Energy transfer in molecular layer-by-layer films of water-soluble perylene diimides

Multilayer films of water-soluble anionic and cationic perylene diimide (PDI) moieties have been prepared using the molecular layer-by-layer method described in an earlier publication (Tang, T. J.; Qu, J. Q.; Müllen, K.; Webber, S. E. Langmuir 2006, 22, 26-28) and the fluorescence intensity compared with and without a base layer prepared using an anionic terrylene diimide dye (n-TDI), which serves as an energy-trapping layer for the PDI exciton. The fluorescence quenching data could be fit equally well to a modification of a model used by Kuhn to describe energy transfer from a J aggregate or a model developed by Kenkre and Wong to describe excitonic transfer. For both models, we obtain a characteristic energy-transfer distance on the order of 5.4 nm. Fluorescence quenching of the PDI via a single F?rster energy-transfer step to the n-TDI layer is ruled out on the basis of the observed power-law dependence. We also consider a model in which the excitation is trapped at the outermost surface. This model provides a reasonable fit to the data only if the Kuhn relationship is used.     

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.     

Structural basis of fluorescence fluctuation dynamics of green fluorescent proteins in acidic environments

Green fluorescent proteins (GFPs) have become powerful markers for numerous biological studies due to their robust fluorescence properties, site-specific labeling, pH sensitivity, and mutations for multiple-site labeling. Fluorescence correlation spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have been attributed to external proton transfer. Here we present experimental evidence indicating that conformational change in the protein &beta-barrel is a determining step for the external protonation of GFP-S65T (at low pH) using time-resolved fluorescence and polarization anisotropy measurements. While the average anionic fluorescence lifetime of GFP-S65T is reduced by approximately 18% over a pH range of 3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single exponential with a rotational time of phi = 17 +/- 1 ns, which indicates an intact beta-barrel with a hydrodynamic volume of 78 +/- 5 nm3. In contrast, the total fluorescence (525 +/- 50 nm) of the excited neutral state of S65T reveals a strong correlation between the fluorescence lifetime, structural conformation, and pH. The average fluorescence lifetime of the excited neutral state of S65T as a function of pH yields pKa approximately 5.9 in agreement with literature values using steady-state techniques. In contrast to the intact beta-barrel at high pH, the anisotropy of neutral S65T (at pH 相似文献   

Green Synthesis of Red‐Emitting Carbon Nanodots as a Novel “Turn‐on” Nanothermometer in Living Cells

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel “turn‐on” carbon‐dot‐based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave‐assisted method and exhibit red fluorescence (λ_em=615 nm) with high quantum yields (15 %). Then, an on–off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation‐induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs–GSH mixture could behave as an off–on fluorescent probe for temperature. Thus, red‐emitting CNDs can be utilized for “turn‐on” fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3‐E1 cells as an example model to demonstrate the red‐emitting CNDs can function as “non‐contact” tools for the accurate measurement of temperature and its gradient inside a living cell.     

A Comparison of the Fluorescence Spectra of Murine and Bovine Central Nervous System and Other Tissues

We describe a comparison of the fluorescence spectra of bovine tissues with murine tissues in order to determine whether spectral features are conserved and whether an appropriate and practical laboratory small animal model system could be identified to be used for investigation of tissue- and age-related fluorescence signal patterns. Recently it has been shown that spectral signatures of lipofuscin have enabled the detection of bovine central nervous system (CNS) tissue in meat products with high sensitivity (Schönenbrücher, H., Adhikary, R., Mukherjee, P., Casey, T.A., Rasmussen, M.A., Maistrovich, F.D., Hamir, A.N., Kehrli, M.J., Richt, J., Petrich, J.W. [2008] J Agric Food Chem 56 , 6220–6226). We report that brain and spinal cord of mice provide fluorescence spectra similar to those of bovine brain and spinal cord. It is concluded that murine CNS tissue is an appropriate model system for bovine CNS tissue for the development of fluorometric CNS detection assays.     

Photon statistics in blinking fluorescence of single PPV-PPyV molecule

A theoretical six-level model for blinking fluorescence of single PPV-PPyV copolymer molecule excited by CW-laser light is proposed. The model has been chosen in accordance with the following facts found in the Paul Barbara group experiment: (i) alternation of two types of fluorescence with moderate and strong levels of emission, (ii) existence of "dark" states with no fluorescence, (iii) linear dependence of inverse on-interval duration on laser intensity, and (iv) existence of laser intensity independent off-intervals. Relations between the distribution function w'(N, T) for photons emitted by a single molecule, the distribution function w'(N, T) for photons arriving at photomultiplier tube (PMT) and photo-electric pulse distribution w(N, T) created in a PMT are discussed. The theory is able to describe pulse distribution function w(N, T) measured experimentally at signal acquisition time T = 0.1 s. Values of all rate constants of the model have been found from comparison of the theory with the experiment. Distributions w(on, off)(t) of on- and off-times and distribution w(N, T) of pulses have been calculated for infrequent and frequent inter-conformational jumps in single copolymer molecule.     

Heterogeneous diffusion in thin polymer films as observed by high-temperature single-molecule fluorescence microscopy

Single-molecule fluorescence microscopy was used to investigate the dynamics of perylene diimide (PDI) molecules in thin supported polystyrene (PS) films at temperatures up to 135 °C. Such high temperatures, so far unreached in single-molecule spectroscopy studies, were achieved using a custom-built setup which allows for restricting the heated mass to a minimum. This enables temperature-dependent single-molecule fluorescence studies of structural dynamics in the temperature range most relevant to the processing and to applications of thermoplastic materials. In order to ensure that polymer chains were relaxed, a molecular weight of 3000 g/mol, clearly below the entanglement length of PS, was chosen. We found significant heterogeneities in the motion of single PDI probe molecules near T(g). An analysis of the track radius of the recorded single-probe molecule tracks allowed for a distinction between mobile and immobile molecules. Up to the glass transition temperature in bulk, T(g,bulk), probe molecules were immobile; at temperatures higher than T(g,bulk) + 40 K, all probe molecules were mobile. In the range between 0 and 40 K above T(g,bulk) the fraction of mobile probe molecules strongly depends on film thickness. In 30-nm thin films mobility is observed at lower temperatures than in thick films. The fractions of mobile probe molecules were compared and rationalized using Monte Carlo random walk simulations. Results of these simulations indicate that the observed heterogeneities can be explained by a model which assumes a T(g) profile and an increased probability of probe molecules remaining at the surface, both effects caused by a density profile with decreasing polymer density at the polymer-air interface.     

肿瘤乏氧靶向响应的罗丹明荧光探针及其成像介导手术治疗

基于罗丹明的良好荧光性能, 经化学偶联反应制备并表征了一个偶氮乏氧特异响应的“Off-On”型荧光成像探针(FY-4). 从分子层面证实了其荧光“Off-On”性能和响应机制; 在L02正常细胞及4T1, HeLa和A549肿瘤细胞层面考察了其对受试细胞株的毒性和不同乏氧时间的荧光成像性能; 再利用4T1肿瘤模型, 分别以肿瘤原位注射和尾静脉注射的方式考察了其荧光成像性能, 并探究了其荧光成像介导切除肿瘤性能, 最后还考察了FY-4的生物安全性. 结果表明, FY-4有高的肿瘤乏氧靶向特异“关-开”响应的荧光成像差异显影及荧光成像介导切除肿瘤的潜能, 结合其良好的光物理性能、 生物安全性和明晰的给药时间等特性, 有望为生物医学荧光成像介导肿瘤切除提供新的研究工具.     

Selective two-step labeling of proteins with an off/on fluorescent probe

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.     

Excited-State Properties of Thymidine and Their Relevance to Its Heterogeneous Emission in Double-Stranded DNA

Published results on synthetic polynucleotides point to T as the major emitting fluorophore in DNA. We have reported also that the bases of the nonalternating polynucleotide poly(dA).poly(dT), in which T was selectively excited, undergo large-amplitude motions on the picosecond-nanosecond time scales (S. Georghiou et al., Biophys. J. 70, 1909-1922, 1996). In that study, the fluorescence decay profile of the T bases of this polynucleotide was found to contain a number of components; these may be considered to be the result of the motions of the bases that give rise to a distribution of stacked geometries of varying rigidity as well as dispersion and polar interactions. Here, we report the results of a study that we have undertaken in order to test this hypothesis. To this effect, we have studied the photophysical properties of thymidine (1) in aqueous buffer and in a number of organic solvents and (2) in aqueous sucrose solutions of viscosity extending to 149 cP. The results suggest that the fluorescence quantum yield decreases with an increase in the polarizability of the solvent, whereas it increases with an increase in the solvent polarity (on the basis of the empirical parameter of solvent polarity ETN) or viscosity. These findings suggest the following for the photophysical properties of the T bases in DNA: (1) Base stacking results in two antagonistic effects, namely it causes a reduction in fluorescence as a result of dispersion interactions and an enhancement as a result of a reduction in the motions of the bases and (2) exposure of the bases to the aqueous environment results in fluorescence enhancement.     

Energy transfer in unsymmetrical phenylene ethynylene dendrimers

We have applied the fluorescence upconversion technique to explore the electronic excitation energy transfer in unsymmetrical phenylene ethynylene dendrimers. Steady-state emission spectra show that the energy transfer from the dendrons to the core is highly efficient. Ultrafast time-resolved fluorescence measurements are performed at various excitation wavelengths to explore the possibility of assigning absorption band structures to exciton localizations. We propose a kinetic model to describe the time-resolved data. Independent of the excitation wavelength, a typical rise-time value of 500 fs is measured for the fluorescence in the dendrimer without an energy trap, indicating initial delocalized excitation. While absorption is into delocalized exciton states, emission occurs from localized states. When an energy trap such as perylene is introduced on the dendrimer, varying the excitation wavelength yields different energy-transfer rates, and the excitation energy migrates to the trap through two channels. The interaction energy between the dendrimer backbone and the trap is estimated to be 75 cm(-1). This value is small compared to the vibronic bandwidth of the dendrimer, indicating that the monodendrons and the energy trap are weakly coupled.     

Kinetics of acid-induced spectral changes in the GFPmut2 chromophore

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.     

Extracting the time scales of conformational dynamics from single-molecule single-photon fluorescence statistics

The quenching rate of a fluorophore attached to a macromolecule can be rather sensitive to its conformational state. The decay of the corresponding fluorescence lifetime autocorrelation function can therefore provide unique information on the time scales of conformational dynamics. The conventional way of measuring the fluorescence lifetime autocorrelation function involves evaluating it from the distribution of delay times between photoexcitation and photon emission. However, the time resolution of this procedure is limited by the time window required for collecting enough photons in order to establish this distribution with sufficient signal-to-noise ratio. Yang and Xie have recently proposed an approach for improving the time resolution, which is based on the argument that the autocorrelation function of the delay time between photoexcitation and photon emission is proportional to the autocorrelation function of the square of the fluorescence lifetime [Yang, H.; Xie, X. S. J. Chem. Phys. 2002, 117, 10965]. In this paper, we show that the delay-time autocorrelation function is equal to the autocorrelation function of the square of the fluorescence lifetime divided by the autocorrelation function of the fluorescence lifetime. We examine the conditions under which the delay-time autocorrelation function is approximately proportional to the autocorrelation function of the square of the fluorescence lifetime. We also investigate the correlation between the decay of the delay-time autocorrelation function and the time scales of conformational dynamics. The results are demonstrated via applications to a two-state model and an off-lattice model of a polypeptide.     

Photon counting histogram: one-photon excitation.

The photon counting histogram (PCH) analysis is a fluorescence fluctuation method that is able to characterize the brightness and concentration of different fluorescent species present in a liquid sample. We find that the PCH model using a three-dimensional Gaussian observation volume profile is inadequate for fitting experimental data obtained from a confocal setup with one-photon excitation. We propose an imoroved model, which is based on the correction to the observation volume profile for the out-of-focus emission. We demonstrate that this model is able to resolve different species present under a wide range of conditions. Attention is given to how this model allows the examination of the effects of different instrumental setups on the resolvability.     

Coherent effects in energy transport in model dendritic structures investigated by ultrafast fluorescence anisotropy spectroscopy

Measurements of ultrafast fluorescence anisotropy decay in model branched dendritic molecules of different symmetry are reported. These molecules contain the fundamental branching center units of larger dendrimer macromolecules with either three (C(3))- or four (T(d), tetrahedral)-fold symmetry. The anisotropy for a tetrahedral system is found to decay on a subpicosecond time scale (880 fs). This decay can be qualitatively explained by F?rster-type incoherent energy migration between chromophores. Alternatively, for a nitrogen-centered trimer system, the fluorescence anisotropy decay time (35 fs) is found to be much shorter than that of the tetramers, and the decay cannot be attributed to an incoherent hopping mechanism. In this case, a coherent interchromophore energy transport mechanism should be considered. The mechanism of the ultrafast energy migration process in the branched systems is interpreted by use of a phenomenological quantum mechanical model, which examines the two extreme cases of incoherent and coherent interactions.     

Self-assembly of α-6T Molecule on Ag(100) and Related STM Induced Luminescence

We have investigated the self-assembly and light emission properties of organic α-sexithiophene (α-6T) molecules on Ag(100) under different coverage by scanning tunneling microscopy (STM). At very low coverage, the α-6T molecules form a unique enantiomer by grouping four molecules into a windmill supermolecular structure. As the coverage is increased, α-6T molecules tend to pack side by side into a denser stripe structure. Fur-ther increase of the coverage will lead to the layer-by-layer growth of molecules on Ag(100) with the lower-layer stripe pattern serving as a template. Molecular fluorescence for α-6T molecules on Ag(100) at a coverage of five monolayers has been detected by light excitations,which indicates a well decoupled electronic states for the top-layer α-6T molecules. How-ever, the STM induced luminescent spectra for the same sample reveal only plasmonic-like emission. The absence of intramolecular fluorescence in this case suggests that the elec-tronic decoupling is not a sufficient condition for generating photon emission from molecules. For intramolecular fluorescence to occur, the orientation of the dynamic dipole moment of molecules and the energy-level alignment at the molecule-metal interface are also important so that molecules can be effectively excited through efficient dipolar coupling with local plasmons and by injecting holes into the molecules.     

Ultrafast dynamics in multibranched structures with enhanced two-photon absorption

The understanding of the mechanism of the enhanced two-photon absorption (TPA) in multibranched chromophore systems is of importance to the design of materials with the large TPA cross-sections and for future applications. In this communication, the mechanism of enhanced TPA properties is investigated. For a dendritic model system, the excited-state dynamics for both population (T1-process) and phase relaxation (T2-process) processes involved are investigated by a combination of time-resolved spectroscopic techniques. The results of time-resolved fluorescence anisotropy are compared with previous results obtained from other branched chromophore systems. It is found that the PRL-701 trimer system, which possesses the large enhancement of two-photon absorption cross-section, gives a faster anisotropy decay (fluorescence upconversion and transient absorption), a longer population relaxation time (fluorescence lifetime), and a weaker coupling to the solvent (a larger photon echo peak shift initial value). New strategies for rational design of large TPA materials can be achieved based on a better understanding of the mechanism of the enhancement.     

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.     

Reversible surface electronic traps in PbS quantum dot solids induced by an order-disorder phase transition in capping molecules

The electronic properties of semiconductor quantum dots (QDs) are critically dependent on the nature of the ligand molecules on their surfaces. Here we show the reversible formation of surface electronic trap states in the model system of solid thin films of PbS QDs capped with thiol molecules. As the temperature was increased from cryogenic to room temperature, we discovered a phase transition in the fluorescence spectra from excitonic emission to trap emission. The critical temperature (T(c)) of the phase transition scales with molecular length and in each case is close to the bulk melting temperature of the capping molecules. We conclude that an order-disorder transition in the molecular monolayer above T(c) introduces surface mobility and the formation of a disordered atomic lead layer at the QD/capping molecule interface, leading to electronic trap formation.