Probing the local environment of green fluorescent protein (GFP) with fluorescence lifetime imaging (FLIM) and time- resolved fluorescence anisotropy imaging (tr-FAIM) K. Suhling, D.M. Davis Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College of Science, Technology & Medicine, London SW7 2AZ, UK. k.suhling@ic.ac.uk D. Phillips Department of Chemistry, Imperial College of Science, Technology & Medicine, London SW7 2AY, UK. J. Siegel, S. Lévêque-Fort 1 , S.E.D. Webb, P.M.W. French Photonics Group, Physics Department, Imperial College of Science, Technology & Medicine, London SW7 2BW, UK. 1 present address: Laboratoire de Photophysique Moleculaire, 91405-Orsay cedex, France. Abstract: Wide-field time-domain fluorescence lifetime imaging and time-resolved fluorescence anisotropy imaging of green fluorescent protein can be used to probe the biophysical environment of specific proteins. 2001 Optical Society of America OCIS codes: (170.2520) Medical optics and biotechnology; (180.2520) Microscopy; (260.2510) Physical Optics; (300.2530,300.6280) Spectroscopy 1. Introduction The green fluorescent protein (GFP) of the jellyfish Aequorea victoria and its variants are widely used in cell imaging applications to reveal the location of proteins [1]. However, such imaging of fluorescence intensity does not generally provide information about the biophysical environment of the protein. Fluorescence lifetime imaging (FLIM) [2] is a technique that, in addition to position and intensity, also provides information concerning the average time a fluorophore remains in its excited state after excitation. FLIM of the fluorescence decay of GFP has, for example, been used to report on fluorescence resonance energy transfer (FRET) [3]. We demonstrate here that the GFP fluorescence lifetime depends on the biophysical environment of the fluorophore and so can be used to directly probe the GFP environment. In particular, it can report local variations in refractive index. We also report what is, to our knowledge, the first demonstration of wide-field time-resolved fluorescence anisotropy imaging (tr- FAIM) which may be used to report local variations in viscosity. Recently, we presented preliminary time-correlated single photon counting studies to identify the parameter that affects the fluorescence lifetime of GFP [4]. We found this to be the refractive index of the environment of GFP, and we showed that in mixtures of water and glycerol, the inverse GFP fluorescence lifetime scales with the square of the refractive index, as predicted by the Strickler Berg formula [5]: v d v v v d v v I v d v I n ~ ~ ) ~ ( ~ ~ ) ~ ( ~ ) ~ ( 10 88 . 2 1 3 2 9 0 ∫ ∫ ∫ × = - - ε τ (1) where τ 0 is the natural fluorescence lifetime, n the refractive index, I the fluorescence emission, ε the extinction coefficient and ν ~ the wavenumber. In this paper, we extend our study of the dependence of the GFP fluorescence lifetime on the refractive index to imaging with time-domain FLIM. We note that changes in viscosity have also been observed to perturb the fluorescence lifetime in FLIM maps, e.g. [6], but the GFP fluorescence lifetime does not depend on the local viscosity [7], since the GFP fluorophore is rigidly attached to a barrel-shaped cage which prevents internal twisting and protects it from collisional encounters with oxygen [1]. Thus, the GFP fluorescence lifetime cannot be used to probe the viscosity of its environment, although this parameter would be of interest in cell biology. The time-resolved fluorescence anisotropy r(t), however, does depend on the mobility of the protein, and this in turn is strongly influenced by the viscosity. The time-resolved fluorescence anisotropy r(t) is defined as [8]