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Biophys. J. BioFAST: First Published May 20, 2005. doi:10.1529/biophysj.104.054502
© 2005 by the Biophysical Society.

A more recent version of this article appeared on August 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Two-photon activation and excitation properties of PA-GFP in the 720-920 nm region

Marc Schneider 1, Sara Barozzi 2, Ilaria Testa 1, Mario Faretta 2 and Alberto Diaspro 3*

1 INFM, University of Genoa
2 European Institute of Oncology
3 MicroScoBio, University of Genoa, IFOM, INFM

* To whom correspondence should be addressed. E-mail: diaspro{at}fisica.unige.it.

Submitted on October 14, 2004
Revised on November 22, 2004
Accepted on 16 May 2005


  Abstract
This report covers the two-photon activation and excitation properties of the PA-GFP, a photo-activatable 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 following irradiation at {lambda} ~ 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. Following 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 {lambda} = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.

Key Words: PA-GFP, activation cross-section, fluorescence imaging, fluorescence spectroscopy, two-photon activation, two-photon microscopy




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Copyright © 2005 by the Biophysical Society.