Fluorescent proteins that allow researchers to switch back and forth between their fluorescent and nonfluorescent states can contribute to a number of applications, including protein tracking, Förster resonance energy transfer imaging and subdiffraction resolution microscopy. However, currently available monomeric reversibly switchable fluorescent proteins (RSFPs) exhibit properties similar to one another, severely limiting the potential of using multiple labels in imaging experiments.A recent report suggests that this could change. In the September issue of Nature Biotechnology, a team at Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, described two bright green RSFPs with distinct absorption and switching characteristics that enable live-cell fluorescence microscopy using multiple labels with a single detection color.The researchers generated the RSFPs – bsDronpa and Padron – through extensive mutagenesis of the Dronpa fluorescent protein, which offers favorable switching behavior and a large dynamic range between the “on” and “off” fluorescent states. The reversibly photoswitchable fluorescent proteins also enable dual-color fluorescence nanoscopy, as shown here with cryosections of E. coli expressing the proteins bsDronpa and Dronpa, anchored to the membrane. The top left and bottom left panels show, respectively, a reconstructed wide-field image and a nanoscopy image of cells expressing M13-bsDronpa. The top right and bottom right panels show a mixed sample of cells expressing M13-bsDronpa or M13-Dronpa. Although color separation is not possible in the diffraction-limited reconstructed wide-field image (top right) without any a priori information about the sample, discrimination of the two labels (bsDronpa: green; Dronpa: red) is allowed in the nanoscopy image. Reprinted with permission of Nature Biotechnology.“We were trying to change the properties of Dronpa,” said Stefan Jakobs, principal investigator of the study. “As a result, we got several new switchable proteins. Padron, especially, was very exciting because it has completely different switching properties as compared to its ancestor, Dronpa. We needed such a completely different photoswitchable protein in order to implement monochromatic multilabel imaging.” Researchers have reported monochromatic multilabel imaging using novel reversibly photoswitchable fluorescent proteins. They demonstrated the efficacy of the technique through three-dimensional confocal time-lapse imaging of budding yeast cells expressing two of the proteins – rsFastLime and Padron – discriminating between the proteins’ fluorescent signals by using reversible switching. Reprinted with permission of Nature Biotechnology.Name changeThey began by performing mutagenesis using Stratagene’s QuikChange Site Directed Mutagenesis kit. This yielded a variant with positive switching characteristics – blue light switched the protein to the on state and excited the fluorescence – but weak fluorescence. Furthermore, even after switching it off, 50 percent of the on-state fluorescence signal remained. To address these shortcomings, they performed a series of comprehensive error-prone mutagenesis, taking turns with site-directed saturation mutagenesis at particular amino acid positions. This gave them a variant with a high-fluorescence signal and a large dynamic range in the switchable signal, but also with positive-switching characteristics. Because it exhibits reverted switching behavior with respect to that of Dronpa, they named the variant Padron.Padron is a bright green fluorescent protein in the on state. Irradiating it with blue light yields fluorescence and switches it from the off to the on state; irradiating it with ultraviolet light switches it off again. It exhibits a dynamic range of 150:1 – among the highest of RSFPs – and it almost maintains the switching speed of rsFastLime, a variant of Dronpa and the parent of Padron.Generation of the new RSFP may have been unexpected, but ultimately it was very welcome. “I was surprised by the discovery of Padron,” Jakobs said. “That discovery just opened the way to monochromatic multi-label imaging.”While screening for Padron, the researchers identified a mutant that was negatively switchable – that is, irradiation excites fluorescence but also switches the protein from the fluorescent on state to the nonfluorescent off state – but also demonstrated a broad asymmetric absorption spectrum. Further improvements gave them a variant that they termed bsDronpa, for broad-absorption-spectrum Dronpa, which offers both efficient excitation with ultraviolet light and good switching characteristics.Having generated the new RSFPs, they applied them to monochromatic multilabel imaging as well as to dual-color subdiffraction resolution microscopy. Usually, with multicolor microscopy, researchers distinguish fluorophores based upon their absorption and emission spectra. This tends to produce artifacts resulting from chromatic aberrations, however. In the Nature Biotechnology study, the investigators demonstrated monochromatic multilabel imaging by switching Padron and rsFastLime/bsDronpa using two irradiation wavelengths (405 and 496 nm) and one spectral detection window (500 to 520 nm), which allowed them to avoid such aberrations.No aberrations detectedThey confirmed this by comparing the degree of chromatic aberrations in both conventional confocal multicolor microscopy and monochromatic multilabel imaging. They labeled 40-nm beads with rsFastLime, Padron or the red fluorescent protein EqFP611 and imaged them with a Leica Microsystems beam-scanning confocal microscope outfitted with a UV-corrected 1.4-NA, 63× oil-immersion lens. When imaged with conventional multicolor microscopy, the beads labeled with EqFP611 appeared displaced from the Padron- and rsFastLime-labeled beads by about 150 nm along the optical axis. When they discriminated Padron and rsFastLime by alternate switching, however, no such chromatic aberrations were detected.They then demonstrated the technique’s utility by imaging a sample containing E. coli cells expressing bsDronpa, rsFastLime or Padron. They began by irradiating the cells with 405-nm light, bringing bsDronpa and rsFastLime into the fluorescent on state and Padron into the nonfluorescent off state. Following this, they read out the bsDronpa fluorescence by irradiating the sample with 405-nm light and then probed rsFastLime fluorescence by irradiating it with 496-nm light – choosing the intensity so as to achieve a sufficiently high fluorescence signal while minimizing activation of Padron and inactivation of rsFastLime. Irradiation with 496-nm light with high intensity then switched off rsFastLime and bsDronpa and switched on Padron. And, finally, they read out the Padron fluorescence by irradiating the sample again with 496-nm light.Jakobs and colleagues also showed how the RSFPs can allow subdiffraction resolution microscopy – or nanoscopy. Here, they labeled the cytoplasmic membranes of 200-nm-thick cryosections of E. coli with bsDronpa and imaged them. They obtained resolution of approximately 45 nm, which proved a sevenfold improvement over the resolution of a conventional, diffraction-limited wide-field image.They currently are working to identify more and, possibly better, RSFPs. “In fact, we also tried to generate red fluorescent RSFPs,” Jakobs said. “To generate them, we had to start from a different protein, namely the red fluorescent Cherry.” The investigators published this research in the Sept. 15, 2008, issue of Biophysical Journal. They also are looking more closely at applications of the RSFPs in cell biology.