Coherent manipulation of free amino acids fluorescence

Coherent manipulation of molecular wavepackets in biomolecules might contribute to the quest towards label-free cellular imaging and protein identification. We report the use of optimally tailored UV laser pulses in pump-probe depletion experiments that selectively enhance or decrease fluorescence between two aromatic amino acids: tryptophan (Trp) and tyrosine (Tyr). Selective fluorescence modulation is achieved with a contrast of ~35%. A neat modification of the time-dependent fluorescence depletion signal of Trp is observed, while the Tyr transient trace remains unchanged. The mechanism invoked for explaining the change of the depletion of Trp is a less efficient coupling between the fluorescing state and the higher non-radiative excited states by the optimally shaped pulse, than by the reference pulse.     

Photostability of amino acids: internal conversion versus dissociation

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

The principles of infrared-x-ray pump-probe spectroscopy. Applications on proton transfer in core-ionized water dimers

In this paper we derive the basic physics underlying infrared-x-ray pump-probe spectroscopy (IR, infrared). Particular features of the spectroscopy are highlighted and discussed, such as dependence on phase of the infrared pulse, duration and delay time of the x-ray pulse, and molecular orientation. Numerical applications are carried out for the water dimer using wave packet techniques. It is shown that core ionization of the donor oxygen of the water dimer results in a drastic change of the potential with the global minimum placed in the proton transfer region. The results of the modeling indicate that IR-x-ray pump-probe spectroscopy can be used to study the dynamics of proton transfer in this core-ionized state, and that, contrary to conventional core level photoelectron spectroscopy, x-ray core-ionization driven by an IR field is a proper method to explore the proton transfer in a system like the water dimer. We observe that the trajectory of the nuclear wave packet in the ground state potential well is strongly affected by the absolute phase of the IR pulse.     

Non-linear fluorescence lifetime imaging of biological tissues

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues.     

The study of cell wall structure and cellulose–cellulase interactions through fluorescence microscopy

The efficient decomposition of biomass into carbohydrates for the sustainable generation of biofuels has become the focus of much research. Yet, limited understanding exists on how the enzymes that catalyze the biochemical conversion of biomass, such as cellulases, interact with cellulose microfibrils and how cellulose structure is changed by cellulolytic enzymes. This has spurred the application of high-resolution imaging techniques, such as atomic force microscopy or fluorescence microscopy, to visualize the biomolecular interactions and structural changes that occur at the micro/nanoscale. In particular, fluorescence microscopy offers advantages such as high sensitivity and the ability to monitor species under biologically relevant conditions. Furthermore, the introduction of techniques, such as single molecule or super-resolution fluorescence microscopy, has allowed imaging biomolecules and macromolecular structures with near molecular resolution. These advantages make fluorescence microscopy ideally suited for the study of cell wall structure and cellulose–cellulase interactions. The application of fluorescence microscopy has already yielded key insights into the arrangement of structural polysaccharides in the plant cell wall, the reversibility and binding kinetics of cellulases, their molecular motion on crystalline cellulose, and the structural changes that occur as cellulose is depolymerized by cellulases. Yet, the application of fluorescence to study cellulose–cellulase interactions remains limited. This review aims at (1) providing an overview of fluorescence microscopy techniques suitable for the study of cellulose–cellulase interactions; (2) the applications of these techniques to date and the key insights obtained; and (3) the opportunities for future studies of the interaction of cell wall degrading enzymes with cellulosic materials.     

Transient excited-state absorption and gain spectroscopy of a two-photon absorbing probe with efficient superfluorescent properties

The synthesis, linear photophysical properties, two-photon absorption (2PA), excited-state transient absorption, and gain spectroscopy of a new fluorene derivative tert-butyl 4,4'-(4,4' (1E,1'E)-2,2'-(9,9-bis(2- (2-ethoxyethoxy)ethyl)-9H-fluorene-2,7-diyl)bis(ethene-2,1-diyl)bis(4,1 phenylene)]dipiperazine-1-carboxylate (1) are reported. The steady-state linear absorption and fluorescence spectra, along with excitation anisotropy, fluorescence lifetimes, and photochemical stability of 1 were investigated in a number of organic solvents at room temperature. The 2PA spectra of 1 with a maximum cross-section of ~ 300 GM were obtained with a 1 kHz femtosecond laser system using open-aperture Z-scan and two-photon-induced fluorescence methods. The transient excited-state absorption (ESA) and gain kinetics of 1 were investigated by a femtosecond pump-probe methodology. Fast relaxation processes (~1-2 ps) in the gain and ESA spectra of 1 were revealed in ACN solution, attributable to symmetry-breaking effects in the first excited state. Efficient superfluorescence properties of 1 were observed in a nonpolar solvent under femtosecond excitation. One- and two-photon fluorescence microscopy imaging of HCT 116 cells incubated with probe 1 was accomplished, suggesting the potential of this new probe in two-photon fluorescence microscopy bioimaging.     

ANALYSIS OF FLUORESCENCE KINETICS AND ENERGY TRANSFER IN ISOLATED α SUBUNITS OF PHYCOERYTHRIN FROM Nostoc sp.

Abstract— The fluorescence decay profiles, relative quantum yield, and transmission of the phycoerythrin a subunit, isolated from the photosynthetic antenna system of Nostoc sp., were measured using single picosecond laser excitation. The fluorescence decay profiles were found to be intensity independent for the intensity range investigated (4 × 10¹³ and 4 × 10¹⁵ photons-cm-² per pulse). The decay profiles were fitted to a model assuming both chromophores absorb and fluoresce. The inferred total deactivation rates for the two chromophores, in the absence of energy transfer and when the effects of the response time of the streak camera and the finite pulse width are properly included, are 1.0 × 10¹⁰s' and 1.0 × 10⁹ s ¹ for the s and f chromophores. respectively, whereas the transfer rate between the two fluorophorcs is estimated to be 1.0 × 10¹⁰ s⁻¹ giving a s→ f transfer rate on the order of (100 ps)⁻¹. Steady-atate polarization measurements were found to be equal to those calculated using the rate parameters inferred from the kinetic model fit to the fluorescence decays. The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is suggestive of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated α subunits for the intensity range investigated (4 × 10¹³ to 4 × 10¹⁵ photons-cm ² per pulse).     

Fluorescence kinetics of emission from a small finite volume of a biological system

The fluorescence decay, apparent quantum yield and transmission from chromophores constrained to a microscopic volume using a single picosecond laser excitation were measured as a function of incident intensity. The β subunit of phycoeryhthrin aggregate isolated from the photosynthetic antenna system of Nostoc sp. was selected since it contains only four chromophores in a volume of less than 5.6×10⁴ ų. The non-exponential fluorescence decay profiles were intensity independent for the intensity range studied (5 × 10¹³ - 2 × 10¹⁵ photon cm^?2 per pulse). The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is attributed to the combined effects of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated β subunits.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.     

Photodissociation dynamics of hydroxybenzoic acids

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

Live‐Cell Stimulated Raman Scattering Imaging of Alkyne‐Tagged Biomolecules

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.     

Resolving Electronic Transitions in Synthetic Fluorescent Protein Chromophores by Magnetic Circular Dichroism

The detailed electronic structures of fluorescent chromophores are important for their use in imaging of living cells. A series of green fluorescent protein chromophore derivatives is examined by magnetic circular dichroism (MCD) spectroscopy, which allows the resolution of more bands than plain absorption and fluorescence. Observed spectral patterns are rationalized with the aid of time‐dependent density functional theory (TDDFT) computations and the sum‐over‐state (SOS) formalism, which also reveals a significant dependence of MCD intensities on chromophore conformation. The combination of organic and theoretical chemistry with spectroscopic techniques also appears useful in the rational design of fluorescence labels and understanding of the chromophore's properties. For example, the absorption threshold can be heavily affected by substitution on the phenyl ring but not much on the five‐member ring, and methoxy groups can be used to further tune the electronic levels.     

氧芴三苯胺多枝分子的双光子吸收与电化学行为

研究了3个氧芴/三苯胺衍生物: E-2,8-双(4-二苯胺基苯乙烯基)氧芴(简称OT-G1)、E-2,8-双[4-(二苯基氨基-二苯乙烯基)(4’-溴苯基)氨基-苯乙烯基]氧芴(简称OT-G1.5)和E-2,8-双-[4’,4″-二-(二苯胺基苯乙烯基)-4-二苯胺基苯乙烯基]氧芴(简称OT-G2)的双光子吸收和电化学行为. 研究结果表明, 分子“代数”从1→1.5→2增高, 氧芴三苯胺多枝分子的HOMO能级升高、双光子荧光强度和双光子吸收截面明显增大. 由于HOMO能级的升高有利于分子的电荷转移, 因而分子表现出强的双光子吸收能力, 这表明可通过电化学行为来推断出分子的双光子吸收性能.     

Matrix-induced intensity fluctuations in the fluorescence from single oligo(phenylenevinylene) molecules

Single molecule spectroscopy on oligo(phenylenevinylene) (OPV) chromophores shows that the fluorescence intermittency strongly correlates to the rigidity of the environment surrounding the molecules. For OPV single molecules, environmental rigidity inhibits twisting about the vinyl linkages, the molecular motion associated with the observed "off" (nonabsorbing) state. By increasing the rigidity of a single molecule's environment, we can tune its room temperature fluorescence from rapid, sub-millisecond "blinking" fluctuations (fluid polymer environment) to completely "on" with no blinking observed (molecules adsorbed to a rigid bare glass substrate). The difference in fluorescence intermittency from environment to environment is immediately apparent and explicit in single molecule intensity trajectories under cw (continuous wave) excitation, demonstrating the sensitivity of these chromophores to their surroundings and emphasizing the importance of morphological control in real-world applications involving phenylenevinylene-based materials.     

Observation of slow charge redistribution preceding excited-state proton transfer

The photoacid 8-hydroxy-N,N,N',N',N',N'-hexamethylpyrene-1,3,6-trisulfonamide (HPTA) and related compounds are used to investigate the steps involved in excited-state deprotonation in polar solvents using pump-probe spectroscopy and time correlated single photon counting fluorescence spectroscopy. The dynamics show a clear two-step process leading to excited-state proton transfer. The first step after electronic excitation is charge redistribution occurring on a tens of picoseconds time scale followed by proton transfer on a nanosecond time scale. The three states observed in the experiments (initial excited state, charge redistributed state, and proton transfer state) are recognized by distinct features in the time dependence of the pump-probe spectrum and fluorescence spectra. In the charge redistributed state, charge density has transferred from the hydroxyl oxygen to the pyrene ring, but the OH sigma bond is still intact. The experiments indicate that the charge redistribution step is controlled by a specific hydrogen bond donation from HPTA to the accepting base molecule. The second step is the full deprotonation of the photoacid. The full deprotonation is clearly marked by the growth of stimulated emission spectral band in the pump-probe spectrum that is identical to the fluorescence spectrum of the anion.     

Coherent control of pump-probe signals of helical structures by adaptive pulse polarizations

The simplification of the pump-probe spectrum of excitons by pure-phase-polarization pulse shaping is investigated by a simulation study. The state of light is manipulated by varying the phases of two perpendicular polarization components of the pump, holding its total spectral and temporal intensity profiles fixed. Genetic and iterative Fourier transform algorithms are used to search for pulse phase functions that optimize the ratio of the signal at two frequencies. New features are extracted from the congested pump-probe spectrum of a helical pentamer by selecting a combination of Liouville space pathways. Tensor components which dominate the optimized spectra are identified.     

Confocal fluorescence and Raman microscopy in industrial research

 Modern chemical and pharmaceutical industrial research benefits from improved spectroscopic tools. New developments in confocal fluorescence and Raman microscopy lead to an increase in sensitivity, selectivity and speed of microscopic imaging and fluctuation analysis resulting in a better understanding of structure–property relationships essential for targeted development. In this paper we report on the application of fluorescence and Raman microscopy for characterizing the morphology of polymeric multiphase solid-state samples and on new developments in the corresponding correlation spectroscopies for the characterization of the dynamics of complex colloidal systems in the liquid state. In the case of fluorescence new technological opportunities are gained by two-photon excitation. Received: 5 February 1998 Accepted: 16 February 1998     

Ultrafast energy migration in chromophore shell-metal nanoparticle assemblies

A multifunctional ligand-coated nanoparticle system containing approximately 2000 highly two-photon absorptive chromophores has been investigated by means of steady-state and femtosecond time-resolved spectroscopy. This system with a high local concentration of chromophores showed remarkably low self-quenching and a high fluorescence quantum yield, which is important for a variety of two-photon sensing and imaging applications. We have observed evidence for ultrafast energy migration in these chromophore shell-metal nanoparticle systems. Time-resolved experiments also showed non-zero residual anisotropy after the initial fast decay, which can be interpreted as due to the formation of the specific domains on the metal surfaces. This investigation opens new avenues toward the development of multi-chromophoric efficient TPA fluorescence sensing/imaging systems with large numbers of chromophores per one metal particle nanoparticle.