Pulsed fluorescence measurements of trapped molecular ions with zero background detection

Sensitive methods have been developed to measure laser-induced fluorescence from trapped ions by reducing the detection of background scattering to zero levels during the laser excitation pulse. The laser beam diameter has been reduced to approximately 150 microm to eliminate scattering on trap apertures and the resulting laser-ion interaction is limited to a volume of approximately 10(-5) cm which is approximately 0.03-0.15 of the total ion cloud volume depending on experimental conditions. The detection optics collected fluorescence only from within the solid angle defined by laser-ion interaction volume. Rhodamine 640 and Alexa Fluor 350 ions, commonly used as fluorescence resonance energy transfer (FRET) fluorophores, were generated in the gas phase by using electrospray ionization and injected into a radiofrequency Paul trap where they were stored and exposed to Nd:YAG laser pulses at 532 and 355 nm for times up to 10 m. Fluorescence emitted by these ions was investigated for several trap q(z) values and ion cloud temperatures. Analysis of photon statistics indicated an average of approximately 10 photons were incident on the PMT detector per 15 ns pulse for approximately 10(3) trapped ions in the interaction volume. Fluorescence measurements displayed a dependence on trapped ion number which were consistent with calculations of the space charge limited ion density. To investigate the quantitative capability of these fluorescence techniques, the laser-induced fragmentation of trapped Alexa Fluor 350 ions was measured and compared with a rate equation model of the dynamics. Decay of the fluorescence signal as well as the parent ion number compared closely with quantitative predictions of the photofragmentation model.     

Simultaneous time- and wavelength-resolved fluorescence microscopy of single molecules

Single fluorophores and single-pair fluorescence resonance energy transfer were studied with a new confocal fluorescence microscope that allows, for the first time, the wavelength and emission time of each detected photon to be simultaneously measured with single molecule sensitivity. In this apparatus, the photons collected from the sample are imaged through a dispersive optical system onto a time and position sensitive photon detector. For each detected photon the detection system records its wavelength, its emission time relative to the excitation pulse, and its absolute emission time. A histogram over many photons can generate a full fluorescence spectrum and correlated decay plot for a single molecule for any time interval. At the single molecule level, this approach makes possible entirely new types of temporal and spectral correlation spectroscopies. This paper presents our initial results on simultaneous time- and wavelength-resolved fluorescence measurements of single rhodamine 6G (R6G), tetramethylrhodamine (TMR), and Cy3 molecules embedded in thin films of poly(methyl methacrylate) (PMMA), and of single-pair fluorescence resonance energy transfer between two Alexa fluorophores spaced apart by a short polyproline peptide.     

LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.     

Photodissociation dynamics of dichlorocarbene at 248 nm

The dynamics of the 248 nm photodissociation of the CCl(2) molecule have been investigated in a molecular beam experiment. The CCl(2) parent molecule was generated in a molecular beam by pyrolysis of CHCl(3), and both CCl(2) and the CCl photofragment were detected by laser fluorescence excitation. The 248 nm attenuation cross sections was estimated from the reduction of the CCl(2) signal as a function of the photolysis laser fluence. The internal state distribution of the CCl photofragment was derived from analysis of laser fluorescence excitation spectra in the A (2)Delta- X (2)Pi band system. The CCl(X (2)Pi, nu = 0) rotational state distribution was found to be bimodal, with maximum populations at N approximately 10 and 85, and was dependent upon the source backing pressure, and hence upon the internal state distribution of the CCl(2) precursor. The 248 nm photodissociation dynamics appears to involve two separate channels, namely nearly impulsive rotational energy release and predissociation with little rotational energy imparted to the CCl fragment.     

PICOSECOND FLUORESCENCE STUDIES OF EXCITON MIGRATION AND ANNIHILATION IN PHOTOSYNTHETIC SYSTEMS. A REVIEW*

Abstract. In this paper we review picosecond fluorescence studies of exciton dynamics in photosynthesis. We discuss some of the exciton interactions that led to artifacts in early picosecond data and outline procedures for avoiding their presence. In the case of high intensity single pulse excitation (> 10¹³ photons cm²), the dominant mechanism is singlet-singlet fusion, manifesting itself by a decrease in the observed lifetime and quantum efficiency of fluorescence. The manner in which excitons interact in vivo provides an indicator of the topology of the photosynthetic unit (PSU). The shape of the fluorescence quenching curve, as a function of intensity, in particular, can be used to test various models. In addition to fluorescence quenching curves, we also report the results of fluorescence decay following ps laser flashes, using an ultrafast streak camera in four types of systems: (1) organic crystal anologues, (2) chromatophores of various mutants of the photosynthetic bacteria, Rhodopseudomonas sphaeroides, (3) intact cells of the green alga, Chlorella and (4) chloroplasts of higher plants (e.g. spinach).     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.     

State-resolved dynamics of the CH(A2Delta) channels from single and multiple photon dissociation of bromoform in the 10-20 eV energy range

Single photon dissociation of bromoform using synchrotron radiation has been investigated by Fourier transform visible fluorescence spectroscopy (FTVIS). The photodissociation of bromoform in the 12-18 eV energy range results in several products, among which are the CH(A2Delta) and CH(B2Sigma) radicals. Vibrational and rotational state distributions of the CH(A2Delta) are determined from their fluorescence spectra. From the threshold photon energy above which emission from the CH(A2Delta) radicals is observed, the most likely process leading to CH(A) formation is CHBr3 --> CH + 3Br rather than CHBr3 --> CH + Br + Br2. The rotational Boltzmann temperatures in the CH(A --> X) emission spectra for v' = 0 and v' = 1 range between 1570 and 3650 K, depending on the excitation photon energy. From the high rotational excitation, the results suggest that the mechanism for the loss of three bromine atoms is most likely sequential. A small negative emission anisotropy of the CH(A) radicals [(Ipar - Iper)/(Ipar + 2Iper) = -0.024 +/- 0.005] is constant across the action spectrum; a small net absorption dipole of CHBr3 in the vacuum ultraviolet is parallel to the 3-fold symmetry axis of the CHBr3 molecule. The state distributions of the CH(A2Delta) radicals from multiphoton dissociation of bromoform using the 266 nm output (three photons) of a femtosecond laser (Boltzmann temperatures: T(v'=0)(rot)= 4250 +/- 300 K; T(v'=1)(rot)= 3100 +/- 550 K) are compared to those from the single photon dissociation results (Boltzmann temperatures: T(v'=0)(rot)= 3650 +/- 150 K; T(v'=1)(rot)= 2400 +/- 200 K) at the same total excitation energy under collision free conditions. The analysis of the CH(A) rotational populations shows hotter rotational populations for the femtosecond experiment, also suggesting sequential dissociation of the bromoform in the femtosecond experiment. The duration of the femtosecond laser pulse is approximately 180 fs, setting a limit on the time scales for the multiple dissociations.     

Three‐peak Autler‐Townes splitting in the photoelectron spectrum of Li2 molecules caused by femtosecond laser pulses

Three‐peak Autler‐Townes (A‐T) splitting in the resonant multiphoton ionization photoelectron spectrum for a rotating Li₂ molecular system in femtosecond pulse laser fields is studied by using two‐dimension time‐dependent quantum wave packet method. The A‐T splitting results from rapid Rabi oscillation caused by intense femtosecond laser pulses. Because of the effects of molecular rotation and alignment, the Rabi oscillation in the population distribution will be damped in a certain degree. The three‐peak A‐T splitting can only be observed for a strongly aligned molecule with rapid Rabi oscillation. The three‐peak A‐T splitting dynamics can be affected by intensity, duration, temporal profile of laser pulse, and initial molecular rotational temperature. The conditions to observe the A‐T splitting are discussed in detail. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Probing lipid vesicles by bimolecular association and dissociation trajectories of single molecules

Vesicles prepared by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid molecules having sizes smaller than the diffraction-limited focused laser beam have been used to confine single molecules in the laser focus. The confinement of single molecules in a volume smaller than the focused laser beam leads to a Gaussian distribution of single molecule fluorescence intensity. The interactions of single Nile Red molecules with DMPC and SOPC lipid bilayers were studied by single molecule fluorescence confocal microscopy. Nile Red molecules were observed to associate with and dissociate from individual DMPC and SOPC vesicles adsorbed on a glass surface, generating on-and-off fluctuations in a fluorescence signal representing a very low noise two-state trajectory. Off-time statistics were used to investigate the mean radius of the vesicles and the size distribution functions. The means of the on-time distributions of Nile Red in DMPC and SOPC vesicles were significantly different. The association and dissociation reactions of single Nile Red molecules with a vesicle have been studied. Features of the bimolecular interaction between the probe Nile Red and the vesicle were evaluated from the uncorrelated mean on-time and vesicle radius distributions, and the linear Nile Red concentration dependence of the mean off-time. Nile Red is shown to be a useful probe of the structural fluctuations and heterogeneity of these membrane structures, and it is a useful model with which to directly study a diffusion-influenced reversible bimolecular reaction.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

用双激光脉冲操纵N2分子取向

利用求解含时薛定谔方程的方法, 研究了双原子N2分子在激光强度为1.5×10¹⁴ W·cm^-2, 脉冲宽度为50 fs激光脉冲作用下的取向行为. 研究结果表明, 保持总激光强度不变, 将一束激光脉冲分成具有同样脉宽, 强度比为0.3636的两束激光脉冲, 当第一束脉冲产生的分子取向即将达到最大时, 加上有一定延迟时间的第二束脉冲能够使N2分子达到最大的取向程度.     

Spectral identification of specific photophysics of cy5 by means of ensemble and single molecule measurements

The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g., as a probe or fluorescence resonance energy transfer acceptor in large biomolecules. However, the unknown spectral properties of the triplet state and the lack of knowledge on the photoisomerization do not allow us to interpret precisely the unexpected single molecule behaviors. This limits the application of Cy5. The laser photolysis experiments demonstrate that the trans triplet state of Cy5 absorbs about 625 nm, the cis ground state absorbs about 690 nm, and the cis triplet state also absorbs about 690 nm. In other words, the T1-Tn absorptions largely overlap the ground-state absorptions for both trans and cis isomers, respectively. Furthermore, the observation of the cis triplet state indicates an important isomerization pathway from the trans-S1 state to the cis-T1 state upon excitation. The detailed spectra presented in this article let us clearly interpret the exact mechanisms responsible for several important and unexpected photophysical behaviors of single Cy5 molecules such as reverse intersystem crossing (RISC), the observation of dim states with a lower emission intensity and slightly red-shifted fluorescence, and unusual energy transfer from donor molecules to dark Cy5 molecules acting as acceptors in single molecule fluorescence resonance energy transfer (FRET) measurements. Spectral results show that the dim state in the single molecule fluorescence intensity time traces originated from cis-Cy5 because of a lower excitation rate, resulting from the red-shifted ground-state absorption of cis-Cy5 compared to that of the trans-Cy5.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

From stochastic pulse optimization to a stereoselective laser pulse sequence: simulation of a chiroptical molecular switch mounted on adamantane

Quantum dynamical simulations for the laser-controlled isomerization of 1-(2-cis-fluoroethenyl)-2-fluorobenzene mounted on adamantane are reported based on a one-dimensional electronic ground-state potential and dipole moment calculated by density functional theory. The model system 1-(2-cis-fluoroethenyl)-2-fluorobenzene supports two chiral and one achiral atropisomers upon torsion around the C-C single bond connecting the phenyl ring and ethylene group. The molecule itself is bound to an adamantyl frame which serves as a model for a linker or a surface. Due to the C3 symmetry of the adamantane molecule, the molecular switch can have three equivalent orientations. An infrared picosecond pulse is used to excite the internal rotation around the chiral axis, thereby controlling the chirality of the molecule. In order to selectively switch the molecules--independent of their orientations-- from their achiral to either their left- or right-handed form, a stochastic pulse optimization algorithm is applied. A subsequent detailed analysis of the optimal pulse allows for the design of a stereoselective laser pulse sequence of analytical form. The developed control scheme of elliptically polarized laser pulses is enantioselective and orientation-selective.     

FLUORESCENCE RELAXATION KINETICS AND QUANTUM YIELD FROM THE PHYCOBILISOMES OF THE BLUE-GREEN ALGA NOSTOC SP. MEASURED AS A FUNCTION OF SINGLE PICOSECOND PULSE INTENSITY*

Abstract— A detailed experimental study of the effect of intensity of a 6 ps excitation pulse on the decay kinetics and yield from phycobilisomes (PBsomes) is presented. The fluorescence from the c-phycoerythrin (PE) emission from PBsomes was found to decay as a single exponential with a time of 31 ± 4ps for an excitation intensity <10¹⁴ photons/cm² per pulse. The risetime of the c-phycocyanin (PC) and allophycocyanin (APC) emission from PBsomes was found to be 34 ± 13 ps. Therefore, at low excitation intensities, the energy transfer time between the constituent phycobiliproteins, PE and PC, is measured to be 34 ± 13ps from the fluorescence decay time of PE and the fluorescence risetime of the PC and APC emission. The fluorescence yield from the PE emission component in PBsomes was found to be intensity dependent for excitation intensities >10¹⁴ photons/cm². The decrease in yield with increased intensity in this case occurred at a higher intensity than in the isolated phycobiliprotein PE. The fluorescence yield of the PC and APC emission component was also found to decrease markedly with increasing excitation intensity. This is in contrast to the case of the isolated phycobiliprotein APC which showed only a slight quenching of the fluorescence. The higher quenching observed for the APC emission in the PBsome evidences the higher effective absorption of APC via energy transfer from PE to PC and APC.     

Conventional and Laser Photolysis of a Bisdiazo Compound

Entirely different product distributions were observed when the bisdiazo compound 1 was irradiated by a conventional lamp (254 nm) on the one hand and by a pulsed excimer laser (248 nm) on the other. Continuous photolysis gave a complex reaction mixture (Scheme 2), of which eight products were identified. Pulsed laser photolysis gave (11E)-dibenzocyclooctatriene 20 as a new product in 49% yield (Scheme 4). We postulate that compound 20 is formed via the biscarbene intermediate 18 which results from the sequential absorption of two photons by a single molecule within a single laser pulse.     

FLUORESCENCE DECAY KINETICS and CHARACTERISTICS OF BIMOLECULAR EXCITON ANNIHILATION IN CHLOROPLASTS

Abstract— The decay profiles of the fluorescence of dark-adapted spinach chloroplasts (0^A°C) excited with single 30 ps 532 nm laser pulses of varying intensities were measured with a low-jitter streak camera system. By comparing the decay profiles of the fluorescence at low and high laser fluences, i.e. in the absence and presence, respectively, of dynamic bimolecular exciton-exciton annihilation effects, the duration of such dynamic annihilation events can be estimated. A simple model suggests that the influence of bimolecular annihilation events on the fluorescence decay kinetics should disappear within a time interval corresponding to the low intensity, unimolecular lifetime of the exciton population which is subject to exciton-exciton annihilation. The low intensity fluorescence decay profiles are characterized by three to four lifetimes (Reviewed by A. R. Holzwarth, Photochem. Photobiol. 43,707–725, 1986); it is shown here that only the shortest fluorescence components are subject to exciton annihilation, since the kinetics of the fluorescence decay are influenced by annihilations only within the initial 150–200 ps time interval after the excitation pulse. The amplitudes (but not the decay kinetics) of the longer-lived fluorescence components are decreased at high levels of laser pulse excitations, suggesting that these components are derived from the shorter-lived fluorescence decay components. The implications of these results are*discussed within the contexts of current models of the fluorescence in chloroplasts.     

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.