Fluorescence lifetime imaging microscopy (FLIM)
The Wolfson Bioimaging Facility provides fluorescence lifetime imaging microscopy by addition of specialised equipment and software to one of its Leica SP8 CLSM systems.
Monitoring the fluorescence lifetime of a fluorophore (the average time between excitation and photon emission) rather than its intensity provides concentration-independent information on local microenvironment.
Potential FLIM applications include FRET (Fluorescence Resonant Energy Transfer) analysis of fluorophore interactions, measurement of ions and signalling molecules, and analysis of the physical environment of the fluorophore.
Our FLIM system is part of the Leica SP8X system also equipped for STED and FCS and uses a pulsed white light laser which can be tuned precisely within 470-670nm excitation range. Specialised single molecule detectors and SymPhoTime software (PicoQuant) facilitate TSCPC (time correlated single photon counting) FLIM.
Although FLIM acquisition is relatively straightforward with our system, FLIM experiments require specialised expertise and are relatively analysis-intensive.
Recent publications including data acquired with our FLIM system:
- Stan, Shoemark, Alibhai, Hanley (2022) Ca 2+ regulates dimerization of the bar domain protein pick1 and consequent membrane curvature. Front Mol Neurosci. doi.org/10.3389/fnmol.2022.893739
- Leung, Fung, Gallio, Blore, Alibhai, Raven, Hudson (2021) Unravelling the mechanisms controlling heme supply and demand. Proc Natl Acad Sci U S A. 118(22):e2104008118.
- Zhao, Alibhai, Sun, Khalil, Hutchinson, Olzak, Williams, Li, Sessions, Cross, Seager, Aungraheeta, Leard, McKinnon, Phillips, Zhang, Poole, Banting, Mundell (2021) Tetherin/BST2, a physiologically and therapeutically relevant regulator of platelet receptor signalling. Blood Adv. 13: 1884-1898
- Tian, Zhang, Du, He, Jin, Pearce, Eloi, Harniman, Alibhai, Ye, Phillips, Manner (2020) Tailored self-assembled photocatalytic nanofibers for vsisible-light-driven hydrogen production. Nature Chemistry 12(12):1150-1156