Abstract
Photoactivatable fluorescent proteins represent an innovative tool for the direct observation of time dependent macromolecular events in living systems. The possibility of switching on a selected and confined subset of the expressed target proteins allows to follow biological processes reaching high signal to noise ratios. In particular, use of non-linear interactions to bring the molecules in the activated fluorescent form make it possible to extend the advantages of photoactivation to events that requires 3D spatial localization. In this work, we show the possibility to realize confined activated volumes in living cells, by employing photoactivatable green fluorescent protein (paGFP) in two-photon microscopy. The analysis of the kinetics of two-photon paGFP activation in dependence of the wavelength, the laser intensity and the exposure time is provided. This study allowed to assess the optimal conditions to induce photoactivation in living samples and to track the behaviour of tagged histone H2B during cellular division. Furthermore we investigate paGFP photoactivation under evanescent wave illumination. Total internal reflection set-up has been used to selectively activate subresolved distribution of proteins localized in the basal membrane surroundings. These two photoactivation methods provide a suitable tool for many biological applications, combining subresolved surface and in-depth three-dimensionally confined investigations.
Similar content being viewed by others
References
Diaspro A (2006) Shine on proteins. Microsc Res Tech 69:149–151
Diaspro A (ed) (2001) Confocal and two-photon microscopy. Wiley-Liss, New York
Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C (2006) Multi-photon excitation microscopy. Biomed Eng Online 5:36
Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224
Schneider M, Barozzi S, Testa I, Faretta M, Diaspro A (2005) Two-photon activation and excitation properties of PA-GFP in the 720–920-nm region. Biophys J 89:1346–1352
Shimomura O (2005) The discovery of aequorin and green fluorescent protein. J Microscopy 217:3–15
Testa I, Mazza D, Barozzi S, Faretta M, Diaspro A (2007) Blue-light (488 nm)-irradiation-induced photoactivation of the photoactivatable green fluorescent protein. Appl Phys Lett 91:133902
Testa I, Parazzoli D, Barozzi S, Garrè M, Faretta M, Diaspro A (2008) Spatial control of paGFP photoactivation in living cells. J Microscopy 230:48–60
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544
Patterson GH, Lippincott-Schwarz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877
Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW (2006) Nanoscale resolution in GFP-based microscopic nature. Methods 3:721–723
Acknowledgments
This work was supported by IFOM (FIRC Institute of Molecular Oncology, Milan, Italy), by Fondazione Compagnia di San Paolo (Turin, Italy), University of Genoa and PRIN2006-MiUR (2006028909) (Ministry of University and Research). The authors are indebted to George Patterson and Jennifer Lippincott-Schwartz for paGFP availability.
Author information
Authors and Affiliations
Corresponding author
Additional information
Regional Biophysics Conference of the National Biophysical Societies of Austria, Croatia, Hungary, Italy, Serbia, and Slovenia.
Rights and permissions
About this article
Cite this article
Testa, I., Garrè, M., Parazzoli, D. et al. Photoactivation of pa-GFP in 3D: optical tools for spatial confinement. Eur Biophys J 37, 1219–1227 (2008). https://doi.org/10.1007/s00249-008-0317-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-008-0317-9