T2P-GFP: two-photon photo-activation of PA-GFP in the 720-840 nm spectral region.

We report about a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm(1). The aim in this work was to evaluate the use of two-photon interactions for activation of the molecules 2. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The latter objects were used to investigate the ability of different wavelengths to activate the paGFP due to the anticipated more homogeneous density distribution. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation with a single photon at 413 nm and two-photons demonstrates clearly the advantages using non linear processes: much smaller volume in the cell can be activated unlike to a whole cell activation in single photon excitation regime.