Excitation Light Intensity Research Articles

Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics.

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Emission variation in CdSe/ZnS quantum dots conjugated to Papilloma virus antibodies

ABSTRACTThe paper presents a comparative study of the photoluminescence (PL) and Raman scattering spectra of core-shell CdSe/ZnS quantum dots (QDs) in nonconjugated states and after the conjugation to the anti-human papilloma virus (HPV), HPV 16-E7, antibodies. All optical measurements are performed on the dried droplets of the original solution of nonconjugated and bioconjugated QDs located on the Si substrate. CdSe/ZnS QDs with emission at 655 nm have been used. PL spectra of nonconjugated QDs are characterized by one Gaussian shape PL band related to the exciton emission in the CdSe core. PL spectra of bioconjugated QDs have changed essentially: the core PL band shifts into the high energy spectral range (“blue” sift) and becomes asymmetric. A set of physical reasons has been proposed for the “blue” shift explanation of the core PL band in bioconjugated QDs. The variation of PL spectra versus excitation light intensities has been studied to analyses the exciton emission via excited states in QDs. Finally the PL spectrum transformation for the core emission in bioconjugated QDs has been attributed to the electronic quantum confined effects stimulated by the electric charges of bioconjugated antibodies.

Exciton migration dynamics between phenylene moieties in the framework of periodic mesoporous organosilica powder

To understand the excitation migration process between the phenylene (Ph) moieties in the framework of periodic mesoporous organosilicas (PMOs), the exciton–exciton annihilation behavior between the Ph moieties is studied. The dependence of the excitation light intensity on the fluorescence decay curve of the Ph exciton was successfully analyzed with a two-dimensional exciton diffusion and collision model. The quenching distance and diffusion length of the Ph exciton are estimated to be 0.4–0.8 nm and at least 4 nm, respectively. It is suggested that the Ph exciton is delocalized between up to three Ph moieties and moved between approximately nine Ph moieties in the pore wall of Ph–PMO.

Parameters affecting electron transfer dynamics from semiconductors to molecular catalysts for the photochemical reduction of protons

The aim of this work is to use transient absorption spectroscopy to study the parameters affecting the kinetics and efficiency of electron transfer in a photocatalytic system for water reduction based on a cobalt proton reduction catalyst (CoP) adsorbed on a nanocrystalline TiO2 film. In the first approach, water is used as the proton and electron source and H2 is generated after band gap excitation of TiO2 functionalised with CoP. The second system involves the use of a sacrificial electron donor to regenerate the TiO2/CoP system in water at neutral pH. The third system consists of CoP/TiO2 films co-sensitised with a ruthenium-based dye (RuP). In particular, we focus on the study of different parameters that affect the kinetics of electron transfer from the semiconductor to the molecular catalyst by monitoring the lifetime of charge carriers in TiO2. We observe that low catalyst loadings onto the surface of TiO2, high excitation light intensities and small driving forces strongly slow down the kinetics and/or reduce the efficiency of the electron transfer at the interface. We conclude that the first reduction of the catalyst from CoIII to CoII can proceed efficiently even in the absence of an added hole scavenger at sufficiently high catalyst coverages and low excitation densities. In contrast, the second reduction from CoII to CoI, which is required for hydrogen evolution, appears to be at least 105 slower, suggesting it requires efficient hole scavenging and almost complete reduction of all the adsorbed CoP to CoII. Dye sensitisation enables visible light photoactivity, although this is partly offset by slower, and less efficient, hole scavenging.

Near Infrared Fluorescence Enhancement by Local Surface Plasmon Resonance from Arrayed Gold Nanoblocks

The near infrared (NIR) fluorescence enhancement by local surface plasmon resonanoce from arrayed gold (Au) nanoblocks was investigated by NIR fluorescent dyes, IR780, immobilized in hydrophobic DNA thin film on glass substrates, to clarify the gap mode effect on the fluorescence enhancement. In the substrate with Dimer type Au nanoblock arrangement, average total fluorescence intensity was larger by 10.0, 2.4, and 12.4 times for non-polarized, P- and S- polarization as compared with that on a glass substrate alone, respectively. These findings suggested that enhancement of excitation light intensity at nanogap in the Dimer type Au nanoblock arrangement affected the fluorescence intensity. Average total fluorescence intensity, on the other hand, was smaller by 0.63 times as compared with that on a glass substrate alone in the checkerboard type Au array. It is suggested that the fluorescence quenching was caused by the energy transfer from the excited state of IR780 to Au nanoblocks or by the increased deactivation of excited dye molecules induced by resonance with Au nanoblocks at the checkerboard arrangement. We have firstly achieved the NIR fluorescence enhancement by LSPR due to the gap mode.

A Quantum Dot-Immunofluorescent Labeling Method to Investigate the Interactions between a Crinivirus and Its Whitefly Vector.

Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor)-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV) virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs), into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605) for biotin-conjugated anti-LIYV IgG (B-αIgG). IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated approx. seven- to eight-fold slower than that of Alexa Fluor.

Excited-state Dynamics of Fullerene Nanoparticles Dispersed in Pure Water

Abstract Femtosecond transient absorption dynamics of C60 nanoparticles in pure water revealed a strong quenching of the singlet excited state, independent of the excitation light intensity on a 10-ps time scale, and an extremely low yield of the triplet state of C60 nanoparticles upon photoexcitation.

Reversible Photoinduced Formation and Manipulation of a Two-Dimensional Closely Packed Assembly of Polystyrene Nanospheres on a Metallic Nanostructure

Nanostructure-enhanced optical trapping of polymer beads was investigated by means of fluorescence microspectroscopy. It was found that trapping behavior was quite sensitive to the particle size as well as excitation light intensity. We present a 2D closely packed assembly of polystyrene nanospheres on a gold nanostructure that is triggered by gap-mode localized surface plasmon (LSP) excitation. We discuss the trapping mechanism from the viewpoints of not only the radiation force but also of the thermal force (thermophoresis and thermal convection) induced by near-infrared laser irradiation. Thermophoresis worked as a repulsive force whose direction was opposed to that of the radiation force. On the other hand, thermal convection acted in favor of trapping: It supplied nanospheres toward the LSP excitation area. By suppressing the repulsive force, the assembled trapped nanospheres took the form of hexagonal shapes on a gold nanostructure. By optimizing irradiation parameters, we achieved 2D manipulation o...

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

Influence of excitation light intensity and leaf age on the slow chlorophyll fluorescence transient in radish

The temporal characteristics of the slow phase of chlorophyll fluorescence induction-T0.5 (half-decay time) and tmin (an integral-based index of the variable emission rate)-as well as the popular amplitude index FP/FS were determined at different excitation light intensities (Iex [400–500 nm] of 20–80 W/m2) in dark-adapted leaves of different age (3–24 days) taken from radish plants grown under continuous light of 100 W/m2 PAR. All the profiles thus obtained were mutually consistent, and the age-related variations were minimized at Iex > 40 W/m2; at that the age-averaged temporal indices proved to be more light-responsive than the standard amplitude ratio.

EXPRESSION OF YEAST HEM1 CONTROLLED BY ARABIDOPSIS HEMA1 PROMOTER ENHANCES LEAF PHOTOSYNTHESIS IN TRANSGENIC TOMATO

A gene encoding 5-aminolevulinic acid synthase (ALA-S) in Saccharomyces cerevisiae (Hem1) under the control of an Arabidopsis thaliana HemA1 promoter (HemA1 promoter) was introduced into the genome of tomato (Solanum lycopersicum). All transgenic tomato plants were able to transcribe the Hem1 gene and the capacity to synthesize ALA was increased. The transgenic plants had 18% higher levels of chlorophylls, as compared to wild-type plants, indicating that part of the additional ALA from yeast ALA-S was metabolized to chlorophylls. Determination of gas exchange suggested that the net photosynthetic rate (Pn) in the transgenic tomato was about 60% higher than that of wild type. The apparent quantum yield (AQY) in the transgenic plants was 0.0363, significantly higher than 0.0313 of wild type. The Chlorophyll-a fast fluorescence measurement and JIP-test at continuous intensities of excitation light (90-2100 μmol m-2 s-1) showed that the genetic transformation caused a significant reduction in Vj (the relative variable fluorescence at the J-step) but addition in Vi (the relative variable fluorescence at the I-step). The maximum photochemical efficiency (φPo=Fv/Fm) did not vary with the light intensity, which demonstrated that φPo was a stable index for leaf photosynthesis. ψo (the probability that a trapped exciton moves an electron into the electron transport chain beyond QA-) and φEo (the quantum yield of electron transport) decreased with the light intensity increased, especially in wild type. Furthermore, the photosynthetic performance index on absorption basis (PIABS) or cross section basis (PICS) in the transgenic plants was also increased obviously in transgenic tobacco. Thus, the results obtained in this work approved that ALA improved photosynthetic capacity and the activity of PSII reaction center.

Application of PARAFAC to a two-component system exhibiting Fluorescence Resonance Energy Transfer: from theoretical prediction to experimental validation

One of the conventionally accepted requirements for parallel factor analysis (PARAFAC) to handle the fluorescence excitation emission matrices (EEMs) is the independence of each component's absorption and emission spectra in simple mixtures of fluorophores. EEMs of samples in which Förster resonance energy transfer (FRET) occurs between fluorophores seem to fail to meet this requirement. A rigorous theoretical treatment of the steady-state kinetics in the present work indicates that the fluorescence in the presence of FRET, excited by relatively weak excitation light intensity, can be reasonably separated into additive contributions from three parts: donors, acceptors and FRET. This prediction is for the first time verified experimentally in sodium dodecyl sulfate micellar solutions containing biphenyl as the energy donor and 2,5-diphenyloxazole as the energy acceptor. The experimental EEMs were well fitted to three components as predicted. A well accepted diagnostic test called core consistency (CC), specifically designed for modeling simple mixtures of fluorophores with PARAFAC, was found to be negative for the 3-component model in the present study. The simultaneous occurrence of good model fit and significantly negative CC when modeling fluorophore mixtures by conventional PARAFAC would be indicative of the presence of physical/chemical processes (e.g., FRET) that deviate from the conventional working requirements for PARAFAC. The extent of FRET has been independently measured or calculated by three methods: 1) decrease in steady state fluorescence of donor; 2) lifetime measurements with population analysis; and 3) Poisson statistics based on PARAFAC-determined distribution constants. The results of the three methods are consistent. The normalized scores of the three components found by PARAFAC also agree to within a few percent with relative concentrations in aqueous and micelle phases determined from distribution constants for the solutions prepared with nine different combinations of total donor and acceptor concentrations. Our theoretical treatment also for the first time spells out in detail the relationship between the PARAFAC scores and concentrations of components, in terms of photophysical constants of the components and spectral shape factors.

Theoretical Modeling of Measured Photocurrent Dynamics in Dye-Sensitized Nanostructured TiO2 Solar Cells

The general solutions describing the buildup and decay processes of photocurrent in dye-sensitized nanocrystalline TiO2 thin film solar cells have been derived by solving the diffusion equations that include charge transport and recombination parameters of electron lifetime and diffusion coefficient. One analytical solution is obtained under the condition of steady light illumination that may be used to interpret the buildup process of photocurrent. The other solution is derived with the condition that the illumination light is turned off after the photocurrent reaches its steady value. The latter solution can be used to describe the recombination process of charge carriers in the thin films. The two solutions have also been used to simulate experimentally measured results to extract electron lifetime, diffusion coefficient, and diffusion length of electrons for the two conditions under different light intensities. The fitted results show that the lifetime and diffusion coefficient vary with light intensity in opposite senses and the diffusion length only slightly depends on light intensity. In the recombination process, the dark diffusion coefficient is independent of excitation light intensity. These theoretical results confirm the observations reported by other researchers.

Multiphoton generation of electron–hole pairs in crystals with deep impurities II Cascade processes ∗∗The first part of this article, “Multiphoton generation of electron–hole pairs in crystals with deep impurities I Two-photon band–impurity transition probabilities,” was published in Opticheskiĭ Zhurnal No 10, 3 (2010) [J Opt Technol 77, 593 (2010)

This paper discusses the generation of nonequilibrium electron–hole pairs in broad-band crystals as a result of a cascade of two-photon valence-band–deep-impurity and deep-impurity–conduction-band transitions. The balance equations are formulated and solved for the electron and hole populations in the band and impurity states. The proposed mechanism, even in the case of moderate deep-impurity concentrations and light frequencies less than a quarter of the crystal’s band gap, makes it possible to obtain an appreciable free-carrier concentration at exciting-light intensities of j≈100 MW/cm<sup>2</sup>.

Micellar carrier for triplet–triplet annihilation-assisted photon energy upconversion in a water environment

In this paper, we demonstrate energetically conjoined triplet–triplet annihilation-assisted photon energy upconversion (UC) operating in an aqueous environment. Obtained micellar structures show very efficient UC emission in a water environment under extremely low excitation light intensity, down to 10 mW cm−2. The demonstrated sub-linear intensity dependence of the UC emission is of crucial importance for life science applications, allowing upconverted photons to be generated even at low intensity that then serve as a local, in situ, optical excitation source for subsequent light-triggered processes.

Light-Induced Conformational Change and Transient Dissociation Reaction of the BLUF Photoreceptor Synechocystis PixD (Slr1694)

The light-induced reaction of the BLUF ( blue light photoreceptor using flavin adenine dinucleotide) photoreceptor PixD from Synechocystis sp. PCC6803 (Slr1694) was investigated using the time-resolved transient grating method. A conformational change coupled with a volume contraction of 13 mL mol − 1 was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in volume and diffusion coefficient ( D). The determined D-value (3.7 × 10 − 11 m 2 s − 1 ) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, we observed a large increase in D with a time constant of 350 ms following the volume contraction reaction. The D-value of this photoproduct species (7.5 × 10 − 11 m 2 s − 1 ) is close to that of the PixD dimer. Combined with transient grating and size-exclusion chromatography measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control the regulation of the downstream signal transduction by the PixD photoreceptor.

The influence of interface states and bulk carrier lifetime on the minority carrier behavior in an illuminated metal/insulator/GaN structure

An influence of electronic states at an insulator/GaN interface on the behavior of excess holes in an ultraviolet-illuminated metal/ SiO 2/n-GaN structure has been studied by numerical simulations for weak (gate bias of −0.1 V ) and strong (−1 V ) depletion, in a wide range of excitation light intensities (from 10 10 to 10 20 photons cm −2 s −1) and for various bulk carrier lifetimes (from 1 to 100 ns). It has been found that the interface states with densities of 10 12 eV −1 cm −2 dramatically reduce the total (integrated in the whole GaN layer) density of photogenerated holes and thus degrade the sensitivity of the metal/insulator/GaN-based photodetector.

Deep UV resonant Raman spectroscopy for photodamage characterization in cells

We employed deep UV (DUV) Raman spectroscopy for characterization of molecular photodamage in cells. 244 nm light excitation Raman spectra were measured for HeLa cells exposed to the excitation light for different durations. In the spectra obtained with the shortest exposure duration (0.25 sec at 16 µW/µm2 irradiation), characteristic resonant Raman bands of adenine and guanine at 1483 cm−1 and tryptophan and tyrosine at 1618 cm−1 were clearly visible. With increasing exposure duration (up to 12.5 sec), these biomolecular Raman bands diminished, while a photoproduct Raman band at 1611 cm−1 grew. By exponential function fitting analyses, intensities of these characteristic three bands were correlated with sample exposure duration at different intensities of excitation light. We then suggest practical excitation conditions effective for DUV Raman observation of cells without photodamage-related spectral distortion.

Sensitivity enhancement of fluorescence detection in CE by coupling and conducting excitation light with tapered optical fiber

This paper reports the enhancement of sensitivity of detection for in-column fiber optic-induced fluorescence detection system in CE by tapered optical fiber (TOF). Two types of optical fiber, TOF and conventional cylindrical optical fiber (COF), were employed to construct the CE (TOF-CE and COF-CE) and were compared for sensitivity to riboflavin (RF). The fluorescence intensities from a RF sample with excitation light sources and fibers at various coupling angles were investigated. The fluorescence signal from TOF-CE was ca. ten times that of COF-CE. In addition, the detection performance of four excitation light source-fiber configurations including Laser-TOF, Laser-COF, LED-TOF, and LED-COF were compared. The LODs for RF were 0.21, 0.82, 0.80, and 7.5 nM, respectively, for the four excitation light source-fiber configurations. The results demonstrate that the sensitivity obtained by LED-TOF is close to that of Laser-COF. Both Laser-TOF and LED-TOF can greatly improve the sensitivity of detection in CE. TOF has the major attribute of collecting and focusing the excitation light intensity. Thus, the sensitivity obtained by LED-TOF without focusing lens is just same as that of LED-COF with a focusing lens. This demonstrates that the CE system can be further simplified by eliminating the focusing lens for excitation light. LED-TOF-CE and LED-COF-CE system were applied to the separation and determination of RF in real sample (green tea), respectively. The tapered fiber optic-induced fluorescence detection system in CE is an ideal tool for trace analysis.

Deceleration of the E1P-E2P Transition and Ion Transport by Mutation of Potentially Salt Bridge-forming Residues Lys-791 and Glu-820 in Gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P(IIC)-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK(a) modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K(+)-induced stationary currents. In Rb(+) uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E(2)-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E(1)P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E(1)P ↔ E(2)P conformational transition, thus explaining the E(1)P shift and the reduced Rb(+) transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb(+) transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E(2)P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H(+) pumping P-type ATPases.