3D localized photoactivation of pa-GFP in living cells using two-photon interactions

Abstract

We report about two-photon activation of 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. The aim in this work was to evaluate the use of two-photon interactions for confining the molecular switching of pa-GFP in the bright state. 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 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 and two-photon mode demonstrates clearly the better threedimensional (3D) confinement and the possibility of selection of cell volumes of interest. This enables molecular trafficking studies at high signal to noise ratio.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

“…This drawback hampers the rational choice of the region of interest for further photoactivation. Nevertheless, due to its monomeric nature, high contrast and high brightness of the activated form, PA-GFP is one of the most powerful PAFPs and has gained popularity in a variety of applications (29,66,88,91,144,161,165,258,269,272,274,297,320,335,377,426,427,461).…”