Time-Resolved Fluorescence Techniques

Time-correlated single photon counting allows us to measure the time that a molecule stays in its excited state following excitation. The technique works as follows: a laser pulse hits the sample.  After the molecules in the sample are excited, they can decay back to the ground state via several pathways, such as internal conversion, fluorescence, and intersystem crossing to the triplet state.  The sample goes back to the ground state at a rate at the sum of all the possible decay pathways; the reciprocal of this rate is the excited state lifetime. Even if fluorescence is not the fastest decay pathway, the relaxation is a discrete, probabilistic process.  As a result, there is some Poisson probability of detecting fluorescence photons.  We can detect these photons using a PMT and build up an exponential decay.  Fitting this decay gives us the excited state, or fluorescence, lifetime.  In photosynthetic systems, excited chlorophylls can decay back to the ground state by transferring their excited energy to other chlorophylls.  As a result, a measurement of the fluorescence lifetime of photosynthetic systems gives information on the time that an excitation remains in the antenna before it gets quenched.  Because sample scattering is much less of a problem in this technique, it is very useful for studying energy transfer and quenching dynamics on intact photosynthetic systems, such as live cells and leaves. 

Helpful Background Reading:

  1. Models and measurements of energy-dependent quenching, J. Zaks*, K. Amarnath*, E. J. Sylak-Glassman*, G. R. Fleming, Photosynthesis Research, 116, 389-409, 2013. (asterix indicates equal contribution) [PDF]
  2. Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging, E. Auksorius, B. R. Boruah, C. Dunsby,  P. M. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, Opt. Exp., 33, 113-156 (2008).
  3. Nanoscale Resolution in the Focal Plane of an Optical Microscope, V. Westphal and S. W. Hell, Phys. Rev. Lett., 94, 143903-149307 (2005).