Single-photon Excitation Research Articles

Sub-cycle strong-field interferometry

A nonlinear interferometry scheme is described theoretically to induce and resolve electron wavefunction beating on time scales shorter than the optical cycle of the time-delayed pump and probe pulses. By employing two moderately intense few-cycle laser fields with a stable carrier-envelope phase, a large range of the entire electronic level structure of a quantum system can be retrieved. In contrast to single-photon excitation schemes, the retrieved electronic states include levels that are both dipole- and non-dipole-accessible from the ground electronic state. The results show that strong-field interferometry can reveal both high-resolution and broad-band spectral information at the same time with important consequences for quantum-beat spectroscopy on attosecond time scales.

Conjugates of folic acids with BSA-coated quantum dots for cancer cell targeting and imaging by single-photon and two-photon excitation

Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10(4) KB cells, and was about 3times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1μM) for 40min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis.

High-speed focal modulation microscopy using acousto-optical modulators

Focal Modulation Microscopy (FMM) is a single-photon excitation fluorescence microscopy technique which effectively rejects the out-of-focus fluorescence background that arises when imaging deep inside biological tissues. Here, we report on the implementation of FMM in which laser intensity modulation at the focal plane is achieved using acousto-optic modulators (AOM). The modulation speed is greatly enhanced to the MHz range and thus enables real-time image acquisition. The capability of FMM is demonstrated by imaging fluorescence labeled vasculatures in mouse brain as well as self-made tissue phantom.

Dissociation dynamics of ion-pair states of Cl2at principal quantum numbers beyond 1500

Long-lived ion-pair states of Cl{sub 2} have been observed by delayed pulsed-field ionization in the vicinity of the Cl{sup -}({sup 1}S{sub 0})+Cl{sup +}({sup 3}P{sub 2}) dissociation threshold following single-photon excitation from the X {sup 1}{Sigma}{sub g}{sup +}(v=0) ground state with a tunable vacuum-ultraviolet laser. The field-ionization spectra reveal a series of resonances corresponding to ion-pair states with effective principal quantum number n* between 1858 and 1876 belonging to a series converging to the Cl{sup -}({sup 1}S{sub 0})+Cl{sup +}({sup 3}P{sub 0}) dissociation threshold. These states are observed by forced predissociation into the Cl{sup -}({sup 1}S{sub 0})+Cl{sup +}({sup 3}P{sub 2}) ion-pair channel. This process is the ion-pair analog of the process of forced autoionization observed in Rydberg states. The analysis of the spectra and of the field-ionization behavior provides information on the couplings between the relevant ionization and dissociation channels and has enabled the determination of the ion-pair dissociation threshold [E{sub IPD}(Cl{sup -}({sup 1}S{sub 0})+Cl{sup +}({sup 3}P{sub 2}))=95 449.8{+-}1.0 cm{sup -1}] and of the dissociation energies of Cl{sub 2} [D{sub 0}(X {sup 1}{Sigma}{sub g}{sup +})=19 998.4{+-}1.1 cm{sup -1}] and Cl{sub 2}{sup +} [D{sub 0}(X{sup +} {sup 2}{Pi}{sub u})=31 942.1{+-}1.5 cm{sup -1}].

The two-photon absorption spectrum of ruby and its role in distributed optical fibre sensing

The two-photon absorption spectrum and cross-section (δ) for ruby (Cr3+:sapphire) have been measured over the 0.8–1.2 μm wavelength range at 25°C. Absolute values of δ were obtained by comparing the fluorescence yield for two-photon excitation with that for single-photon excitation of the same ionic transition under identical illumination conditions. The maximum values of δ are 4.6×10−3 GM for o-polarisation and 5.9×10−3 GM for e-polarisation at 840 nm. The relevance of these measurements to distributed optical fibre sensing is briefly discussed.

Electron transfer processes in double lanthanide activated YPO 4

This work reviews electron transfer processes in double lanthanide doped YPO 4 studied using a diversity of optical techniques. The aim is to identify those transfer processes and to relate them with the location of lanthanide electron donor and lanthanide electron acceptor states within the bandgap of YPO 4. Electron transfer from the valence band to a trivalent lanthanide impurity or to the conduction band was studied by traditional single photon excitation on single lanthanide doped YPO 4. This work reports on YPO 4 doped with the electron donor Ce 3+ and co-doped with an electron acceptor Pr 3+, Er 3+, Nd 3+, Ho 3+, Dy 3+, Tm 3+, or Sm 3+ studied by means of thermoluminescence and optical stimulated luminescence techniques. The same samples were studied by pump and probe two photon spectroscopy utilizing synchrotron photons to transfer electrons from Ce 3+ to Ln 3+ defects and 445 nm laser diode to probe the concentration of created Ln 2+.

Fast electrons from multi-electron dynamics in xenon clusters induced by inner-shell ionization

Fast electrons emitted from xenon clusters in strong femtosecond 90 eV pulses have been measured at the Free-electron Laser in Hamburg (FLASH). Energy absorption occurs mainly through atomic inner-shell photo-ionization. Photo-electrons are trapped in the strong Coulomb potential of the cluster ions and form a non-equilibrium plasma with supra-atomic density. Its equilibration through multiple energy-exchanging collisions within the entire cluster volume produces electrons with energies well beyond the dominant emission line of atomic xenon. Here, in contrast to traditional low-frequency laser plasma heating, the plasma gains energy from electrons delivered through massive single-photon excitation from bound states. Electron emission induced by thermalization of a non-equilibrium plasma is expected to be a general phenomenon occurring for strong atomic x-ray absorption in extended systems.

Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina

Cell type-specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon-excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.

Temperature and species measurement in a quenching boundary layer on a flat-flame burner

A detailed understanding of transport phenomena and reactions in near-wall boundary layers of combustion chambers is essential for further reducing pollutant emissions and improving thermal efficiencies of internal combustion engines. In a model experiment, the potential of laser-induced fluorescence (LIF) was investigated for measurements inside the boundary layer connected to flame-wall interaction at atmospheric pressure. Temperature and species distributions were measured in the quenching boundary layer formed close to a cooled metal surface located parallel to the flow of a premixed methane/air flat flame. Multi-line NO-LIF thermometry provided gas-phase temperature distributions. In addition, flame species OH, CH2O and CO were monitored by single-photon (OH, CH2O) and two-photon (CO) excitation LIF, respectively. The temperature dependence of the OH-LIF signal intensities was corrected for using the measured gas-phase temperature distributions. The spatial line-pair resolution of the imaging system was 22 μm determined by imaging microscopic line pairs printed on a resolution target. The experimental results show the expected flame quenching behavior in the boundary layer and they reveal the potential and limitations of the applied diagnostics techniques. Limitations in spatial resolution are attributed to refraction of fluorescence radiation propagating through steep temperature gradients in the boundary layer. For the present experimental arrangements, the applied diagnostics techniques are applicable as close to the wall as 200 μm with measurement precision then exceeding the 15–25% limit for species detection, with estimates of double this value for the case of H2CO due to the unknown effect of the Boltzmann fraction corrections not included in the data evaluation process. Temperature measurements are believed to be accurate within 50 K in the near-wall zone, which amounts to roughly 10% at the lower temperatures encountered in this region of the flames.

Neutral Dissociation of Superexcited Nitric Oxide Induced by Intense Laser Fields

Superexcited states of NO molecule and their neutral dissociation processes have been studied both experimentally and theoretically. Neutral excited N* and O* atoms are detected by fluorescence spectroscopy for the NO molecule upon interaction with 800 nm intense laser radiation of duration 60 fs and intensity 0.2 PW/cm2. Intense laser pulse causes neutral dissociation of superexcited NO molecule by way of multiphoton excitation, which is equivalent to single photon excitation in the extreme-ultraviolet region by synchrotron radiation. Potential energy curves (PECs) are also built using the calculated superexcited state of NO+. In light of the PECs, direct dissociation and pre-dissociation mechanisms are proposed respectively for the neutral dissociation leading to excited fragments N* and O*.

Ramsey spectroscopy of bound atomic states with extreme-ultraviolet laser harmonics

We report the experimental measurement of Ramsey interference fringes in the single-photon excitation to a high-lying bound state of atomic argon by pairs of phase-locked, time-delayed, extreme UV high-order-harmonic pulses at 87 nm. High-visibility Ramsey fringes are observed for delays larger than 100 ps, thus demonstrating a potential resolving power >10(5) at this wavelength.

Metal-Enhanced Multiphoton Absorption Polymerization with Gold Nanowires

Metal-enhanced multiphoton absorption polymerization (MEMAP) is studied using gold nanowires with a set of three different photoresists. Photoresists that employ radical and cationic polymerization are investigated. In all cases, MEMAP is strongly correlated with multiphoton-absorption-induced luminescence (MAIL) of the nanowires. Wavelength-dependent studies indicate that the dominant mechanism for MEMAP is not field-enhanced two-photon absorption of the photoinitiator, but rather single-photon excitation of the photoinitiator by the broadband MAIL emission.

Styryl Dyes as Two-Photon Excited Fluorescent Probes for DNA Detection and Two-Photon Laser Scanning Fluorescence Microscopy of Living Cells

Spectral-fluorescent properties of benzothiazole styryl monomer (Bos-3) and homodimer (DBos-21) dyes in presence of DNA were studied. The dyes enhance their fluorescence intensity in 2-3 orders of magnitude upon interaction with DNA. Studied styrylcyanines in DNA presence demonstrate rather high values of two-photon absorption (TPA) cross-section, which are comparable with the values of TPA cross section of the rhodamine dyes. An applicability of the styrylcyanines as probes for the fluorescence microscopy of living cells was studied. It was shown that both dyes are cell-permeable but homodimer dye DBos-21 produces noticeably brighter staining of HeLa cells comparing with monomer dye Bos-3. Molecules of DBos-21 initially bind to the nucleic acids-containing cell organelles (presumable mitochondria) and are able to penetrate into the cell nucleus. Thus, homodimer styryl DBos-21 dye is viewed as efficient stain for single-photon and two-photon excitation fluorescence imaging of living cells.

Fluorescence advantages with microscopic spatiotemporal control.

We present a clever design concept of using femtosecond laser pulses in microscopy by selective excitation or de-excitation of one fluorophore over the other overlapping one. Using either a simple pair of femtosecond pulses with variable delay or using a train of laser pulses at 20-50 Giga-Hertz excitation, we show controlled fluorescence excitation or suppression of one of the fluorophores with respect to the other through wave-packet interference, an effect that prevails even after the fluorophore coherence timescale. Such an approach can be used both under the single-photon excitation as well as in the multi-photon excitation conditions resulting in effective higher spatial resolution. Such high spatial resolution advantage with broadband-pulsed excitation is of immense benefit to multi-photon microscopy and can also be an effective detection scheme for trapped nanoparticles with near-infrared light. Such sub-diffraction limit trapping of nanoparticles is challenging and a two-photon fluorescence diagnostics allows a direct observation of a single nanoparticle in a femtosecond high-repetition rate laser trap, which promises new directions to spectroscopy at the single molecule level in solution. The gigantic peak power of femtosecond laser pulses at high repetition rate, even at low average powers, provide huge instantaneous gradient force that most effectively result in a stable optical trap for spatial control at sub-diffraction limit. Such studies have also enabled us to explore simultaneous control of internal and external degrees of freedom that require coupling of various control parameters to result in spatiotemporal control, which promises to be a versatile tool for the microscopic world.

A high-peak-power UV picosecond-pulse light source based on a gain-switched 155 μm laser diode and its application to time-resolved spectroscopy of blue-violet materials

We generated sub-kilowatt peak-power and 6-ps duration 390-nm optical pulses via the fourth harmonic generation of amplified optical output from a gain-switched 1.55-microm laser diode. We obtained a power-conversion-efficiency of 12% from 1.55-microm to 390-nm light, and subsequently applied the ultraviolet pulses to time-resolved spectroscopy of blue-violet luminescent materials, including a Coumarine dye solution and nitride semiconductor materials using single-photon and two-photon excitation schemes.

Two-Photon Optogenetic Control of Neuronal Activity with Single Synapse Precision by Sculpted Light

Recent advances in optogenetic techniques have provided new tools for controlling neuronal activity, opening up the way to a range of studies in neuroscience. The most widely used approach has been the optical activation of the genetically expressed light-gated ion channel Channelrhodopsin-2 (ChR2) to initiate population activity in neuronal circuits. However, single cell resolution of optogenetic activation has remained challenging. This is because neither single-photon nor conventional two-photon excitation provides the necessary combination of high spatial selectivity and the simultaneous stimulation of a sufficiently large membrane area necessary to induce fast and significant depolarizations by ChR2 in a single neuron.The presented work reports on two-photon excitation of ChR2 allowing the generation of fast and large ChR2-mediated currents in single cells with high spatial and temporal resolution by using temporally focused beams. It is demonstrated that this technique efficiently induces strong depolarization and reliable action potential firing in single ChR2-expressing neurons in rat and mouse hippocampal slices. It is further shown that subcellular compartments such as dendrites and large presynaptic terminals can be activated by the TF-2P technique. The superb spatial and temporal resolution provided by this technique allows so far unattainable precision for fine manipulation of neuronal activity to study and control the function of neuronal microcircuits in vitro and in vivo.

Non-resonant two-photon photochemistry of a Barton ester, N-phenylacetyloxy-2-pyridinethione

N-Acetyloxy-2-pyridinethiones, otherwise known as Barton esters, are a class of molecules that can be easily photolysed via single-photon excitation to facilitate the controlled release of carbon or oxygen-centred radicals. In the present work, we investigate the two-photon chemistry of a simple Barton ester, and show that this material can be photolysed via two-photon excitation, with a two-photon bleaching cross section value of 0.13 +/- 0.01 GM.

Neutral Dissociation of Superexcited Oxygen Molecules in Intense Laser Fields

Superexcited states (SESs) of oxygen molecules and their neutral dissociation processes have been studied both experimentally and theoretically using intense femtosecond laser. We find that at the laser intensity of approximately 2 x 10(14) W/cm(2), ultrashort laser pulse causes neutral dissociation of oxygen molecule by way of SESs. The dissociation products are the excited neutral oxygen atoms, which are observed through fluorescence spectroscopy. Laser power dependence of the fluorescence intensity shows that each molecule effectively absorbs an average of ten laser photons. The total energy absorbed is sufficient to stimulate the molecule to many of the SESs. The effect is equivalent to single photon excitation in the extreme-ultraviolet (XUV) region by synchrotron radiation (SR). Morse potential energy curves (PECs) are constructed for the SESs of O(2) molecules. In light of the PECs, predissociation mechanism is proposed for the neutral dissociation. Quasi-classical trajectory (QCT) calculations show that the predissociation time is as short as 100 fs, which is consistent with our experimental measurement using ultrafast pump-probe technique.

High-Photoluminescence-Yield Gold Nanocubes: For Cell Imaging and Photothermal Therapy

Gold nanocubes demonstrate unique optical properties of the high photoluminescence (PL) quantum yield and a remarkably enhanced extinction band at 544 nm. The 4 x 10(-2) PL yield, which is about 200 times higher than that of gold nanorods, allows gold nanocubes to be successfully used in cell imaging of human liver cancer cells (QGY) and human embryo kidney cells (293T) with a common method of single-photon excitation. The high extinction coefficients of gold nanocubes also facilitate them carrying out the photothermal therapy of QGY and 293T cells, showing similar photokilling efficiency as compared to gold nanorods.

A comparison of tissue penetrations between single and two-photon-excitations

We use the penetration constants, which are independent of the excitation power density, to evaluate the penetration abilities of single-photon-excitation (SPE) at 405 nm and two-photon-excitation (TPE) at 800 nm in porcine tissues. The penetration constant of TPE is smaller than that of SPE although the transmission loss of 405 nm is much larger than 800 nm in skin tissues. On the other hand, the penetration of SPE in blood-contained systems is obviously worse than that of TPE due to the strong absorption of hemoglobin molecules at 405 nm.