MRI: Development of an Advanced Two-Photon Microscope for Five-Dimensional Imaging of Macromolecular Systems in Living Cells

University Of Wisconsin-Milwaukee, Milwaukee WI

Investigators

Abstract

The proper function and survival of any living organism depends critically upon the ability of its proteins to interact with one another to form short- or long-lived macromolecular complexes. Recent investigations by the Principal Investigator and his collaborators have demonstrated that it is possible to determine the number and relative disposition of protein monomers in macromolecular complexes in living cells, which is one of the major challenges of contemporary science. The experimental setup developed for conducting those experiments consisted of a novel two-photon microscope which provided entire emission spectra at the level of single image pixels . This technology permits pixel-level measurements of Foerster resonance energy transfer (FRET), that is, the non-radiative transfer of energy from an excited fluorescent molecule - called a 'donor' (D) to a non-excited 'acceptor' (A) that resides nearby. The quantity of particular interest is the FRET efficiency, which is determined for every pixel from the constituent spectra of D and A as obtained by spectral un-mixing. The proposed technology will take the existing technology to a whole new level, by allowing unmatched tracking of fluorophore-bearing proteins and dynamic monitoring of the protein-protein interactions in living cells. More specifically, the new instrument will acquire images orders of magnitude faster than its predecessor - which itself remains unique in the field and do so in three spatial dimensions rather than two. The development of this cutting edge technology will allow cellular and molecular biologists, biochemists, and other life-scientists to investigate dynamic features of multiple protein populations, including co-localization and trafficking, protein-complex trafficking, and ligand-induced changes in conformation and oligomeric status. The design of the instrument will be made available to other researchers and to microscope manufacturing companies, while the instrument itself will be made available for use by interested research groups. In addition to impacting the research programs of numerous principal investigators, the proposed instrument development will provide exquisite training opportunities for over forty undergraduate, graduate, and postgraduate trainees at UW-Milwaukee, UW-Madison and other research institutions around the nation and abroad.