Single Photon Counting Experiments Research Articles

Copillar[5]arene Appended Pyrene Schiff Base: Photophysics, Aggregation Induced Emission and Picric Acid Recognition.

Herein, we report the synthesis of copillar[5]arene-based pyrene Schiff base 1 and its characterization by using 1H, 13C NMR, FT-IR and mass spectrometry. UV-vis absorption, steady-state fluorescence and time-resolved fluorescence are done to elucidate the photophysical behaviors of 1. To understand the electronic structure of 1, density functional theory (DFT) calculations are performed. Owing to the presence of pyrene via a Schiff base linkage, compound 1 exhibits aggregation-induced emission (AIE) characteristics. It shows aggregation in aqueous THF and DMF. The aggregation behavior is successfully demonstrated by steady-state fluorescence, dynamic light scattering (DLS) and time-correlated single-photon counting (TCSPC) experiments. Experimental findings reveal that hydrophobic effect is the driving force in the formation of aggregates. As application, the aggregated state of 1 in aqueous THF fluorimetrically recognizes picric acid (PA) selectively over a series of nitro- and nonnitroaromatics with a detection limit of 1.62 x 10-7 M. The emission of the aggregated state is fully quenched upon interaction with PA.

Theoretical analysis of argon 2p states' density ratios for nanosecond plasma optical emission spectroscopy

A theoretical analysis of excited argon state densities responsible for optical emission spectra of atmospheric pressure argon plasma is presented for its use in plasma diagnostics. Nanosecond pulsed barrier discharges are simulated using spatially one- and two-dimensional fluid-Poisson models using the reaction kinetics model presented by Stankov et al. [1], which considers all ten argon 2p states (Paschen notation) separately. The very first (single) discharge and repetitive discharges with frequencies from 5 kHz to 100 kHz are considered. A semi-automated procedure is utilized to find appropriate 2p states for electric field determination using an intensity ratio method, which is based on a time-dependent collisional-radiative model. The fluid simulations in combination with the semi-automated procedure are used to quantify the sensitivity of selected 2p-state ratios to given preionization of the gas. A highly sensitive time-correlated single photon counting experiment shows clearly that the selected ratio is sensitive to the electric field variation in the streamer head, yet additional calibration is needed for absolute values determination. Different approaches for effective lifetime determination are tested and applied also to measured data. The influence of radial and axial 2p state density integration on the intensity ratio method is discussed. The above mentioned models and procedures result in a flexible theory-based methodology applicable for development of new diagnostic techniques.

Dual-Coreactants Enhanced Electrochemiluminescence.

The electrochemiluminescence (ECL) of ruthenium(II) tris(2,2'-bipyridyl) (Ru(bpy)3 2+) with tri-n-propylamine (TPrA) as the good coreactant can be unexpectedly enhanced by a weak coreactant, such as triethanolamine (TEOA). First, the intensity of ECL emitted by Ru(bpy)3 2+/TPrA can be remarkably amplified by 10.8-fold after adding some amount of TEOA. Moreover, the ECL layer thickness, measured by self-interference spectroscopy, is also doubled. The enhancement far exceeding the superposition of respective contribution of TPrA and TEOA was elucidated by a "chemical oxidation mechanism", in which TEOA+⋅ acts as a chemical enhancer to oxidize TPrA in solution and to accelerate significantly the ECL reaction kinetics. This mechanism was proved by single-photon counting experiment and finite element simulations. In addition, the dual-coreactants strategy works well not only in solution with freely diffusive Ru(bpy)3 2+, but also on Ru(bpy)3 2+-functionalized microbeads, suggesting that ethanolamines could act as cheap, easily available and low-background enhancers for ECL-based bioanalysis and microscopy.

Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.

Managing photoinduced electronic interactions on a molybdenum disulfide/diblock copolymer/anionic porphyrin nanoensemble

Exploring the modular nature of MoS2 and its ability to be easily functionalized, both in covalent and non-covalent ways and with a variety of chemical species, a MoS2/diblock copolymer system was conceptualized. As the constituents of this diblock copolymer, the poly(benzyl methacrylate), abbreviated as pBzMA, block was designed to serve as the immobilization point of the polymeric chains onto the MoS2 lattice, taking advantage of the S-π non-covalent interactions between phenyl rings and ample S atoms, while the quaternized poly(dimethylamino-ethyl methacrylate), abbreviated as QpDMAEMA, was incorporated as the second block to capture the tetrasulfo-tetraphenyl anionic porphyrin, abbreviated as TPPS4 via electrostatic interactions with the ammonium groups of the quaternized polymeric chain. The so-formed MoS2/QpDMAEMA-b-pBzMA/TPPS4 ensemble was characterized and assessed via spectroscopic (UV-Vis), electron microscopy imaging (TEM) and dynamic light scattering (DLS) techniques. Markedly, efficient quenching of the characteristic fluorescence of the porphyrin species by the MoS2 nanosheets was observed, suggesting the development of photoinduced charge-transfer phenomena within the MoS2/QpDMAEMA-b-pBzMA/TPPS4 ensemble. This was further corroborated by time-correlated single photon counting (TCSPC) and blank experiments, signifying the role of Coulombic interactions for bringing into electronic communication the porphyrin moieties with MoS2. Overall, this rationally designed nanoensemble based on non-covalently modified MoS2 with polymers and photoactive species sets the foundations for inexpensive yet potent systems to be used in energy-related applications.

Photo-response of Fe-doped ZnO electron transport interlayer on Poly(3-hexylthiophene-2,5-diyl): Phenyl-C61-butyric acid methyl ester based thin film device for photodiode application

An electron transport material - Fe doped ZnO layer with atomic ratio Zn: Fe = 90:10 was prepared for enhancing the photocurrent between an optically active poly(3-hexylthiophene-2,5-diyl): phenyl-C61-butyric acid methyl ester film and an indium tin oxide film used as an anode. The crystallite size of ZnO decreases with increasing Fe doping as estimated from major X-ray diffraction (100), (002), (101), and (110) peaks using the Scherrer formula. ZnO particle size reduction on Fe doping was also observed by field emission scanning electron microscopy. The photoluminescence study on Fe-doped ZnO shows interstitial oxygen defects band at ∼2.28 eV and the bound exciton caused by oxygen vacancy in ZnO. The X-ray photoelectron spectra show shift of the Zn2p3/2, and Zn2p1/2 peaks to lower binding energy due to Fe3+ doping in the ZnO. Time-correlated single-photon counting experiment reveals faster decay of lifetime of Fe-doped ZnO than undoped material at excitation wavelength of 650 nm due to higher electron transport rate through the interface according to Langevin recombination formula. The photoresponse property of the films was measured under solar light illumination (10 mW/cm2), and the results show that Fe-doped ZnO can be a better than undoped ZnO based multilayer device for photodiode application.

Single photon multiclock lock-in detection by picosecond timestamping

Extracting signals at low single-photon count rates from large backgrounds is a challenge in many optical experiments and technologies. Here, we demonstrate a single-photon lock-in detection scheme based on continuous photon timestamping to improve the SNR by more than two orders of magnitude. Through time-resolving the signal modulation induced by periodic perturbations, 98% of dark counts are filtered out and the < 1 c o u n t / s contributions from several different nonlinear processes identified. As a proof-of-concept, coherent anti-Stokes Raman measurements are used to determine the vibrational lifetime of few molecules in a plasmonic nanocavity. This detection scheme can be applied to all single-photon counting experiments with any number of simultaneous modulation frequencies, greatly increasing SNR and resolving physical processes with picosecond time resolution while keeping the photon dosage small. The open instrumentation package provided here enables low-cost implementation.

Excitation-independent and fluorescence-reversible N-GQD for picomolar detection of inhibitory neurotransmitter in milk samples ‒ an alleyway for possible neuromorphic computing application

N‒GQDs with an average size of ca. 20–30 nm are utilized for the picomolar detection of inhibitory neurotransmitters, glycine (Gly), in pH ca. 7.0. The crystalline nature, morphology, elemental composition, and chemical state of N–GQDs are investigated by XRD, FE-SEM, HR-TEM, XPS, and FT-IR techniques. The addition of Gly (100 × 10−9 M; 0 → 1.0 mL) steadily quenches the fluorescence intensity of N–GQD (1 × 10−6 M) at 432 nm (λex 333 nm) due to inner filter effect (IFE) through the formation of ground-state complex, N–GQD•Gly. The excitation-independent N‒GQDs showed an outstanding selectivity and sensitivity towards Gly with binding constant (Ka = 8.97 × 10−3 M−1) and LoD (21.04 pM; S/N = 3). Time-correlated single-photon counting experiment confirms the static quenching of N–GQD (8.77 → 8.85 ns) in the presence of Gly. The interference of other amino acids on the strong binding of the N–GQD•Gly complex in H2O is examined. Combinatorial Ex-OR and NOT gate logic circuits that could be useful in neuromorphic computing are developed based on the reversible fluorescence intensity changes of N–GQD upon the addition of Gly (ФF 0.54 → 0.39). The real-time application of N–GQD was investigated using commercially available relevant milk samples. Remarkably, not less than 99% cytotoxic reactivity of N–GQDs is attained against HeLa cells.

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

DNA Hairpin Hybridization under Extreme Pressures: A Single-Molecule FRET Study.

Organisms have evolved to live in a variety of complex environments, which clearly has required cellular biology to accommodate to extreme conditions of hydraulic pressure and elevated temperature. In this work, we exploit single-molecule Forster resonance energy transfer (FRET) spectroscopy to probe structural changes in DNA hairpins as a function of pressure and temperature, which allows us to extract detailed thermodynamic information on changes in free energy (ΔG°), free volume (ΔV°), enthalpy (ΔH°), and entropy (ΔS°) associated with DNA loop formation and sequence-dependent stem hybridization. Specifically, time-correlated single-photon counting experiments on freely diffusing 40A DNA hairpin FRET constructs are performed in a 50 μm × 50 μm square quartz capillary cell pressurized from ambient pressure up to 3 kbar. By pressure-dependent van't Hoff analysis of the equilibrium constants, ΔV° for hybridization of the DNA hairpin can be determined as a function of stem length (nstem = 7-10) with single base-pair resolution, which further motivates a simple linear deconstruction into additive stem (ΔV°stem = ΔV°bp x nstem) and loop (ΔV°loop) contributions. We find that increasing pressure destabilizes the DNA hairpin stem region [ΔV°bp = +1.98(16) cm3/(mol bp)], with additional positive free volume changes [ΔV°loop = +7.0(14) cm3/mol] we ascribe to bending and base stacking disruption of the 40-dA loop. From a van't Hoff temperature-dependent analysis of the DNA 40A hairpin equilibria, the data support a similar additive loop/stem deconstruction of enthalpic (ΔH° = ΔH°loop + ΔH°stem) and entropic (ΔS° = ΔS°loop + ΔS°stem) contributions, which permits insightful comparison with predictions from nearest-neighbor thermodynamic models for DNA duplex formation. In particular, the stem thermodynamics is consistent with exothermically favored (ΔH°stem < 0) and entropically penalized (ΔS°stem < 0) hydrogen bonding but with additional enthalpic (ΔH°loop > 0) and entropic (ΔS°loop > 0) contributions due to loop bending effects consistent with distortion of dA base stacking in the 40-dA linker.

Panchromatic sensitization of new terpyridine ligated thiocyanate-free Ru-complex

Two new thiocyanato-free Ru sensitizers GS1 and GS2 having substitutions at the central pyridyl group of the terpyridine ligand successfully synthesized. GS1 and GS2 were used as photosensitizers for DSSCs devices. These sensitizers were characterized by 1H NMR, FT-IR, UV–visible, and differential pulse voltammetry studies. The optical and electrochemical properties of these sensitizers were compared with N749 dye. The fabricated device with these complexes portrays an efficient panchromatic sensitization which covers the entire region from visible to NIR. Time-correlated single photon counting (TCSPC) experiment was also performed to study the charge carrier dynamics of both the sensitizers. The average excited state-life time of the GS1 and GS2 sensitizers on TiO2 is 4.04 ns and 3.07 ns respectively, which was much better compared to the reference sensitizer (0.25 ns). Among these two sensitizers, GS2 exhibited a maximum overall power conversion efficiency of 3.25% under standard air mass 1.5 irradiation (100 mW cm−2) with the Jsc = 10.073 mA/cm2, the Voc = 0.451 V, and the FF = 0.714.

A multi-scale time-resolved study of photoactivated dynamics in 5-benzyl uracil, a model for DNA/protein interactions.

We combine fluorescence up-conversion and time correlated single photon counting experiments to investigate the 5-benzyl uracil excited state dynamics in methanol from 100 fs up to several ns. This molecule has been proposed as a model for DNA/protein interactions. Our results show emission bands at about 310 and 350 nm that exhibit bi-exponential sub-ps decays. Calculations, including solvent effects by a mixed discrete-continuum model, indicate that the Franck Condon region is characterized by significant coupling between the excited states of the benzyl and the uracil moieties, mirrored by the short-lived emission at 310 nm. Two main ground state recovery pathways are identified, both contributing to the 350 nm emission. The first 'photophysical' decay path involves a ππ* excited state localized on the uracil and is connected to the ground electronic state by an easily accessible crossing with S0, accounting for the short lifetime component. Simulations indicate that a possible second pathway is characterized by exciplex formation, with partial benzene → uracil charge transfer character, that may lead instead to photocyclization. The relevance of our results is discussed in view of the photoactivated dynamics of DNA/protein complexes, with implications on their interaction mechanisms.

Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.

A highly selective, pH-tolerable and fast-response fluorescent probe for Fe3+ based on star-shape benzothiazole derivative

Abstract A star-shape benzothiazole-based molecule TBB , obtained through a simple condensation reaction of 2-aminothiophenol with 1,3,5-benzenetricarboxylic acid in the presence of available catalyst polyphosphoric acid (PPA), has been exploited as a highly selective fluorescent Fe 3+ probe over other metal ions in aqueous media (H 2 O/THF, v/v = 4/6). For this fast-response probe with a response time of 50 s, its quenching mechanism involving in the combination of static and dynamic quenching is confirmed by the test of time-correlated single photon counting (TCSPC) experiments. Noticeably, the probe TBB is suitable for Fe 3+ detection in a wide pH range (3–12) of aqueous solution, and it can be successfully applied for Fe 3+ quantitative measurement in real water samples.

Excited-State Relaxation and Förster Resonance Energy Transfer in an Organic Fluorophore/Silver Nanocluster Dyad.

A single-stranded DNA-based (ssDNA) dyad was constructed comprising 15 silver atoms stabilized by a ssDNA scaffold (DNA-AgNC) and an Alexa 546 fluorophore bound to the 5′ end. The Alexa 546 was chosen to function as a Förster resonance energy transfer (FRET) donor for the AgNC. Time-correlated single photon counting (TCSPC) experiments allowed unraveling the excited-state relaxation processes of the purified DNA-AgNC-only system. The TCSPC results revealed slow relaxation dynamics and a red shift of the emission spectrum during the excited-state lifetime. The results from the model systems were needed to understand the more complicated decay pathways present in the collected high-performance liquid chromatography fraction, which contained the dyad (37% of the emissive population). In the dyad system, the FRET efficiency between donor and acceptor was determined to be 94% using TCSPC, yielding a center-to-center distance of 4.6 nm. To date, only limited structural information on DNA-AgNCs is available and the use of TCSPC and FRET can provide information on the center-to-center distance between chromophores and provide positional information in nanostructures composed of AgNCs.

Self-assembled structures of N-alkylated bisbenzimidazolyl naphthalene in aqueous media for highly sensitive detection of picric acid

A 2,6-dibenzimidazole-appended naphthalene derivative flanking with two N-alkyl chains (sensor 4) was designed and applied for highly sensitive detection of picric acid (PA) in aqueous media. Driven by the hydrophobicity of alkyl chain and π-π stacking effect of aryl, sensor 4 can undergo self-assembly to form an orderly rod-like structure in H2O/THF (v/v, 90/10) solution, as shown by the dynamic light scattering (DLS) and scanning electron microscopy (SEM) studies. Sensor 4 shows high selectivity and sensitivity toward PA over other nitroaromatic explosives. DFT calculations and 1H NMR, the time-correlated single photon counting (TCSPC) experiments confirm that the quenching mechanism is due to both electron and energy transfer from the electron-rich sensor 4 to the electron-deficient PA. Sensor 4 can detect as low as 0.57 ppb PA in aqueous media and 11.46 ag cm−2 PA by contact mode. Importantly, sensor 4 exhibits low interference against common solvents, metal ions and anions. Thus, it is practically applicable for sensing PA in real environmental samples and vapor phase.

Photophysics of Pyrenyl-Functionalized Poly(isobutylene-alt-maleic anhydride) and Poly(isobutylene-alt-maleic N-alkylimide). Influence of Solvent, Degree of Substitution, and Temperature

A series of polymers derived from poly(isobutylene-alt-maleic anhydride) (PIMA) with ca. 1% to >90% of the anhydride units randomly substituted with 1-pyrenylmethylimido groups (Py-PIMA) has been synthesized and characterized. The remaining anhydride units in the Py-PIMA with 10% pyrenyl substitution have been converted to N-hexyl and N-decyl imides. The photophysical properties of these polymers, as obtained from steady-state fluorescence intensities, time-correlated single photon counting experiments, and time-resolved emission spectra, have provided insights into the dependence of the polymer conformations and their labilities on the degree of pyrenyl substitution, the nature of the appended alkyl chains, temperature, and solvent properties according to the Flory interaction coefficient and Hansen solubility parameters. None of the solvent characteristics, alone, can account for all aspects of the polymer behavior. The interactions of the medium with the polymer chains and the excited singlet states of...

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B.

It is often convenient to know the minimum amount of data needed to obtain a result of desired accuracy and precision. It is a necessity in the case of subdiffraction-limited microscopies, such as stimulated emission depletion (STED) microscopy, owing to the limited sample volumes and the extreme sensitivity of the samples to photobleaching and photodamage. We present a detailed comparison of probability-based techniques (the maximum likelihood method and methods based on the binomial and the Poisson distributions) with residual minimization-based techniques for retrieving the fluorescence decay parameters for various two-fluorophore mixtures, as a function of the total number of photon counts, in time-correlated, single-photon counting experiments. The probability-based techniques proved to be the most robust (insensitive to initial values) in retrieving the target parameters and, in fact, performed equivalently to 2-3 significant figures. This is to be expected, as we demonstrate that the three methods are fundamentally related. Furthermore, methods based on the Poisson and binomial distributions have the desirable feature of providing a bin-by-bin analysis of a single fluorescence decay trace, which thus permits statistics to be acquired using only the one trace not only for the mean and median values of the fluorescence decay parameters but also for the associated standard deviations. These probability-based methods lend themselves well to the analysis of the sparse data sets that are encountered in subdiffraction-limited microscopies.

Dispersed dynamics of solvation in G-quadruplex DNA: comparison of dynamic Stokes shifts of probes in parallel and antiparallel quadruplex structures

G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1 × 105 M−1. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

Surface Nanobubbles Studied by Time-Resolved Fluorescence Microscopy Methods Combined with AFM: The Impact of Surface Treatment on Nanobubble Nucleation.

The impact of surface treatment and modification on surface nanobubble nucleation in water has been addressed by a new combination of fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM). In this study, rhodamine 6G (Rh6G)-labeled surface nanobubbles nucleated by the ethanol-water exchange were studied on differently cleaned borosilicate glass, silanized glass as well as self-assembled monolayers on transparent gold by combined AFM-FLIM. While the AFM data confirmed earlier reports on surface nanobubble nucleation, size, and apparent contact angles in dependence of the underlying substrate, the colocalization of these elevated features with highly fluorescent features observed in confocal intensity images added new information. By analyzing the characteristic contributions to the excited state lifetime of Rh6G in decay curves obtained from time-correlated single photon counting (TCSPC) experiments, the characteristic short-lived (<600 ps) component of could be associated with an emission at the gas-water interface. Its colocalization with nanobubble-like features in the AFM height images provides evidence for the observation of gas-filled surface nanobubbles. While piranha-cleaned glass supported nanobubbles, milder UV-ozone or oxygen plasma treatment afforded glass-water interfaces, where no nanobubbles were observed by combined AFM-FLIM. Finally, the number density of nanobubbles scaled inversely with increasing surface hydrophobicity.