Researchers have devoted a great deal of effort to probing the photophysical and photochemical properties of green fluorescent protein (GFP). Many are especially interested in the transitions between GFP's fluorescent and nonfluorescent (bright and dark) states that reversibly and quickly turn it on and off (known as blinking or flickering) and lead to photobleaching. Control of these transitions could open the door to a number of new applications of GFP.Recent studies have demonstrated that irradiation with blue/UV radiation may enable the recovery of fluorescence emission after photobleaching in certain GFP mutants. Now a team at the Universities of Milan, Genoa and Parma and at Scuola Normale Superiore in Pisa, all in Italy, has demonstrated controlled photoswitching between the bright and dark states in one of the mutants with two-photon microscopy. The work points to a variety of applications, especially in optical data storage architectures.The researchers introduced the mutant E2GFP in 2001 to take advantage of various biological and fluorescence properties of the GFP family. Currently, they are developing it for applications in molecular biology and in nanophotonics for information storage. In the recent study, they sought to determine whether they could achieve photoswitching of E2GFP using two-photon rather than one-photon excitation.To this end, they performed a series of experiments on GFP with an optical setup based on a TE300 inverted microscope and PCM2000 scanning head, both made by Nikon Corp. of Japan. A mode-locked Ti:sap-phire laser from Spectra-Physics of Mountain View, Calif., served as the illumination source. A dielectric beamsplitter allowed simultaneous use of the scanning port for imaging and the epifluorescence ports for spectroscopy.The researchers found that two-photon excitation can effectively induce controlled photoswitching, said Fabio Beltram of Scuola Normale Superiore. They also discovered that it offers unexpected and attractive benefits. For example, there was a clear separation of excitation bands for switching on and off, centered at 780 and 870 nm. Because of this, the photoswitching is much more efficient than one-photon excitation.The findings suggest a variety of applications, including data storage. Being able to toggle and detect the bright and dark states of the protein by optical means may enable the storage of information at the "ultimate limit" of a single molecule -- that is, with extremely high density."Proteins are stable at normal conditions and can be easily targeted to nanopatterned substrates, thanks to molecule recognition," Beltram said. "By exploiting these key features, we are currently investigating possible architectures for biophotonic memory devices."He noted two issues that remain to be addressed. First is the question of how blinking affects the reliability of the "reading" process, the measurement of whether the protein is in a bright or dark state. In the bright state, the protein exhibits bursts of fluorescence, interposed with periods of darkness on a millisecond timescale. However, because the switching off of the fluorescence during blinking is photoinduced, careful calibration of the laser source intensity during such measurement could solve the problem. Another option, Beltram said, might be to employ several proteins in a building block so that the blinking may be washed out without significantly affecting performance.The second issue involves the optimization of the switching times for fast writing and erasing of data. In this case, the use of two-photon excitation is crucial because of the significantly improved efficiency of the switching processes.