New 3D imaging infrastructure available at CRC
Optical Projection Tomography (OPT) is an optical imaging tool that creates high resolution 3D images of large biological samples in the range of 1mm - 30 mm across. It was created to fill 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 imaging modality is that it avoids the need to physically section the sample.
NOD mouse pancreas stained for insulin and quantified for different size categories
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. Mouse embryos are ideal specimens for OPT as they are naturally transparent . The otherwise cleared sample is embedded in a block of agarose gel and placed on a motor through a magnetic mount. It undergoes a complete rotation within the scanner through angular steps of 0.9 degrees (ca. 400 images) while suspended in a quartz cuvette filled with the same solution used for optical clearing.
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). 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.
Furthermore, OPT is able to take advantage of fluorescent dyes, and different wavelength channels can be used. This allows the observation of autofluorescence of the tissue (to inform on tissue structure), along with the mapping of gene and protein expression. The range of species successfully imaged so far includes human (1), mouse (2,3), chick (4), reptile species (5), zebrafish (6), Drosophila (7) and Arabidopsis (8). Successes with bigger specimens such as whole organs taken from the adult mouse have also now been reported, for brain (9), spinal cord (10), pancreas (11), kidney (11) and lungs (12). This opens up exciting new applications such as preclinical disease research, like whole pancreas imaging performed quantitatively to compare the mass of β-cell tissue in normal versus diabetic specimens, using the NOD mouse model (11) or imaging progressive inflammation of spinal cord and optical nerve in a mouse model of multiple sclerosis (10).
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