Recent investigations have shown that spectrally resolved fluorescence microscopy, in conjunction with Fluorescence Resonance Energy Transfer Spectrometry (or FRET Spectrometry), is an effective technique for uncovering the quaternary structure of membrane protein complexes in living cells. To realize the full potential of this approach and determine the supramolecular structures of protein complexes, as well as their relative concentrations, dynamics and spatial distributions Inside a cell or tissue, one must obtain three pieces of critical information at image pixel level: the concentrations of donor and acceptor molecules, and the FRET efficiency occurring between the two. Acquiring this information necessitates exciting the sample at two distinct wavelengths. Existing laser-scanning microscopes (including confocal, two-photon, and FLIM microscopes) perform two-wavelength excitation scans in a serial fashion. This leads to a long time delay (10-100 s) between the two successive scans on a pixel level. As membrane proteins can diffuse in and out of a pixel within 0.100 s, the two excitations are scanning different molecules, thereby compromising the molecular-level resolution. Furthermore, reducing the number of molecules per pixel requires increased spatial resolution, which is not available using current FLIM, confocal, or two-photon microscope technologies. Availability of this cutting-edge technology will open up a new avenue of research in cell signaling and provide an untapped source of pharmacological targets, which in turn may improve public health. The design of the instrument will be made available to other researchers, while the instrument itself will be made available for use by interested research groups. We will also partner with US companies to bring this instrument to the market. Besides to impacting the research programs of investigators around the nation and abroad, the proposed instrument will provide exquisite training opportunities for undergraduate, graduate, and postgraduate trainees in interdisciplinary research across the boundaries between biology, biochemistry, pharmacology and physics. This instrument also will be used for new education initiatives developed by the PI, which bring practical applications of physical and mathematical concepts to elementary and high school students as well as college freshmen, including underrepresented minorities.
In this proposed instrument development project, we will design and construct a two-photon optical micro-spectroscope capable of quasi-parallel excitation of tens of focal spots spread in one or two dimensions, and rapid switching between two different excitation wavelengths. This instrument will present several advantages over existing ones: (1) The parallel excitation of multiple sample voxels will lead to dramatically increased signal-to-noise ratio of 20X to 100X compared with single point-scan microscopes. (2) Spectrally resolved fluorescence will be collected at two excitation wavelengths separated by 10 ms; this switching time is 100-1000X faster than existing technology and will enable the currently unattainable determination of the localization of differently sized oligomeric species together with their size distribution (e.g., monomers, dimers, tetramers) on a pixel level. (3) Finally, this instrument will present both reduced out-of-focus blur and increased axial resolution (by about 2X) compared to existing two-photon microscopes. The dramatic improvement in temporal and spatial resolution as well as data accuracy will provide biologists with a means to determine how protein oligomerization and function affect one another. The multidisciplinary team assembled by the PI is ideally suited to develop and validate this technology, since it combines exquisite technical expertise, facilities, experience with developing and running an imaging facility, and close collaborations between physicists, biologists and other life scientists.
