Genetically encoded fluorescent protein tags are excellent tools for directly visualizing specific cell populations, ideally suited for studying multi-cellular events such as tissue differentiation, organ growth and the establishment of neural connectivity dispersed over large regions. Using fluorescent protein technologies to study large samples in their entirety at high resolution will provide unprecedented insight into the interactions and interdependencies that occur throughout the sample. Unfortunately, the field of view of current high-resolution imaging tools is severely restricted, making it difficult to study large samples. Extended volume imaging techniques allow multiple, overlapping regions to be imaged in order to reconstruct a high-resolution, composite, three dimensional volume much larger than a single microscopic field of view. However, the best currently available techniques are not compatible with fluorescent protein signal, because they employ an organic solvent-based embedding procedure that can denature fluorescent proteins that ruins their fluorescence and prevents direct visualization.
To fill this unmet need, an automated, integrated aqueous tissue sectioning microscope stage will be developed and deployed. Compatibility with fluorescent proteins is maintained by eliminating all organic-solvent processing steps. Furthermore, by miniaturizing the device, it will be made compatible with the standard upright laser scanning microscopes that biologists, neuroscientists and other practitioners are currently using.
The features, design and implementation of the aqueous tissue sectioning microscope stage for three dimensional, extended volume imaging will be publicly disseminated in both publications and on web pages of the Caltech Biological Imaging Center. In addition, optimized protocols for mounting, sectioning and imaging samples from a variety of model organisms will be freely distributed to facilitate widespread adoption of the new device. The eventual widespread availability of this modular microscope accessory will allow researchers from all over the world to modify an existing laser scanning microscope to perform three dimensional extended volume imaging of fluorescent protein labeled biological samples. Undergraduate students will be involved in the multidisciplinary instrument development project through the Summer Undergraduate Research Fellowship program, and representation of underrepresented students in science and engineering will be increased through the Minority Undergraduate Research Fellowship program.
