Long-lived Dark States Research Articles

All-optical fluorescence image recovery using modulated Stimulated Emission Depletion.

Fluorescence modulation for selective recovery of desired fluorescence signals has to date required careful fluorophore selection combined with repeated optical recovery from long-lived photoinduced dark states. Adapting an all-optical scheme, modulated Stimulated Emission Depletion generalizes such modulation schemes by eliminating the need for dark state residence by directly optically depopulating the emissive state at any externally applied frequency. Using two overlapped Gaussian laser spots with the depletion beam being intensity-modulated, fluorescence modulation is readily achieved with a depletion ratio governed by the intensity of the depleting laser. Selective image recovery of otherwise unmodulatable fluorophore signals is directly achieved through this all-optical modulation, and common STED-degrading multiphoton-excited background is readily discriminated against. Both beads and dyes in solution as well as fluorophores bound within fixed cells are readily imaged in this manner.

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

Metastable Dark States Enable Ground State Depletion Microscopy of Nitrogen Vacancy Centers in Diamond with Diffraction-Unlimited Resolution

Current far-field optical nanoscopy schemes overcome the diffraction barrier by ensuring that adjacent features assume different states upon detection. Ideally, the transition between these states can be repeated endlessly and, if performed optically, with low levels of light. Here we report such optical switching, realized by pairing the luminescent triplet and a long-lived dark state of diamond color centers, enabling their imaging with a resolution >10 times beyond the diffraction barrier (<20 nm).

Photoactivatable Fluorescent Protein mEos2 Displays Repeated Photoactivation after a Long-Lived Dark State in the Red Photoconverted Form

Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP) mutants that have gone into a long-lived dark state. The reported behavior is the standard for the reverse photoswitchable protein Dronpa and its mutants. However, conventional knowledge regarding the mEos2 photoactivatable fluorescent protein (PA-FP) is that, once bleached, this fluorophore is hardly reactivated, aside from a minority population that might display this behavior. Here we show that, in a typical experiment, approximately 50% of the investigated single molecule time traces display multiple reactivations, making this a seemingly inherent feature of the mEos2 PA-FP. These results hint to some similarities between mEos2 and other reversibly photoactivatable probes such as Dronpa. We investigate the consequences of this phenomenon in the framework of photoactivated localization microscopy (PALM) experiments.

FRET-enabled optical modulation for high sensitivity fluorescence imaging.

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Diphenylhexatrienes as Photoprotective Agents for Ultrasensitive Fluorescence Detection

Given the particular importance of dye photostability for single-molecule investigations, fluorescence fluctuation spectroscopy, and laser-scanning microscopy, refined strategies were explored for enhancing the fluorescence signal by selectively quenching the first excited triplet state of the laser dye Rhodamine 123 (Rh123). The strategy is to quench the T(1) state by Dexter triplet energy transfer, while undesired quenching of the singlet state via Forster or Dexter singlet energy transfer and the generation of free radicals through electron transfer should be avoided. Diphenylhexatrienes (DPHs) were tested in ethanol for their beneficial effects as a novel class of photoprotective agents using fluorescence correlation spectroscopy. A library of DPHs with electron-donating (dimethlyamino) and withdrawing substituents (e.g., trifluormethyl) was synthesized to optimize the electronic properties. Quantum chemical calculations, optical spectroscopy, and cyclic voltammetry were used to determine the electronic properties. The computed T(1) emission energy of Rh123 and the T(1) excitation energies of all DPHs allow for exergonic triplet energy transfer to the quencher. The parent compound quenches the T(1) state of Rh123 nearly diffusion controlled (4.9 x 10(9) M(-1) s(-1)). All electron-deficient DPHs significantly increase (3x) the fluorescence rate of Rh123 by reducing the triplet state population and by avoiding the formation of other long-lived dark radical states. The quenching constants are reduced by more than a factor of 2, if substituents with increasing size or electronegativity are introduced. The beneficial effect of triplet quenching of substituted DPHs is governed by a delicate interplay of steric, electronic, and intermolecular Coulombic effects.

Intersystem Crossing and Characterization of Dark States in the Pyrimidine Nucleobases Uracil, Thymine, and 1-Methylthymine

The ground and low-lying excited states of the pyrimidine nucleo bases uracil, thymine, and 1-methylthymine have been characterized using ab initio coupled-cluster with approximate doubles (CC2) and a combination of density functional theory (DFT) and semiempirical multireference configuration interaction (MRCI) methods. Intersystem crossing rate constants have been determined perturbationally by employing a nonempirical one-center mean-field approximation to the Breit-Pauli spin-orbit operator for the computation of electronic coupling matrix elements. Our results clearly indicate that the S(2)((1)pi-->pi*)-->T(2)((3)n-->pi*) process cannot compete with the subpicosecond decay of the S(2) population due to spin-allowed nonradiative transitions, whereas the T(1)((3)pi-->pi*) state is populated from the intermediate S(1)((1)n-->pi*) state on a subnanosecond time scale. Hence, it is very unlikely that the S(1)((1)n-->pi*) state corresponds to the long-lived dark state observed in the gas phase.

Fluorescence Imaging Of Ryanodine Receptor And Caveolin Distribution In Cardiac Myocytes At 30 nm Resolution

In cardiac ventricular muscle excitation-contraction coupling arises from Ca2+ release via clusters of ryanodine receptors (RyRs) in regions of close apposition between the sarcoplasmic reticulum and surface membranes. The local Ca2+ signalling in these regions depends on the nanometer organization of RyRs and other protein clusters. Until recently, however, optical imaging was thought to be limited to a resolution of ∼250 nm set by the diffraction of light. We have overcome this limitation using a new technique that allows imaging of conventionally labelled fluorescent samples at much higher resolution. Isolated rat cardiac ventricular myocytes were labelled with specific antibodies against cardiac ryanodine receptors and caveolin-3 and secondary antibodies conjugated to the organic fluorochrome Alexa 488. To visualize the distribution of the labelled proteins at the surface sarcolemma cells were imaged on a total internal reflection fluorescence microscope. We observed that Alexa 488 (and several other organic fluorophores) exhibit reversible photobleaching with recovery times >10s. This observation provided the basis for a new imaging technique we term reversible photobleaching microscopy (RPM). RPM relies on intense illumination to reversibly induce a very long-lived dark state from which single fluorochromes slowly return stochastically. These single molecule fluorescence events were detected with a sensitive camera and each event could be localized with an accuracy of better than 30 nm. From several thousand frames we thus reconstructed ultra-high-resolution images of the distribution of RyRs and caveolin at the surface sarcolemma of the stained cells. Both proteins formed dense clusters with diameters that were generally much smaller than the diffraction limit. Our new approach is applicable to measure the nanoscale distribution of many proteins of interest in excitation-contraction coupling or other signalling processes.

Light-Induced Dark States of Organic Fluochromes Enable 30 nm Resolution Imaging in Standard Media

We show that high quantum efficiency fluorophores can exhibit reversible photobleaching. This observation provides the basis for an imaging technique we call reversible photobleaching microscopy. We demonstrate applicability of this technique using antibody labeled biological samples in standard aqueous (or glycerol based) media to produce far-field images at ∼30 nm resolution. Our novel method relies on intense illumination to reversibly induce a very long-lived (>10 s) dark state from which single fluorochromes slowly return stochastically. As in other localization microscopy methods, reversible photobleaching microscopy localizes single fluorochromes, but has the advantage that specialized photoactivatible and photoswitchable molecules or special immersion/embedding media are not required.

On fluorescence blinking of single molecules in polymers

Fluorescence blinking of single DiI molecules was studied in matrices: polymethylmetacrylate (PMMA), polystyrene (PS), and polyvinyl alcohol (PVA). The triplet lifetimes of DiI were significantly different in PMMA, PS, and PVA. We found that short triplet lifetimes give short lifetimes of photobleaching, which is attributed to fluorescence quenching by molecular oxygen. We also found, that the intersystem crossing yield changes, for the same chromophore, when the polymer host is changed; a feature which could be attributed to different internal conversions for different polymer matrices. Long-lived dark states were often observed in PVA, a feature which suggests that dark state formation is due to a radical formation by intermolecular electron transfer.

Photoswitching microscopy with standard fluorophores

We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2–10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ∼20 nm. The method requires wide-field illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

Influence of the Dark Exciton State on the Optical and Quantum Optical Properties of Single Quantum Dots

The dark exciton state strongly affects the optical and quantum optical properties of flat InP/GaInP quantum dots. The exciton intensity drops sharply compared to the biexciton with rising pulsed laser excitation power while the opposite is true with temperature. Also, the decay rate is faster for the exciton than the biexciton and the dark-to-bright state spin flip is enhanced with temperature. Furthermore, long-lived dark state related memory effects are observed in second-order cross-correlation measurements between the exciton and biexciton and have been simulated using a rate-equation model.

Calculation of quantum-dot blinking using the Gillespie Monte Carlo algorithm

We calculate the stochastic photoluminescence intensity of a single colloidal quantum dot in order to calculate the blinking behaviour of the optical emission. The combination of the long-lived dark state with a lifetime of the order of seconds together with the radiative recombination lifetimes in the nanosecond regime make the stochastic model of the system incredibly computer intensive. The stochastic behaviour of the single quantum dot using a system of rate equations for the probabilities of occupancy of the confined energy levels is calculated. A standard Monte Carlo algorithm is modified by introducing a time step that is dependent on the transitions possible in the state that the system occupies. The model is verified by comparison with the calculation using the standard algorithm and from the calculated probability densities. The improvement in computational time of this algorithm over the standard Monte Carlo method is also determined.

Determination of energetics and kinetics from single-particle intermittency and ensemble-averaged fluorescence intensity decay of quantum dots

Quantification of energetics and kinetics for the band-edge exciton states of quantum dots and the long-lived dark state is important for better understanding of the underlying mechanism for single-particle intermittency and ensemble fluorescence intensity decay. Based on a multistate diffusion-reaction model by extending our previous studies, we analyze experimental data from ensemble measurements and fluorescence intermittency of single quantum dots and determine important molecular-based quantities such as Stokes shift, free energy gap, activation energy, reorganization energy, and other kinetic parameters.

Power-Law-Distributed Dark States are the Main Pathway for Photobleaching of Single Organic Molecules

We exploit the strong excitonic coupling in a superradiant trimer molecule to distinguish between long-lived collective dark states and photobleaching events. The population and depopulation kinetics of the dark states in a single molecule follow power-law statistics over 5 orders of magnitude in time. This result is consistent with the formation of a radical unit via electron tunneling to a time-varying distribution of trapping sites in the surrounding polymer matrix. We furthermore demonstrate that this radicalization process forms the dominant pathway for molecular photobleaching.

Exponential and Power-Law Kinetics in Single-Molecule Fluorescence Intermittency

Perylenemonoimide chromophores, attached to a small dendron arm embedded in the polymer hosts Zeonex, poly(vinylbutyral), and poly(methyl methacrylate), are studied by means of laser-induced confocal fluorescence detection. Transient fluorescence intensity exhibits dark periods on two different time scales: on a 100 μs time scale and on a considerably longer time scale, ranging from 100 ms to as much as tens of seconds under high-vacuum conditions. The exponentially distributed short “off” times are attributed to triplet excursions of the molecule. The long-lived dark states follow a power-law distribution and are discussed in terms of the formation of radical anions/cations via electron tunneling processes.

Photoblinking of Rhodamine 6G in Poly(vinyl alcohol): Radical Dark State Formed through the Triplet

We investigate the fluorescence intensity of rhodamine 6G in poly(vinyl alcohol) as a function of excitation intensity, illumination time, the presence of oxygen, and temperature. The variations in emissivity (or fluorescence brightness) are attributed to a dark state, which shows populating kinetics resembling those of the triplet state, but a much longer lifetime. We simulate the observed kinetics by a four-level model, in which a long-lived dark state is formed through the triplet as an intermediate state. The weak temperature dependence of the lifetime of the dark state points to electron tunneling as the main recovery process. This intermolecular mechanism also explains the observed broad distribution of lifetimes. An electron-spin-resonance experiment confirms the assignment of the dark state to a radical. For the first time, photoinduced charge transfer is identified as a source of blinking in single-molecule measurements.

The role of long-lived dark states in the photoluminescence dynamics of poly(phenylene vinylene) conjugated polymers. II. Excited-state quenching versus ground-state depletion

The two pulse fluorescence bleaching experiments reported in an earlier paper [J. Chem. Phys. 117, 454 (2002)] are reanalyzed in the context of additional experiments. The fluorescence bleaching observed in that paper was originally ascribed to ground-state depletion. By analyzing the absorption saturation behavior and the magnitude of the pump–probe signal, we find that the absorption cross section of poly(phenylene vinylene) at 400 nm is two orders of magnitude too small to significantly deplete the ground state given the pump fluences used in that experiment. Instead, the observed depletion is due to a combination of exciton–exciton annihilation at early times and dark state luminescence quenching at later times. Different experiments have different sensitivities to the fluence-dependent quenching, with time-resolved experiments like transient emission and fluorescence decay time consistently underestimating the exciton–exciton annihilation rate. Experiments that measure the integrated fluorescence, such as fluorescence saturation and bleaching, result in a consistent value for the exciton–exciton annihilation constant of 6×10−9 cm3/s, while the dark state quenching constant is estimated to be at least 1.2×10−8 cm3/s. Indirect evidence based on the wavelength and sample dependence of the dark state formation suggest that the dark states are charge-separated polarons. The relatively large quenching constants are consistent with what has been observed in other conjugated polymers and suggest that the long-lived dark states in particular are effective quenchers, capable of quenching thousands of surrounding chromophores.

The role of long-lived dark states in the photoluminescence dynamics of phenylene vinylene conjugated polymers

The role of intermediate nonluminescent states in the relaxation of singlet excitons in the conjugated polymer poly(phenylene vinylene) (PPV) and its soluble derivative poly[(2-methoxy-5-hexyloxy-p-phenylene) vinylene] (MH-PPV) is investigated. Time-resolved luminescence and absorption measurements provide evidence for a long-lived, weakly emissive species in PPV at 17 K, in addition to the luminescent singlet state. Ground state recovery times at this temperature provide evidence that up to 40% of the initially excited chromophores end up in a state that does not relax back to the ground state on the 5 ns time scale of the experiment. As the temperature is raised from 17 K to 290 K, the redshifted emission disappears, the fluorescence decay becomes more rapid, and the magnitude of the long-time bleach decreases. These results can be understood in terms of a three-level model where the initially excited singlet state decays nonradiatively via two separate channels: thermally-activated direct relaxation to the ground state, and nanosecond relaxation into a third, long-lived dark state. As the temperature increases, the thermally-activated process increases at the expense of both the fluorescence and the intermediate dark state population. Using this model, a temperature-independent dark state formation time of 1.8 ns was found for PPV, and 1.1 ns for MH-PPV. Our data and modeling provide no evidence for a subpicosecond relaxation channel in the decay of the luminescent excitons in these phenylene vinylene polymers.

Looking at the photodynamics of individual fluorescent molecules and proteins

Abstract The photodynamics of individual molecules and fluorescent proteins has been investigated in real time. In the case of organic molecules, both the triplet state lifetime and intersystem crossing yield appear to vary in time and space. In the case of autofluorescent proteins, light-driven "on-off" fluorescence emission has been observed, with a behavior that is consistent with the existence of a long-lived dark state.