Andrea Ursic
Two-Photon Conversion of the Fluorescent Protein EYQ1
Master Thesis – University of Pisa, 2009Abstract
EYQ1 is a photochromic fluorescent protein from the family of the YFPs (yel-low fluorescent proteins) derived by site-directed mutations from Aequorea
vic-toria GFP (green fluorescent protein). The photochromic properties involve the
switching off (bleaching) of its fluorescence by irradiation at 515 nm and the switching on by rapid irradiation at 405 nm. Both processes are reversible and repeatable many times. Photoactivatable (PAFPs) and reversibly switchable fluorescent proteins (RSFPs) like EYQ1 can be employed in advanced fluor-escence microscopy techniques on biological samples, thanks to the fact that they can highlight with their activation restricted and controllable regions of the sample. By this way, for instance, monitoring the temporal evolution of cell structures and the diffusion of proteins to which PAFPs are fused, are made simpler.
Photochromic properties in single-photon excitation mode have already been studied. In this thesis work are presented results of an analysis of the same properties in two-photon excitation mode. Indeed, Two-photon excitation tech-niques have the advantage of producing a better three-dimensional confinement of excitation and, therefore, of activation.
Firstly, two-photon excitation spectra of EYQ1 (in both states with an-ionic or protonated chromophore, respectively) were determined in acqueous solutions. Subsequently, in experiments with immobilized proteins in a polyac-rylamide gel, the shape of the two-photon excitation band from which reactiv-ation originates was also determined. Stability and fatigue of the protein in re-peated one-photon off-switching and two-photon on-switching were determined in experiments in gel and compared with previous single-photon experiments. Additionally, the vertical thickness of the two-photon reactivated volume was evaluated as a function of total irradiation time.
The orders of magnitude of two-photon reversible and irreversible bleaching were extimated from measurements of fluorescence decay under irradiation of the immobilized protein in thin layers of polyvinyl alcohol (PVA). Finally, two-photon reactivation experiments were carried on also on live and fixed cells. From the results appears that EYQ1 photochromism is conserved also in two-photon excitation conditions. In one-two-photon off- and two-two-photon on-switching cycles at lower powers, EYQ1 behaviour resulted comparable to the single-photon activation case. Bleaching and reactivating at a higher speed and irra-diation power results in a decrease of protein stability, though it is still possible to perform some cycles. It was noticed that the irradiation power in the single-photon (bleaching) phase affects protein fatigue more than does the irradiation power in the (two-photon) reactivation phase. In vitro observations were not contradicted by the in vivo experiments. Finally, the vertical localization of reactivation was observed to be very good.
