Time-Resolved Confocal Fluorescence Microscope with Single Molecule Sensitivity

项目总结

PROJECT SUMMARY This application seeks funds to purchase a time-resolved confocal fluorescence microscope with single molecule sensitivity. This instrument will support NIH-funded users in three departments and three colleges within the Texas A&M University community and will provide numerous single molecule fluorescence (SMF) capabilities currently unavailable to a dozen laboratories via a new shared user facility. The requested five-line pulsed laser system will enable numerous multi-color correlation, fluorescence lifetime, and polarization applications through its time-correlated single photon counting (TCSPC) capabilities that are required by the user group. The most common application will be fluorescence resonance energy transfer (FRET) measurements used to determine intra- and inter-molecular distance information in a wide-range of macromolecular complexes. While ensemble FRET measurements are often compromised by various artifacts and limitations, the single molecule FRET (smFRET) capabilities of the requested microscope system substantially improve the accuracy of such measurements and provide unique ways to quantify molecular dynamics. An additional key application of the proposed instrument will be the development of a novel approach for measuring rotational mobility termed single molecule rotational diffusion microscopy on the microsecond timescale (µs-SiMRoD). Rotational mobility is an underutilized experimental read-out useful for probing the effects of macromolecular crowding, or other similar situations where the molecular assembly influences probe mobility. SiMRoD has no corresponding ensemble analog, emphasizing the importance of the instrument’s single molecule sensitivity. Though the majority of users will examine well-controlled in vitro systems, in cellulo FRET imaging will benefit from the fluorescence lifetime microscopy (FLIM) capability of the system, which will generate more accurate measurements (FLIM-FRET) than are available from a typical confocal microscope. The Major Users will examine fundamental and diverse cell biological and mechanistic biochemistry questions focused on phosphoinositide binding proteins, nucleosomes, molecular chaperones, membrane fission, nuclear pores, and mitochondrial integrity. All the colleges and departments represented in the user group will contribute to instrumentation costs, emphasizing the fundamental importance of the new microscope capabilities in the growth of current research programs. The instrument will be housed in the College of Medicine by the Department of Molecular and Cellular Medicine, which has donated substantial equipment and space for the microscope facility. Altogether, the identified users have planned new research directions that will require over 93% of the total accessible user time, indicating the substantial demand for both existing and new projects. In total, the requested time-resolved confocal fluorescence microscope with single molecule sensitivity will provide substantial and fundamental infrastructural support for a wide range of projects important for understanding and improving human health.