Developments in imaging technologies have traditionally contributed to breakthroughs in scientific understanding. While the earliest imaging tools were primarily directed to resolving gross anatomical structures, modern imaging techniques allow visualization of very fine structural details as well as molecular information in some cases.
The development of a number of new imaging modalities, along with evolution of molecular and cellular biology techniques, has created a need for improved methods and devices to maximize the advantages offered by these technologies. For example, genetically modified animals are readily produced by molecular biology methods. Systematic imaging of genetically modified animals offers an opportunity to add significantly to an understanding of the function of particular genes. Additionally, it is now feasible to spatially correlate data from various imaging modalities, in order to glean still further information from genetically modified animals, as well as other animals and specimens. Imaging techniques amenable to correlation illustratively include confocal optical microscopy (COM), positron emission tomography (PET), magnetic resonance imaging (MRI), X-ray computed tomography (CT), and single photon emission computed tomography (SPECT). However, in spite of the opportunities presented, there remain limitations in imaging technologies.
While gene targeting potentially allows unprecedented insight into the function of genes and their roles in patterning the mammalian embryo (Capecchi, M. R., Nat Rev Genet 6, 507-12, 2005), the number of animals which must be imaged in such a project is large. A full understanding of mammalian development by this means, using the gene targeting approach for every one of the ˜25,000 or more mouse genes, may seem like a daunting task. Nevertheless, more than ten percent of known mouse genes have already undergone disruption by gene targeting, while the National Institutes of Health is leading an effort to create a collection of mouse lines with disruption of every known gene (Austin, C. P. et al., Nat Genet 36, 921-4, 2004). The challenge laid before developmental biologists will be to systematically analyze morphological phenotypes, and where possible, determine the quantitative contribution of each gene to patterning of the embryo. Attributes associated with an imaging tool for this type of phenomic analysis include rapid scan speed, low-cost, and accessible high-throughput methods of high resolution anatomical imaging as well as stage-specific, statistically-averaged wild type morphological atlases that can be used to discern normal variation from mutant phenotype (Jacobs, R. E., et al., Comput Med Imaging Graph 23, 15-24, 1999). Traditional histological methods of screening embryonic animals or other specimens for anatomical and/or molecular variation are time intensive, such that processing and analysis of large numbers of specimens is generally impractical.
Pre-clinical studies are also becoming increasingly reliant on multiple imaging modalities for sophisticated evaluation of various parameters to allow for accurate and largely non-invasive assessment and/or monitoring of phenotype resulting from various stressors such as mutation, tumor size and growth rate, effect of drug or other treatment, and drug localization. This effort is frustrated by multiple mode imaging owing to difficulties in attaining image registration due to positioning inconsistency. Currently, animals tend to move over time in a holder and often, the animal must be moved to a different holder to accommodate the particularities of various imaging techniques.
A limitation of existing animal holders is that close contact of the animal with the animal holder is visible in the resulting images, making image post-processing tedious. An image of animal tissues in proximity with the holder is often obscured or poorly resolved. Image quality and efficiency suffer since the holder must be identified and subtracted in subsequent image processing. To date, an animal holder for acquisition of microCT images has not been identified that mitigates the shortcomings of existing holders, allowing the specimen to be distinguished clearly from the holder.
X-ray microscopic computed tomography (microCT) represents an attractive imaging choice, alone or as part of an imaging battery, owing to the suitability of the technique to semi-automated or fully automated methods of analysis. This attribute is important in achieving high-throughput phenomic studies or clinical pathology results. A comparison of CT and magnetic resonance methods, applications, and costs shows that microCT-based virtual histology offers a potentially higher resolution mode of morphometrics that is simple to implement, relatively inexpensive, and more rapid than comparable methods of phenotyping embryo anatomy.
MicroCT virtual histology would be an even more attractive imaging technique if limitations associated with specimen staining and mounting could be overcome. Current staining processes, such as that described in M. D. Bentley et al., National Synchotron Light Source Activity Report 1998 Beamline X2B, struggle to yield an adequate signal to noise ratio in order to achieve high resolution of anatomical structures. MicroCT virtual histology throughput has also been hampered by a lack of specimen holders capable of containing multiple specimens. In addition, microCT virtual histology of whole embryos results in voluminous data. The true value of such data will only be fulfilled if systems and methods for retrievably storing and analyzing data are developed.
Thus, there is a continuing need for improved staining processes for producing a microCT image, systems and processes for retrievably storing and analyzing such image data, and specialized devices for holding a stained specimen or living animal to be imaged using one or more imaging modalities in order to take advantage of these opportunities.
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