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Optical projection tomography (OPT) is a new technique for three-dimensional (3D) imaging of large biological samples (of the order of 1 cubic centimetre). It fills the imaging gap between conventional microscopy and MRI. It is particularly suited to study fundamental biological processes using light emitted from inside the organ, via optical fluorescence. The main advantage of this new imaging modality is that it avoids the need to physically section the sample. Furthermore, OPT is able to take advantage of fluorescent dyes, and different wavelength channels can be used. This allows the observation of the autofluorescence of the tissue (to inform on tissue structure), alongside the mapping of gene and protein expression. Our principal aim is to use the OPT scanner installed in CABI for cancer research, using particular fluorophores to stain tumours and study their vasculature.
How does OPT work?
OPT uses visible light for illumination, and in order to reduce scattering and absorption of light, the sample needs to be rendered transparent by the use of optical clearing techniques. The cleared sample is embedded in a block of agarose gel (1% low-melting-point) and placed on a motor through a magnetic mount. It undergoes a complete rotation within the scanner through angular steps of 0.9 or 0.45 degrees (depending on the desired resolution) while suspended in a quartz cuvette filled with the same solution used for optical clearing, in order to reduce light scattering. The most common clearing approach we use is matching the refractive indices within the organism by replacing intra and extracellular water with 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB). Since the refractive index of BABB is matched to that of tissue, scattering through the sample is minimized and the sample is rendered optically transparent. The specimen in the OPT device is rotated about its vertical
axis while under illumination in the IR-UV wavelengths. A series of optics focus the illumination onto the sample and then the fluorescent light to a CCD camera. An image is acquired at a series of angles and tomographic reconstruction is performed using a back-projection algorithm, and this yields a 3D volumetric representation of the specimen.
See the collaborations page for information on access to this system
