Evanescent Wave Induced Fluorescence Microscopy

Andrew Rapson, XiaoTao Hao

We use the evanescent field, generated upon the total internal reflection of light at the interface between two materials of differing refractive index, to induce fluorescence from fluorophores residing in close proximity to the interface. The emission is time resolved which can provide information on the dynamics and local environment of the fluorophore.


Above is a schematic of the evanescent wave experimental setup.  Having two parabolic mirrors after the rotating mirror allows the angle of incidence to be varied (and thus the excitation depth) without causing misalignment of the detection system.  Below we see the decaying evanescent wave away from the surface.  Varying the angle of incidence allows the penetration of this wave to be varied between approximately 10 – 200 nm.



Relevant papers:

  • C.A. Scholes, D.P. Millar, M.L. Gee and T.A. Smith, “Resonance Energy-Transfer Studies of the Conformational Change on the Adsorption of Oligonucleotides to a Silica Interface”, J. Phys. Chem. B (2011) 115 (19), 6329–6339
  • A.C. Rapson, A. Hossain, J.D. Wade, E.C. Nice, A.H.A. Clayton, T.A. Smith and M.L. Gee, “Structural Dynamics of a Lytic Peptide Interacting with a Supported Lipid Bilayer”, Biophysical Journal 100, 1353-1361 (2011)
  • T.A. Smith, C.A. Scholes and M.L. Gee, “Polarized Time-Resolved Evanescent Wave-Induced Fluorescence Measurements”, in Reviews in Fluorescence, C.D. Geddes and J.R. Lakowicz (Eds), Springer Science, pp 245-270, (2005)
  • M.L. Gee, L. Lensun, T.A. Smith and C.A. Scholes, “The Determination of Molecular Conformational Changes of Proteins at an Interface Using Time-Resolved Evanescent Wave-Induced Fluorescence Anisotropy Measurements”, Eur. Biophys. J, (2004), 33(2), 130-139
  • M.L. Gee and T.A. Smith, “Shedding Light on the Conformation of Proteins and Other Macromolecules at an Interface”, Aust. J. Chem. (2003), 56, 1005-1012