Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.     

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.     

Reverse Intersystem Crossing of Single Deuterated Perylene Molecules in a Dibenzothiophene Matrix

Intersystem crossing to the long-lived metastable triplet state is often a strong limitation on fluorescence brightness of single molecules, particularly for perylene in various matrices. In this paper, we report on a strong excitation-induced reverse intersystem crossing (rISC), a process where single perylene molecules in a dibenzothiophene matrix recover faster from the triplet state, turning into bright emitters at saturated excitation powers. With a detailed study of single-molecule fluorescence autocorrelations, we quantify the effect of rISC. The intrinsic lifetimes found for the two effective triplet states (8.5±0.4 ms and 64±12 ms) become significantly shorter, into the sub-millisecond range, as the excitation power increases and fluorescence brightness is ultimately enhanced at least fourfold. Our results are relevant for the understanding of triplet state manipulation of single-molecule quantum emitters and for markedly improving their brightness.     

Single carbon nanotube optical spectroscopy.

This Minireview discusses novel insights into the electronic structure of carbon nanotubes obtained using single-molecule fluorescence spectroscopy. Fluorescence spectra from single nanotubes are well described by a single, Lorentzian lineshape. Nanotubes with identical structures fluoresce with different energies due to local electronic perturbations. Carbon nanotube fluorescence unexpectedly does not-show any intensity or spectral fluctuations at 300 K The lack of intensity blinking or bleaching demonstrates that carbon nanotubes have the potential to provide a stable, single-molecule infrared photon source, allowing for the exciting possibility of applications in quantum optics and biophotonics.     

Singlet-triplet and triplet-triplet interactions in conjugated polymer single molecules

Single molecule fluorescence correlation spectroscopy has been used to investigate the photodynamics of isolated single multichromophoric polymer chains of the conjugated polymers MEH-PPV and F8BT on the microsecond to millisecond time scale. The experimental results (and associated kinetic modeling) demonstrate that (i) triplet exciton pairs undergo efficient triplet-triplet annihilation on the <30 micros time scale, (ii) triplet-triplet annihilation is the dominant mechanism for triplet decay at incident excitation powers > or =50 W/cm(2), and (iii) singlet excitons are quenched by triplet excitons with an efficiency on the order of (1)/(2). The high efficiency of this latter process ensures that single molecule fluorescence spectroscopy can be effectively used to indirectly monitor triplet exciton population dynamics in conjugated polymers. Finally, correlation spectroscopy of MEH-PPV molecules in a multilayer device environment reveals that triplet excitons are efficiently quenched by hole polarons.     

Investigation of an acid-base and redox molecular switch: from bulk to the single-molecule level

In this work we investigate a new fluorescent molecular switch based on the interconversion between the fluorescent zwitterionic form (ZW1) and the non-fluorescent anionic state (MC2) of a spirocyclic Meisenheimer complex of 1,3,5-trinitrobenzene. Density functional theory molecular orbital calculations reveal that photo-induced electron transfer from a guanidine group to the trinitrocyclohexadiene fluorophore of the complex quenches the emission from MC2. Protonation, as well as coordination of other Lewis acids to the guanidine group, suppress the quenching mechanism and allow the complex to fluoresce. In agreement with the calculations, reversible on-off fluorescence switching of the ZW1-MC2 bulk system occurs by protonation-deprotonation of the guanidine moiety upon acid-base addition. Interestingly, spectroelectrochemical ensemble measurements show that switching of the ZW1-MC2 pair can also be attained electrochemically, thus unraveling the versatile functioning of this system. The ultimate limit of monitoring the reversible on-off operation of individual switch molecules is reached by means of single-molecule fluorescence spectroscopy, which demonstrates the potential of the ZW1-MC2 system to be used as a true single-molecule switch on the nanometer scale.     

Excited-state and photoelectrochemical behavior of pyrene-linked phenyleneethynylene oligomer

An oligophenyleneethynylene (OPE), 1,4-bis(phenyleneethynyl)-2,5-bis(hexyloxy)benzene (2), is coupled with pyrene to extend the conjugation and allow its use as a light-harvesting molecule [Py-OPE (1)]. The absorption and emission maxima of 1 are red-shifted compared to those of 2. Similar differences in the singlet and triplet excited-state properties are evident. The fluorescence yield of 2 in toluene is 0.53, which is slightly less than the value for the parent OPE (2) of 0.66. The excited singlet and triplet of 1 as characterized from transient absorption spectroscopy exhibit lifetimes of 1.07 ns and 4.0 micros, respectively, in toluene. When 1 was cast as a film on a glass electrode (OTE) and excited with a 387-nm laser pulse, we observed the formation of excitons that decayed within a few picoseconds. When 1 was cast as a film on a SnO2-modified conducting glass electrode (OTE/SnO2), a small fraction of excitons dissociated to produce a long-lived charge-separated state. The role of the SnO2 interface in promoting charge separation was inferred from the photoelectrochemical measurements. Under visible light excitation, the OTE/SnO2 electrode was capable of generating photocurrent (approximately 0.25 mA/cm2) with an incident photon conversion efficiency (IPCE) of approximately 6%.     

Unexpected Photoluminescence of Fluorinated Naphthalene Diimides

Two new amino core‐substituted naphthalene diimides (cNDIs) bearing fluorinated side chains have been synthesised. Steady‐state and time‐resolved fluorescence spectroscopy reveals unprecedented optical properties for the cNDIs with high quantum yields of ～0.8 and fluorescence lifetimes of ～13 ns in a range of solvents. These properties are apparent at the level of single molecules, where the compounds also show exceptional photostability under pulsed‐laser excitation. Photon emission is remarkably consistent with very few long timescale (millisecond or longer) interruptions with molecules regularly undergoing >10⁷ cycles of excitation and emission. Intermittencies owing to triplet‐state formation occur on a sub‐millisecond timescale with a low yield of 1–2 %, indicating that the presence of the fluorine atoms does not lead to a significant triplet yield through the heavy‐atom effect. These properties make the compounds excellent candidates for single‐molecule labelling applications.     

Monitoring molecular beacon DNA probe hybridization at the single-molecule level

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.     

Hydrogen Bonding-Induced H-Aggregation for Fluorescence Turn-On of the GFP Chromophore: Supramolecular Structural Rigidity

To turn on the fluorescence of the native green fluorescence protein (GFP) chromophore, 4-hydroxybenzylidene-dimethylimidazolinone (HBDI), in an artificial supramolecular system has been a challenging task, because it requires high local environmental rigidity. This work shows that the formation of H-aggregates of an HBDI-containing organogelator results in two orders of magnitude fluorescence enhancement (Φ_f=2.9 vs. 0.02 %), in which the inter-HBDI OH⋅⋅⋅OH H-bonds play a crucial role. The aggregation-induced fluorescence enhancement of HBDI has important implications on the origin of the high fluorescence quantum efficiency of HBDI in the GFP β-barrel and on the supramolecular strategy for a full fluorescence recovery of HBDI. These results reveal a new approach to designing rigid chromophore aggregates for high-performance optoelectronic properties.     

Using single-molecule fluorescence spectroscopy to study electron transfer.

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.     

Oligo(p‐phenylene‐ethynylene)‐Derived Super‐π‐Gelators with Tunable Emission and Self‐Assembled Polymorphic Structures

Linear π‐conjugated oligomers are known to form organogels through noncovalent interactions. Herein, we report the effect of π‐repeat units on the gelation and morphological properties of three different oligo(p‐phenylene‐ethynylene)s: OPE3 , OPE5 , and OPE7 . All of these molecules form fluorescent gels in nonpolar solvents at low critical gel concentrations, thereby resulting in a blue gel for OPE3 , a green gel for OPE5 , and a greenish yellow gel for OPE7 . The molecule–molecule and molecule–substrate interactions in these OPEs are strongly influenced by the conjugation length of the molecules. Silicon wafer suppresses substrate–molecule interactions whereas a mica surface facilitates such interactions. At lower concentrations, OPE3 formed vesicular assemblies and OPE5 gave entangled fibers, whereas OPE7 resulted in spiral assemblies on a mica surface. At higher concentrations, OPE3 and OPE5 resulted in super‐bundles of fibers and flowerlike short‐fiber agglomerates when different conditions were applied. The number of polymorphic structures increases on increasing the conjugation length, as seen in the case of OPE7 with n=5, which resulted in a variety of exotic structures, the formation of which could be controlled by varying the substrate, concentration, and humidity.     

Optical Sensing of Current Dynamics in Organic Light‐Emitting Devices at the Nanometer Scale

Photoluminescence quenching of single dibenzoterrylene (DBT) dye molecules in a polymeric organic light‐emitting diode was utilized to analyze the current dynamics at nanometer resolution. The quenching mechanism of single DBT molecules results from an increase in the triplet‐state population induced by charge carrier recombination on individual guest molecules. As a consequence of the long triplet‐state relaxation time, its population results in a reduced photoluminescence of the dispersed fluorescent dyes. From the decrease in photoluminescence together with photon correlation measurements, we could quantify the local current density and its time‐dependent evolution in the vicinity of the single‐molecule probe. This optical technique establishes a non‐invasive approach to map the time‐resolved current density in organic light‐emitting diodes on the nanometer scale.     

From Graftable Biphotonic Chromophores to Water‐Soluble Organic Nanodots for Biophotonics: The Importance of Environmental Effects

The photophysical and two‐photon absorption (TPA) properties of biphotonic chromophores with one or two phenol pendant units were studied and compared with that of a model biphotonic quadrupolar chromophore. A water‐soluble dendritic structure was then synthesized by using the pendant moieties as starting points for the construction of dendritic branches. We show that the polarity of the environment significantly modulates both the fluorescence and the TPA responses of the different chromophoric derivatives. This extends to more subtle effects that involve phenol pendant moieties that were found to act as discrete solvating units and to modify both the photophysics and the TPA response of the chromophore. This demonstrates the high sensitivity of the TPA response of quadrupolar derivatives to minute alterations in the environment. Moreover, the dendritic branches were found to behave as a peculiar cybotactic environment that was able to tune the fluorescence and TPA response of the inner chromophore by creating a polar environment. This reveals a new direction for exploiting such effects by playing on the dendritic architecture (e.g., the nature and shape of the building blocks, the geometry and position of the chromophore) to modulate the TPA responses.     

Extraction of kinetic information from single-molecule experiments.

We discuss two possible approaches for extracting kinetic information from single-molecule experiments. The first approach is based on computing correlation functions from measured fluorescence signals, and the second on studying the statistics of on and off times of the same fluorescence signal. We show that in both cases it is possible to extract kinetic information about the nature of intramolecular fluctuations of the single molecule. We show that for single-molecule kinetics the intramolecular fluctuations produce stochastic memory effects which lead to new dynamic features that do not exist in traditional chemical kinetics. In particular, we investigate a new type of chemical oscillations in correlation functions observed experimentally by Edman and Rigler (Proc. Natl. Acad. Sci. USA 2000, 97, 8266).     

Single-molecule triplet-state photon antibunching at room temperature

We have probed single-molecule metal-to-ligand charge transfer (MLCT) dynamics of a ruthenium complex at room temperature. Using photon antibunching measurements under continuous wave (CW) laser excitation, nonclassical photon statistics, and excitation power dependent measurements, we were able to selectively measure the single-molecule MLCT state lifetime. This work demonstrated, as the first single-molecule photon antibunching measurement of the triplet excited state, a new application of single-molecule spectroscopy on excited-state dynamics and ground-state recovering dynamics of an important class of chemical species that have often been used and studied in energy conversion and electron transfer.     

Quantitative determination of the single-molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis

Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules is no longer negligible, i.e., no single-molecule detection regime. If the mean number N of molecules is lower than 0.0057, the detection limit of the method is reached, i.e., the fluorescence signal cannot be distinguished from the background. Consequently, the concentration of coumarin 153 characteristic for the single-molecule detection regime lies in the range 13-110 pmol/l for the given experimental conditions. We also investigate the influence of the molecular brightness, i.e., detected photons per fluorophore molecule and sampling time, on the single-molecule detection regime.     

Synthesis and Photophysics of Core‐Substituted Naphthalene Diimides: Fluorophores for Single Molecule Applications

The synthesis and photophysics of two new aminopropenyl naphthalene diimide (SANDI) dyes are reported. A general and convenient method for the synthesis of the precursor mono‐, di‐, and tetrabrominated 1,4,5,8‐naphthalene tetracarboxylic dianhydrides is described. The two core‐substituted SANDIs exhibit many of the photophysical properties required for fluorescence labeling applications including high photostability and high fluorescence quantum yields (>0.5) in the visible region of the spectrum. The emission wavelength is sensitive to the number of substituents on the NDI core, and the fluorescence decay times are in the range of ～8–12 ns for both compounds in the solvents investigated. Preliminary fluorescence emission data from single molecules of the compounds embedded in poly(methyl methacrylate) films are also reported and show that single molecules have very low yields of photobleaching, particularly the di‐substituted system. Furthermore, only a small proportion (<10 %) of the single molecules studied display fluorescence intermittencies or “blinks” in their photon trajectory. The compounds appear to be excellent candidates for applications at the single molecule level, for example, as FRET labels.