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.
Intellectual merit A novel laser scanning microscope, called a micro-spectroscope with two-photon excitation has been designed, built, and characterized, which provides four- five-dimensional fluorescence images of macromolecules in living cells: that is, two or three spatial dimensions, a spectral dimension (i.e., color information), and a temporal dimension, on a scale of 0.05–2 s (depending on image size). The development of this cutting edge technology allows scientists to investigate spatial and dynamic features of multiple protein populations, including changes in co-localization of proteins (see fluorescence micrograph), protein-complex formation and trafficking, and changes induced by binding of ligands such as drugs to various receptor proteins. Broader impacts Such studies had been the purview of very few investigators, if possible at all. The instrument developed as part of this grant (see picture #1) may be used by any scientist interested in biophysical studies of living systems. The technology also provides critical capabilities to researchers in areas outside that of biophysics, which include cellular and molecular biology, biochemistry, pharmacology and other life-sciences. This instrument is part of an imaging facility housed in the Physics Department at UW-Milwaukee and may be used for a reasonable fee (covering the operating costs) by researchers in other departments and at other US institutions. A web page has also been established (http://www4.uwm.edu/collaboratory/optimis.cfm) which provides information and forms for use by potential users. The PI has established a hands-on Physics program in the Shorewood School District (Milwaukee, WI) which enrolls forty students each year from the districtâ€™s two elementary schools. In this program, the students (see picture #2) learn basic physical optics by constructing and using simple instruments (e.g., telescope, microscope, and spectrograph). At the end of each academic year spend one afternoon at UW-Milwaukee to learn about laser-scanning microscopy and spectroscopy, as well as astronomic instruments. The overarching goal of this program, which is in its third year of existence and has quickly become very popular, is to stimulate the creation of a cadre of future scientists.