Visualising individual green fluorescent proteins with a near field optical microscope

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximately 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutants was monitored in time, with 500-micros real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-off behaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements.