Activation and characterization of a GFP variant using two-photon absorption

Ilaria Testa1, Sara Barozzi2, Marc Schneider1, Mario Faretta2 and Alberto Diaspro1

1-INFM Genoa, Dept. of Physics, Via Dodecaneso 33, I-14146 Genoa, Italy// 2-European Institute of Oncology, Dept of Exp. Oncology, Via Ripamonti 435, I-20141 Milan, Italy

We report about a photoactivatable variant of the Aequorea Victoria green fluorescent protein (paGFP). As reported by Patterson and Lippincott-Schwartz1 this special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light with λ = 413 nm. The aim in this work was to evaluate of two-photon photoactivation of the paGFP molecules. Therefore experiments were performed partially purified protein immobilised on microspheres. The spheres were used to investigate the ability of different wavelengths to activate the paGFP. The molecular switches were irradiated with laser light in a range of wavelength of a Chameleon-laser coupled to Leica TCS SP2 system. For this system we measured the concentration variation per sphere and additionally we investigated the activation dependence on the irradiation time in power. From these data one can create a map for the best investigation parameters which also take into account the bleaching sensitivity. Additionally, long exposure times reveal the finite character of the gaussian beam in the focal plane.
(1) Patterson, G. H.; Lippincott-Schwarz, J. Science 2002, 297, 1873.

Email: schneider@ge.infm.it

Address: INFM Genoa, Dept. of Physics, Via Dodecaneso 33, I-14146 Genoa, Italy