REVIEW ARTICLE From EosFP to mIrisFP: structure-based development of advanced photoactivatable marker proteins of the GFP-family Jo ¨ rg Wiedenmann 1 , Susan Gayda 2 , Virgile Adam 3 , Franz Oswald 4 , Karin Nienhaus 2 , Dominique Bourgeois 5 , and G. Ulrich Nienhaus * ; 2; 6 1 National Oceanography Centre, University of Southampton, Southampton, SO14 3ZH, UK 2 Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), 76128 Karlsruhe, Germany 3 Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium 4 Department of Internal Medicine I, 89069 University of Ulm, Germany 5 Institut de Biologie Structurale (IBS) Jean-Pierre Ebel, CEA, CNRS, Universite ´ Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble, France 6 Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Received 23 December 2010, revised 25 January 2011, accepted 26 January 2011 Published online 14 February 2011 Key words: red fluorescent protein, green fluorescent protein, crystal structure, anthozoa, photoactivation. # 2011 by WILEY-VCH Verlag GmbH& Co. KGaA, Weinheim Journal of BIOPHOTONICS Early View publication on www.wileyonlinelibrary.com (issue and page numbers not yet assigned; citable using Digital Object Identifier – DOI) Fluorescent proteins from the GFP family have become indispensable imaging tools in life sciences research. In recent years, a wide variety of these proteins were dis- covered in non-bioluminescent anthozoa. Some of them feature exciting new properties, including the possibility to change their fluorescence quantum yield and/or color by irradiating with light of specific wavelengths. These photoactivatable fluorescent proteins enable many inter- esting applications including pulse-chase experiments and super-resolution imaging. In this review, we discuss the development of advanced variants, using a structure- function based, molecular biophysics approach, of the photoactivatable fluorescent protein EosFP, which can be photoconverted from green to red fluorescence by 400 nm light. A variety of applications are presented that demonstrate the versatility of these marker proteins in live-cell imaging. The photoactivatable fluorescent protein mIrisFP com- bines two forms of photoactivation, reversible on-off photoswitching and irreversible green-to-red photocon- version, which enables pulse-chase experiments with superresolution. (Top) Light-induced structural changes upon on-off switching of the red fluorescent form, (bot- tom) photoactivation localization microscopy (PALM) reconstruction images of the formation of focal adhe- sions using the paxillin-mIrisFP fusion protein. * Corresponding author: e-mail: uli@illinois.edu J. Biophotonics 1–14 (2011) / DOI 10.1002/jbio.201000122