The photophysical properties of chromophores at high (100 mM and above) concentrations in polymers and as neat solids

The absorption, fluorescence, and photostability of five conjugated chromophores: perylene, 2,5,8,11-tetra-t-butyl perylene (TTBP), perylene orange (PO), perylene red (PR), and a zwitterionic Meisenheimer complex (MHC), are studied as a function of concentration in poly(methyl methacrylate) (PMMA). At 1 mM concentrations, all five molecules exhibit properties consistent with unaggregated chromophores. At higher concentrations, perylene and PO both exhibit excimer formation, while TTBP, PR, and the MHC retain their monomeric fluorescent lineshapes. In these three molecules, however, the fluorescence decay times decrease by 10% (TTBP) to 50% (MHC) at concentrations of 100 mM in PMMA. The fluorescence properties of these highly concentrated samples are sensitive to the sample preparation conditions. In the neat solid where the effective concentration is on the order of 1 M, all three molecules exhibit very fast fluorescence decays, on the order of 150 ps or less, despite the fact that they retain their basic monomeric fluorescence lineshape. In addition to the enhanced nonradiative decay at high concentrations, these three molecules also undergo a concentration-dependent photobleaching. The combined effects of intermolecular nonradiative decay channels and photobleaching appear to be a general obstacle to achieving highly concentrated dye-doped solids.     

Spectroscopic investigation of novel donor–acceptor chromophores as specific application agents for opto-electronics and photonics

Donor–acceptor chromophores were synthesized by Knoevenagel condensation and characterized by using spectroscopic measurements (FT-IR, ¹H NMR, ¹³C NMR, and mass) and elemental analyses. UV–Vis and fluorescence spectroscopic studies provided that these dyes are good absorbent and fluorescent. The results of photostability study showed that photobleaching of Dyes 2 and 4 was more significant than that of Dyes 1 and 3, providing that Dyes 1 and 3 are more photostable than Dyes 2 and 4. In addition, all dyes included in this study are found to be sensitive to the polarity of the microenvironment provided by different solvents based on the results of fluorescence polarity studies.     

Intramolecular triplet energy transfer in multi-chromophoric dyes and its influence on the photostability

The photostability of organic dyes plays a very important role in practically all aspects of the development and applications of these dyes. In recent years, intramolecular transfer of triplet excitation energy between isolated chromophores on the same molecule has been a subject of intensive studies. Many multi-chromophoric organic dyes with covalent linkage between the chromophores, one of which can act as a triplet acceptor of the excess energy, have been synthesized and studied. The significant increases in photostability of such assembled dyes have been reported, suggesting that some chromophores can act as internal photostabilizers. These modified dyes have enhanced photostability and hence potential applications in a wide range of areas such as laser dyes, electroluminescent (EL) devices and solar cells.     

Hydrogen-bond locked purine chromophores with high photostability for lipid droplets imaging in cells and tissues

Recently, hydrogen-bonding has attracted extensive attention in the design of chromophores. Here, a new class of hydrogen-bond locked purine chromophores (HOPs) were reported by introducing a hydroxyphenyl group into the C(6) position of purine. The intramolecular hydrogen bond plays a dominant role to light up these probes. As a bonus, HOPs show high photostability. Moreover, HOPs exhibit remarkable capability for the specific lipid droplets imaging in living cells with excellent biocompatibility and are also potential for diagnosing fatty liver diseases. These results bring important new insights into the photophysics of the purine-based chromophores and provide a new scaffold with high photostability for bioimaging.     

Photoblinking and photobleaching of rylene diimide dyes

We investigate photoblinking and photobleaching of perylene diimide (PDI) and its higher homologue terrylene diimide (TDI). Single molecule fluorescence trajectories of the dye molecules embedded in PMMA under ambient conditions exhibit "on"-"off" blinking in the time range from ms to s. Due to the limited statistics of individual trajectories we construct ensemble distributions of "on" and "off" times which follow power laws with similar power law coefficients (m(on) ≈ 1.18, m(off) ≈ 1.31). The blinking is attributed to reversible formation of radical cations which are presumably created by electron transfer from higher excited triplet states T(n) of the molecules to acceptor levels in the PMMA host. This view is corroborated by the properties of TDI, which blinks at an excitation wavelength of 520 nm but does not at lower energy excitation (647 nm). In line with this observation, T(1)-T(n) absorption data of TDI (and PDI) indicate that above a certain illumination wavelength population of higher excited triplet states T(n) does not occur, preventing blinking. It is furthermore argued that the long-lived dark ("off") states, i.e. the radical cations, are precursors for the photobleaching process of the dye molecules. Consequently, the photobleaching quantum yield Y(bl) for TDI is very small at an excitation wavelength of 647 nm (Y(bl) = 2 × 10(-10)) but increases by two orders of magnitude at 520 nm (Y(bl) = 2 × 10(-8)), which lies in the range observed for PDI investigated with an excitation wavelength of 488 nm. Additional studies of a PDI-TDI donor-acceptor dyad give further insights into the blinking and bleaching processes. Important findings include the observation of power law blinking of TDI and PDI (after bleaching of TDI) with similar coefficients as found for the isolated chromophores. Furthermore, in the dyad the photostability of TDI decreases due to efficient population of the states T(n) by singlet-triplet annihilation, while that of PDI (after bleaching of TDI) is the same as for the isolated dye. These findings support the conclusions drawn for the isolated chromophores, in particular the involvement of the triplet manifold in the blinking (and bleaching) behavior.     

Photostability of amino acids: internal conversion versus dissociation

Photodissociation dynamics for various tryptophan chromophores was studied at 193 or 248 nm using multimass ion imaging techniques. The competition between internal conversion to the ground electronic state and dissociation from the repulsive excited state reveals size-dependent photostability for these amino acid chromophores. As the size of chromophore increases, internal conversion to the ground state becomes the major nonradiative process. For tryptophan and larger chromophores, dissociation directly from the repulsive state is completely quenched.     

Antioxidant activity assay based on the inhibition of oxidation and photobleaching of l-cysteine-capped CdTe quantum dots

Quantum dots (QDs) have recently been the focus of attention of many investigators for development of diagnostic tools in many research areas. In this work, we established a new QD-based assay to evaluate the antioxidant/polyphenolic activity. This assay is based on measurement of the inhibitory effect of the antioxidant/polyphenolic compounds on the UV-induced bleaching of CdTe QDs with l-cysteine capping. QDs exhibited excellent photostability without any UV exposure, while they bleached rapidly under UV irradiation. Generation of reactive oxygen species (ROS) under UV irradiation is probably the main cause of the photobleaching of QDs. By comparing the photostability of QDs in buffer solution in the absence and presence of sodium azide, as a known (1)O(2) quencher, the involvement of (1)O(2) in photobleaching of QDs was confirmed. The photobleaching effect induced by ROS could be reduced in the presence of antioxidant/polyphenolic compounds. We tested several antioxidant/polyphenolic compounds as well as known antioxidants such as trolox and 4 different types of tea. The results obtained by the QD-based assay revealed a very good correlation with the data acquired by Folin-Ciocalteu assay. Furthermore, a deeper understanding of the mechanism and the solution for photobleaching of QDs under UV irradiation might be very meaningful in promoting their clinical applications.     

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.     

Photobleaching of bacteriorhodopsin solubilized with triton X-100

In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.     

Reduced blinking and long-lasting fluorescence of single fluorophores coupling to silver nanoparticles

The fluorescence signal of single organic fluorophores is characterized by random blinking and irreversible photobleaching. Photoinduced blinking of Cy5 has posed various limitations of this popular near-infrared (NIR) probe in biological applications. Here we show that fluorophore-metal nanoparticle (NP) complexes greatly suppress Cy5 blinking and noticeably reduce photobleaching events. The blinking behavior of single Cy5 molecules was investigated and compared in the absence and the presence of silver nanostructures. A power-law distribution of off time population was observed for single Cy5 molecules. Average off times were compared to evaluate the plasmonic effect of silver nanoparticles on the triplet decay rates. We furthermore demonstrate enhanced photostability in the presence of silver NPs. The results show that plasmonic-controlled fluorescence can lead to a novel physical mechanism to enhance fluorescence intensity, reduce blinking, and increase photostability.     

Photogeneration and transport of charge carriers in polysilylenes

The attachment of π-conjugated chromophores that absorb the radiation with long wavelengths to poly(methylphenylsilylene) ( 1 ) via reactions of its formylated derivative is described. Some of the polymers obtained show improved photostability and higher quantum photogeneration efficiency in comparison with the parent polymer. Photoconductive ultra-thin layers can be prepared from polar derivatives of ( 1 ) by the Langmuir–Blodgett technique.     

Comparative Photostability Studies of BODIPY and Fluorescein Dyes by Using Fluorescence Correlation Spectroscopy

In single‐molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S₁, in contrast to previous findings.     

Enhanced photostability of ICG in close proximity to gold colloids

Photobleaching of fluorophores frequently limits their detectability or observation time. We examined Indocyanine green (ICG) which is widely used in medical testing and is highly unstable. We showed that spatial localization of ICG near metallic gold colloids resulted in increased photostability. This suggests the use of fluorophore-metal conjugates in situations adversely affected by photobleaching.     

Hybrid materials for solid-state dye laser applications

The quest for a solid-state tunable dye laser can be satisfied by sol-gel prepared organic-inorganic hybrids. A photostability study of porous silica-Rhodamine 6G hybrids prepared via a sol-gel method is presented. The dye molecules can be incorporated into the silica matrix by forming weak or covalent bonds (hybrids of classes I and II, respectively). New class II samples and traditional class I materials prepared by the pre-doping method were synthesized. Samples were characterized by photoluminescence measurements to compare the emission properties and the photostability of the samples. The decay of the fluorescence signal as the cumulative excitation energy increases is reported and interpreted by hypothesizing that the dye molecules can be hosted in different surroundings within the porous glass matrix. The reported photoluminescence and photobleaching features indicate the class II samples as good candidates for solid-state dye lasers.     

Photophysical aspects of single-molecule detection by two-photon excitation with consideration of sequential pulsed illumination.

An important goal in single molecule fluorescence correlation spectroscopy is the theoretical simulation of the fluorescence signal stemming from individual molecules and its autocorrelation function. The simulation approaches developed up to now are based exclusively on continuous-wave (cw) illumination and consequently on cw-excitation. However, this approximation is no longer valid in the case of two-photon excitation, for which pulsed illumination is usually employed. We present a novel theoretical model for the simulation of the fluorescence signal of single molecules and its autocorrelation function with consideration of the time dependence of the excitation flux and thus of all illumination-dependent photoprocesses: two-photon excitation, induced emission and photobleaching. Further important characteristics of our approach are the consideration of the dependence of the photobleaching rate on illumination and the low intersystem-crossing rates of the studied coumarins. Moreover, using our approach, we can predict quantitatively the effect of the laser pulse width on the fluorescence signal of a molecule, that is, the contributions of the photobleaching and saturation effects, and thus we can calculate the optimal laser pulse width. The theoretical autocorrelation functions were fitted to the experimental data, and we could ascertain a good agreement between the resulting and the expected parameters. The most important parameter is the photobleaching constant sigma, the cross section of the transition Sn<--S1, which characterises the photostability of the molecules independent of the experimental conditions. Its value is 1.7 x 10(-23) cm2 for coumarin 153 and 5 x 10(-23) cm2 for coumarin 314.     

Probing the electronic structure of platinum(II) chromophores: crystal structures, NMR structures, and photophysical properties of six new bis- and di- phenolate/thiolate Pt(II)diimine chromophores

A general route for synthesis of six structurally similar Pt(II) diimine thiolate/phenolates chromophores possessing bulky phenolate or thiolate ligands is reported. The Pt chromophores were characterized using an array of techniques including 1H, 13C, and 195Pt NMR, absorption, emission, (spectro)electrochemistry, and EPR spectroscopy. Systematic variation of the electronic structure of the Pt(II) chromophores studied was achieved by (i) changing solvent polarity; (ii) substituting oxygen for sulfur in the donor ligand; (iii) alternating donor ligands from bis- to di-coordination; and (iv) changing the electron donating/withdrawing properties of the ligand(s). The lowest excited state in these new chromophores was assigned to a [charge-transfer-to-diimine] transition from the HOMO of mixed Pt/S (or Pt/O) character on the basis of absorption and emission spectroscopy, UV/vis (spectro)electrochemistry, and EPR spectroscopy. One of the chromophores, Pt(dpphen)(3,5-di-tert-butyl-catecholate) represents an example of a Pt(II) diimine phenolate chromophore that possesses a reversible oxidation centered predominantly on the donor ligand. Results from EPR spectroscopy indicate participation of the Pt(II) orbitals in the HOMO. There is a dramatic difference in the photophysical properties of carborane complexes compared to other mixed-ligand Pt(II) compounds, which includes room-temperature emission and photostability. The charge-transfer character of the lowest excited state in this series of chromophores is maintained throughout. Moreover, the absorption and emission energies and the redox properties of the excited state can be significantly tuned.     

Merocyanine dyes with improved photostability

Merocyanine dyes have proven valuable for live cell fluorescence imaging applications, but many structures have been limited by rapid photobleaching. We show that photostability is substantially enhanced for merocyanines having a cyano group at a specific position in the central polymethine chain. Evidence is presented that this is due to reduction in reactivity of the dyes with singlet oxygen. These results point toward cyano-substitution as a general strategy for improving dye performance in imaging applications.     

Photochemical instability of thiol-capped CdTe quantum dots in aqueous solution and living cells: process and mechanism

The process and mechanism of photochemical instability of thiol-capped CdTe quantum dots (QDs) in aqueous solution were experimentally studied. After laser irradiation, the corresponding Raman bands of the Cd-S bond decreased obviously, indicating bond breaking and thiol detachment from the QD surfaces. Meanwhile, a photoinduced aggregation of QDs occurred with the hydrodynamic diameter increased to hundreds of nanometers from an initial 20 nm, as detected with dynamic light scattering measurements. The bleaching of the photoluminescence of QDs under laser irradiation could be attributed to the enhanced nonradiative transfer in excited QDs caused by increased surface defects due to the losing of thiol ligands. Singlet oxygen (1O2) was involved in the photooxidation of QDs, as revealed by the inhibiting effects of 1O2 quenchers of histidine or sodium azide (NaN3) on the photobleaching of QDs. The linear relationship in Stern-Volmer measurements between the terminal product and the concentration of NaN3 demonstrated that 1O2 was the main pathway of the photobleaching in QD solutions. By comparing the photostability of QDs in C2C12 cells with and without NaN3 treatment, the photooxidation effect of 1O2 on photobleaching of cellular QDs was confirmed.     

Photodissociation dynamics of hydroxybenzoic acids

Aromatic amino acids have large UV absorption cross-sections and low fluorescence quantum yields. Ultrafast internal conversion, which transforms electronic excitation energy to vibrational energy, was assumed to account for the photostability of amino acids. Recent theoretical and experimental investigations suggested that low fluorescence quantum yields of phenol (chromophore of tyrosine) are due to the dissociation from a repulsive excited state. Radicals generated from dissociation may undergo undesired reactions. It contradicts the observed photostability of amino acids. In this work, we explored the photodissociation dynamics of the tyrosine chromophores, 2-, 3- and 4-hydroxybenzoic acid in a molecular beam at 193 nm using multimass ion imaging techniques. We demonstrated that dissociation from the excited state is effectively quenched for the conformers of hydroxybenzoic acids with intramolecular hydrogen bonding. Ab initio calculations show that the excited state and the ground state potential energy surfaces change significantly for the conformers with intramolecular hydrogen bonding. It shows the importance of intramolecular hydrogen bond in the excited state dynamics and provides an alternative molecular mechanism for the photostability of aromatic amino acids upon irradiation of ultraviolet photons.     

Investigating the Photostability of Quantum Dots at the Single‐Molecule Level

Quantum dots (QDs) have shown great potential to provide spatial, temporal, and structural information for biological systems. However, blinking, photobleaching, and spectral blueshift are adverse effects on their practical applications in biomedical research. An investigation of the effects of six reducing agents including cysteine (Cys), 1,4‐dithiothreitol (DTT), ethyl gallate (EG), L ‐glutathione (GSH), mercaptoacetic acid (MAA), and thiourea (TU) on the photostability of single QDs was studied. Our experiments demonstrate that both DTT and EG effectively inhibit blinking, photobleaching, and spectral blueshift. GSH molecules block blinking and photobleaching of QDs. The other reagents, Cys, MAA, and TU, only have the ability to counteract blinking. Possible explanations are given on the basis of research evidence. The results suggest possibilities for significant improvements in QDs for biological applications by adjusting the environmental conditions.