological and biomedical research there is an increasing trend towards 3D cell cultures and in vivo model organisms to provide more physiologically relevant context. The second implementation uses an image relay attached to the camera port of the microscope such that the limiting aperture can be positioned conjugate to the pupil plane, allowing standard OPT to be performed at higher magnifications.
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