Instrumentation for Single Molecule Fluorescence Lifetime Imaging in Living Systems
Abstract
Requested are funds to build a 3D fast-scanning Fluorescence Lifetime Imaging (FLIM)attachment for the adaptive Guide Star single molecule fluorescence microscope developedunder AFOSR grant FA9550-14-1-0272. This attachment will be interfaced with an existingtime correlated single photon counting (TCSPC) and time-tagged time-resolved (TT) datacollection capability to: (1) measure the stochasticity and overall free energy landscape ofmultiple ligands binding to individual Ligand Gated Ion Channels (LGICs) within 10-100 nmlipid rafts, (2) simultaneously keep track of photon dynamics arising from individual probemolecules, and (3) characterize the quantum properties of probe molecules in real time which isneeded to accurately deconvolute the dynamics arising from biophysical and photophysicalprocesses. The combination of TT-TCSPC and fast scanning confocal microscopy will allow usto span the time range from psec to minutes. This will allow us to account for nearly all thedynamics associated with the systems currently under investigation and will greatly enhance thescope and direction of projects proposed as future AFOSR research. In particular, the newinstrumental capability will be used to create new quantum standards for single moleculebiophysical measurements, measure the photon dynamics emitted from fluorescently labeledagonists of a number of LGICs, and to simultaneously measure the binding and unbinding ofmultiple ligands from individual receptors. Specifically, a confocal attachment, fast piezo 3Dscanner,a 150x high NA microscope objective, and a picosecond super continuum laser will beinterfaced with our existing Guide Star microscope and TT-TCSPC instrumentation to create a3D fluorescence lifetime imaging (FLIM) capability with single molecule sensitivity along withits current state-of-the-art super-resolution capability.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 11, 2018
- Source ID
- FA95501810395
Entities
People
- James A Brozik
Organizations
- Air Force Office of Scientific Research
- United States Air Force
- Washington State University