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Two-Photon Activation and Excitation Properties of PA-GFP in the 720–920-nm Region

Marc Schneider ^*, Sara Barozzi , Ilaria Testa ^* , Mario Faretta  and Alberto Diaspro ^*  

^* INFM Genoa, Department of Physics, University of Genoa, I-16146 Genoa, Italy; European Institute of Oncology, Department of Experimental Oncology, 20141 Milan, Italy; LAMBS-MicroScoBio Research Center, University of Genoa, 16146 Genoa, Italy; and IFOM, Istituto FIRC di Oncologia Molecolare, 20139 Milan, Italy

Correspondence: Address reprint requests to Alberto Diaspro, LAMBS-MicroScoBio, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy. Tel.: 39-0103536426-480-309; Fax: 39-010314218; E-mail: diaspro{at}fisica.unige.it.

This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.

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