Journal of Biotechnology 149 (2010) 289–298 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Data storage based on photochromic and photoconvertible fluorescent proteins Virgile Adam a,b,∗ , Hideaki Mizuno a,c , Alexei Grichine d , Jun-ichi Hotta a , Yutaka Yamagata e , Benjamien Moeyaert a , G. Ulrich Nienhaus f,g,h , Atsushi Miyawaki c , Dominique Bourgeois i , Johan Hofkens a a Laboratory of Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium b European Synchrotron Radiation Facility, 6, rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France c Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-City, Saitama 351-0198, Japan d Platform ‘Optical Microscopy – Cell Imaging’ UJF-Grenoble 1, Inserm U823, Institut Albert Bonniot, 38042 La Tronche, France e VCAD System Research Program, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-City, Saitama 351-0198, Japan f Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), 76128 Karlsruhe, Germany g Institute of Biophysics, University of Ulm, 89069 Ulm, Germany h Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, IL 6180, USA i IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, Université Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble, France article info Article history: Received 18 November 2009 Received in revised form 30 March 2010 Accepted 8 April 2010 Keywords: Optical data storage Photoconversion Photochromism Fluorescent proteins Protein crystals 3D data storage abstract The recent discovery of photoconvertible and photoswitchable fluorescent proteins (PCFPs and RSFPs, respectively) that can undergo photoinduced changes of their absorption/emission spectra opened new research possibilities in subdiffraction microscopy and optical data storage. Here we demonstrate the proof-of-principle for read only and rewritable data storage both in 2D and 3D, using PCFPs and RSFPs. The irreversible burning of information was achieved by photoconverting from green to red defined areas in a layer of the PCFP Kaede. Data were also written and erased several times in layers of the photochromic fluorescent protein Dronpa. Using IrisFP, which combines the properties of PCFPs and RSFPs, we performed the first encoding of data in four colours using only one type of fluorescent protein. Finally, three-dimensional optical data storage was demonstrated using three mutants of EosFP (d1EosFP, mEosFP and IrisFP) in their crystalline form. Two-photon excitation allowed the precise addressing of regions of interest (ROIs) within the three-dimensional crystalline matrix without excitation of out- of-focus optical planes. Hence, this contribution highlights several data storage schemes based on the remarkable properties of PCFPs/RSFPs. © 2010 Elsevier B.V. All rights reserved. 1. Introduction In the quest for optical data storage devices of ever-higher den- sity, organic substrates have often been suggested as a potentially Abbreviations: AOTF, Acousto-optic tunable filter; bR, Bacteriorhodopsin; CD, Compact disc; DVD, Digital versatile disc; (d)STORM, (direct) STochastic optical reconstruction microscopy; EDC, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; ESD, ElectroSpray deposition; (F)PALM, (Fluorescence) Photoacti- vated localisation microscopy; FWHM, Full width at half maximum; ITO, Indium tin oxide; NHS, N-Hydroxysulphosuccinimide; OPE, One-photon excitation; PAFP, Photoactivatable fluorescent protein; PCFP, Photoconvertible fluorescent protein; PLL, Poly-l-lysine; RESOLFT, Reversible saturable optical fluorescence transitions; ROI, Region of interest; RSFP, Reversibly switchable fluorescent protein; STED, Stimulated emission depletion microscopy; TPE, Two-photon excitation; WORM, Write-once–read-many; WMRM, Write-many–read-many. ∗ Corresponding author. Laboratory of Photochemistry and Spectroscopy, Depart- ment of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, bus 02404, 3001 Heverlee, Belgium. Tel.: +32 16 327399; fax: +32 16 327990. E-mail address: virgile.adam@chem.kuleuven.be (V. Adam). interesting storage medium. The possibility of using organic dyes displaying photochromic properties (i.e. that can be reversibly pho- toswitched between two states having distinct spectral properties) to design rewritable optical data storage devices was first suggested by Y. Hirschberg more than 50 years ago and further explored by numerous researchers (Berkovic et al., 2000; Irie, 2000; Kawata and Kawata, 2000; Yokoyama, 2000; Irie et al., 2002). The molecules which were first explored are known as spiropyrans. Spiropyrans can be reversibly photocyclised to their merocyanine forms that exhibit spectral properties distinct from those of their native forms (Hirschberg, 1956). While Hirschberg showed that most of the photochromic molecules in solution can be reversibly phototrans- formed after the absorption of a single photon at room temperature, other researchers demonstrated that some molecules retain pho- tochromic properties in a rigid medium (Lewis and Lipkin, 1942). Furthermore it was demonstrated that photochromism can be pro- duced by two-photon excitation (TPE) (Mandzhikov et al., 1973). The probability of simultaneous absorption of two photons is pro- portional to the square of the intensity and, therefore, two-photon excitation only allows the excitation of molecules at the very focus 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.04.001