Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488-and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.photochromism ͉ protonation͞deprotonation ͉ fluorescence microscopy P hotoinduced alteration of chemical and physical properties of photochromic molecules is of great interest because of its potential applications for optoelectronic devices, such as optical memory and optical switches (1). Photoinduced switching of fluorescent properties is one of the most attractive concepts for the realization of a nondestructive read-out system (2-4). Apart from this application, the photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is being recognized as new methodology of optical marking (5, 6). Intracellular dynamics of selected molecules can be followed by activating the fluorescent proteins to their fluorescent state (7-11). Realization of photoswitching at the single-molecule level will open up exciting opportunities in the field of optoelectronics and biological imaging, where it could provide molecular-scale devices as well as detection of fast dynamics of individual proteins in living cells. Reversible photoswitching at the single-molecule level, however, has not yet been well characterized (12-17). Dickson et al. (12) reported reversible photoswitching of a mutant of GFP. Although they demonstrated a few photoswitching events at the single-molecule level, minutes of illumination was required to achieve the switching. Irie and coworkers (13, 14) also reported reversible photoswitching of diarylethene derivatives, which occurred relatively slowly with a response time of seconds. Although the switching can be repeated Ͼ10 4 times at the ensemble level (1), the number of switching events obtained at the single-m...

Four generations of a dendrimer with a fluorescent core consisting of a rubicene moiety are synthesized. The biexponential nature of fluorescence decay in toluene indicates the presence of two emitting conformations. Molecular modeling suggests that a conformation where the dendrons interact with the core is not improbable. The relative weight of the two decay times indicates that the contribution of that conformation in toluene increases as the dendrimer generation increases. In acetone and acetonitrile however the fluorescence decay is, except for the fourth generation, monoexponential. The hydrodynamical volume of the dendrimer is determined in toluene, a good solvent, acetone, a medium quality solvent, and acetonitrile, a poor solvent, with the time-resolved fluorescence depolarization technique. No change of the hydrodynamical volume is found in toluene in a temperature range between 20 and 94 °C. This suggests that the dendrimers of all the generations are fully expanded in this solvent. In acetonitrile however the hydrodynamical volume of the dendrimers is substantially smaller than in toluene and acetone. This effect is most clear for the fourth-generation dendrimer. For this compound the hydrodynamical volume is only a few percentages larger than the excluded volume.

The differences in the fluorescence behavior of a polyphenylene dendrimer with eight peryleneimides chromophores (1) and a single hexaphenylperyleneimide chromophore have been investigated at a single‐molecule level through the combination of ultrasensitive fluorescence detection and microscopy.

The photophysical and hydrodynamic properties of dendrimers (GnPZn and GnTPPH2) with zinc porphyrin (PZn) and tetraphenylporphyrin (TPP) cores are studied in tetrahydrofuran (THF) and dimethylformamide (DMF). UV−vis absorption spectra of GnPZn exhibit a small red shift of the Soret band upon increasing the generation as a result of interactions between the dendrons and the core. All fluorescence decays obtained from global analysis show a monoexponential profile. The intrinsic viscosity obtained for GnPZn from the hydrodynamic volume (V h) passes through a maximum as a function of generation (G) in agreement with earlier experimental findings and calculations suggesting that the internal density profile of dendrimers decrease monotonically outward from the center of the molecule. Within the investigated range (G = 1−3), GnTPPH2 exhibits an approximately constant intrinsic viscosity due to the linear dependence between the hydrodynamic volume and the molecular weight. The differences observed between GnPZn and GnTPPH2 are correlated to structural differences in their cores. The additional phenyl group of the TPP in GnTPPH2 increases the distance between the branches and the porphyrin moiety compared to GnPZn, resulting in a more flexible structure. The enhanced flexibility allows the terminal groups to sample more conformational space and therefore decreases the volume of the dendrimer as compared to the theoretical fully extended structure where V h ∝ G 3. A comparison of the results obtained from analysis of fluorescence anisotropy decays with previously reported viscometry measurements shows a dependence of the structural collapse on the core size.