Tracking of fluorescent molecules diffusing within membranes

A new method is proposed for tracking fluorescing single molecules diffusing within a two-dimensional membrane. It is based on a confocal microscopy setup with a constantly rotating laser focus, which follows the position of the molecule. The optimization and efficiency of the method are theoretically studied for a broad range of experimentally realistic conditions. The proposed method allows for a long-time observation of diffusing molecules while allowing the application of fast spectroscopic techniques such as fluorescence decay time determination or fluorescence anisotropy measurements. Received: 14 January 2000 / Published online: 13 September 2000     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

SPDM: light microscopy with single-molecule resolution at?the?nanoscale

Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the ‘point-like’ components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling (‘spectral signatures’). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th–1/10th of the exciting wavelength.     

Anisotropic properties of carbonaceous material produced in arc discharge

A helium-pressure influence on the structure and properties of carbonaceous material from the inner part of a deposit forming in an arc discharge has been studied. At the helium pressure of about 150 Torr and higher, the material was found to have a layered texture, which is perpendicular to the deposit growth axis. Electron microscopy measurements showed that a sample produced in He gas of 800 Torr consists of ∼80% carbon nanotubes having predominant orientation in the layers. Electric conductivity along and perpendicular to the samples’ surface have been measured from 4.2 to 500 K. The samples synthesised at higher helium pressures have significant anisotropy of conductivity, increasing with the carbon nanotubes content rise. X-ray fluorescence spectra represented the angle dependence that is caused by the anisotropy of chemical bonding in the carbon nanotubes. The valence π- and σ-bands for the carbonaceous material were separated. Received: 26 November 1999 / Accepted: 20 April 2000 / Published online: 10 January 2001     

Optoacoustic sensor head for depth profiling

-1 (for 0.25 mJ/cm²). The response function of the total experimental setup is derived from the optoacoustic signal of a black absorber, which is necessary to correct theoretical calculations with regard to the finite time response of the detection system. In the case of homogeneous samples, the theory of optoacoustic signal generation is verified, qualitative results are achieved with layered samples. Received: 14 October 1998 / Published online: 24 February 1999     

Thermal effect on multiphonon-assisted anti-Stokes excited upconversion fluorescence emission in Yb3+-sensitized Er3+-doped optical fiber

The temperature effect upon infrared-to-visible frequency upconversion fluorescence emission in Yb³⁺-sensitized Er³⁺-doped germanosilicate optical fibers excited with cw radiation at 1.064 μm is investigated. The experimental results revealed an eightfold enhancement in the visible upconversion emission intensity as the fiber temperature was increased from 17 °C to 180 °C. The fluorescence emission enhancement is attributed to the temperature-dependent multiphonon-assisted anti-Stokes excitation process of the ytterbium sensitizer. A theoretical approach that takes into account a sensitizer absorption cross-section, which depends on the phonon occupation number, has proven to agree very well with the experimental data Received: 6 April 1999 / Revised version: 27 August 1999 / Published online: 27 January 2000     

Photochemical effects in the UV laser ablation of polymers: Implications for laser restoration of painted artworks

0 ∝t^1/2. The best results are expected for a circular top-hat beam shape. Received: 15 January 1999 / Accepted: 18 January 1999     

Phases of wave functions and level repulsion

Avoided level crossings are associated with exceptional points which are the singularities of the spectrum and eigenfunctions, when considered as functions of a complex coupling parameter. It is shown that the wave function of one state changes sign but not the other, if the exceptional point is encircled in the complex plane. An experimental setup is suggested where this peculiar phase change could be observed. Received: 7 January 1999 / Received in final form: 15 March 1999     

Optical and waveguiding properties of Nd:KGW films grown by pulsed laser deposition

 Nd: KGd(WO₄)₂ thin films were deposited by KrF laser ablation on MgO, YAP, YAG and Si substrates at temperatures up to 800 °C. Film crystallinity, morphology, stoichiometry (WDX, RBS and PIXE), excitation spectra, fluorescence, refractive index and waveguiding properties were studied in connection with deposition conditions. The best films were crystalline and exhibited losses of approximately 5 dB cm^-1 at a wavelength of 633 nm. Received: 8 January 2001 / Accepted: 7 November 2001 / Published online: 11 February 2002     

Spectroscopic detection of biological NO with a quantum cascade laser

Two configurations of a continuous wave quantum cascade distributed feedback laser-based gas sensor for the detection of NO at a parts per billion (ppb) concentration level, typical of biomedical applications, have been investigated. The laser was operated at liquid nitrogen temperature near λ=5.2 μm. In the first configuration, a 100 m optical path length multi-pass cell was employed to enhance the NO absorption. In the second configuration, a technique based on cavity-enhanced spectroscopy (CES) was utilized, with an effective path length of 670 m. Both sensors enabled simultaneous analysis of NO and CO₂ concentrations in exhaled air. The minimum detectable NO concentration was found to be 3 ppb with a multi-pass cell and 16 ppb when using CES. The two techniques are compared, and potential future developments are discussed. Received: 1 November 2000 / Revised version: 19 January 2001 / Published online: 20 April 2001     

Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting

We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%. Received: 31 March 2000 / Revised version: 31 May 2000 / Published online: 13 September 2000     

Characterization of particulates accompanying laser ablation of pressed polytetrafluorethylene (PTFE) targets

We present observations of sub-micron- to micron-sized particles generated by high fluence (≈2 J/cm²) 248-nm laser ablation of pressed polytetrafluorethylene (PTFE) targets in air at atmospheric pressure. The original target material was hydrostatically compressed ≈7 μm PTFE powder, sintered at 275 °C. Collected ejecta due to laser irradiation consists of four basic particle morphologies ranging from small particles 50–200 nm in diameter to larger particles ≈10 μm in diameter. Many particles formed in air carry electric charge. Using charged electrodes we are able to collect charged particles to determine relative numbers of ± charge. We observe roughly equal numbers of positively and negatively charged particles except for the largest particles which were predominantly negative. For a range of particle sizes we are able to measure the sign and magnitude of this charge with a Millikan-oil-drop technique and determine surface charge densities. The implications of these observations with respect to pulsed laser deposition of PTFE thin films and coatings are discussed. Received: 15 January 1999 / Accepted: 18 January 1999 / Published online: 7 April 1999     

Microstructure evolution in Pr-Co-C-Ti nanophase magnets

The microstructures of nanophase Pr-Co-C-(Ti) materials, which have improved magnetic properties, were investigated by means of transmission electron microscopy (TEM) to reveal their phase assemblage and grain-boundary structure. The phase assemblage was carefully controlled by the introduction of TiC nanoparticles and annealing. The optimal nanostructure contained uniformly distributed PrCo₅ and PrCo₂ nanophases without any magnetically soft phases, resulting in high coercivity and the characteristics of a single, hard magnetic phase. TEM analysis confirmed the presence of an amorphous grain-boundary phase surrounding the grains in alloys without TiC. In contrast, alloys with added TiC showed no amorphous phase and also showed higher coercivity compared to Co-Pr-C. Therefore, the variation of the grain boundary phases may be effective in changing the degree of exchange coupling. Controlling the formation of a uniform nanoscale microstructure, leading to improved magnetic properties, is discussed. Received: 5 September 2002 / Accepted: 10 September 2002 / Published online: 22 January 2003 RID="*" ID="*"Corresponding author. Fax: +1-630/252-7777, E-mail: ytang@anl.gov     

Dipole traps with mode-locked lasers

We investigate the properties of two separate dipole traps, realised using a cw mode-locked Ti:sapphire laser and a cw mode-locked Nd:YVO₄ laser, red-detuned by 25 nm and 284 nm, respectively. Approximately 10³ laser-cooled ⁸⁵Rb atoms were confined in the traps at ≈50 μK, with no observable heating after initial loading. The lifetimes of the traps were consistent with limitations imposed by wavelength-dependent photoassociation losses and collisions with background vapour. Determination of the ac Stark shift of the 780 nm cooling transition using a weak probe beam showed no observable difference between using narrow-bandwidth or mode-locked trapping light. Techniques for trapping and focusing of atoms based on the dipole force of blue and uv light become much more accessible through efficient doubling, tripling and quadrupling of mode-locked sources. This opens up the possibility of manipulating more technologically interesting species. Received: 30 September 1999 / Revised version: 21 December 1999 / Published online: 24 March 2000     

Photophysical properties of a crown ether-bearing [60]fulleropyrrolidine

The photophysical properties of the first crown ether-bearing [60]fulleropyrrolidine (CFP) have been studied. The absorption and fluorescence spectra are hypsochromically shifted by metal ions complexation via intramolecular electron transfer which is confirmed by AMI computation. The third-order optical nonlinearity of CFP is measured by time-resolved degenerate four-wave-mixing experiment. The third-order optical susceptibility χ⁽³⁾ of CFP is 8.97×10⁻¹⁴ esu. The nonlinear optical temporal response is second-order exponential, the fast process is determined as 120 ps. Received: 24 March 1999 / Revised version: 11 May 1999 / Published online: 25 August 1999     

Holographic and photoelectric characterization of a novel photorefractive organic glass

We present a detailed study of the photoelectric as well as the holographic properties of a novel organic photorefractive glass based on triphenylamine. We studied the quantum efficiency Φ of the photogeneration of charges by means of photoinduced discharge measurements. The photoconductivity σ and the charge carrier mobility μ were obtained via dc photoconduction and pulsed time-of-flight experiments, respectively. The holographic characterization was performed by two-wave and degenerate four-wave mixing experiments allowing for the determination of properties such as diffraction efficiency η, modulation of the refractive index Δn, gain coefficient Γ, and phase-shift φ_p of the investigated system. The experimental data for Φ could be successfully described by the Onsager formalism with a thermalization radius of r₀=24 ? and a primary quantum yield of Φ₀=40%. We evaluated the E field and temperature-dependent measurements of μ using the B?ssler formalism yielding a width of the density of states of σ=0.13 eV and a disorder parameter Σ=3.6. On this basis the lifetime and the average drift length of the charge carriers could be estimated from the dc photoconduction experiments. From the photoelectric measurements we also calculated the holographic response time that matched very well to the measured response time and described the E-field dependence satisfactorily. The presented photorefractive system shows outstanding optical properties and stability with respect to degradation. We measured a gain coefficient of Γ=90 cm^-1, and a diffraction efficiency of η=27% at a response time of 30 ms for only 40-μm-thick samples. Orientational enhancement was observed and evaluated quantitatively. To our knowledge, this work presents the first determination of each of the above quantities all in one single organic photorefractive material. Received: 16 November 1998 / Revised version: 4 January 1999 / Published online: 31 March 1999     

Generation of 10 to 50 fs pulses tunable through all of the visible and the NIR

Noncollinearly phase-matched optical parametric amplifiers (NOPAs) pumped by the blue light of a frequency-doubled Ti:sapphire regenerative amplifier are a convenient source of continuously tunable ultrashort pulses in the visible and near infrared for spectroscopic experiments. We present the underlying principles, report recent improvements and describe the experiences gained from the routine use of a number of NOPAs in our laboratories. We find that the setup can easily be optimized for the given experimental requirements. Typical output-pulse energies in the visible are 5 to 10 μJ and a few μJ in the NIR from 200 μJ regenerative-amplifier pulses at 800 nm. From 460 to 700 nm, pulse lengths between 10 and 20 fs are routinely achieved, while the length increases monotonically from about 20 fs at 900 nm to just below 50 fs at 1600 nm. In all cases this corresponds to a dramatic shortening compared to the length of the pump pulses of around 100 fs. First results show that the 700 to 900 nm region can be accessed with sub-50-fs pulse lengths by use of an intermediate white-light generator in a two-stage setup. Received: 29 November 1999 / Published online: 5 July 2000     

Planar laser-induced fluorescence imaging of carbon monoxide using vibrational (infrared) transitions

We report a new imaging diagnostic suitable for measurements of infrared-active molecules, namely infrared planar laser-induced fluorescence (IR PLIF), in which a tunable infrared source is used to excite vibrational transitions in molecules and vibrational fluorescence is collected by an infrared camera. A nanosecond-pulse Nd:YAG-pumped KTP/KTA OPO/OPA system is used to generate 12 mJ of tunable output near 2.35 μm which excites the 2ν band of carbon monoxide (CO); fluorescence resulting from excited CO is collected at 4.7 μm by using an InSb focal plane array. Quantitative, high-SNR PLIF imaging of gas-phase CO is demonstrated at a 10-Hz acquisition rate with a minimum detection limit of 1350 ppm at 300 K. Received: 30 July 1999 / Published online: 16 September 1999     

Iron contamination in silicon technology

This article continues the review of fundamental physical properties of iron and its complexes in silicon (Appl. Phys. A 69, 13 (1999)), and is focused on ongoing applied research of iron in silicon technology. The first section of this article presents an analysis of the effect of iron on devices, including integrated circuits, power devices, and solar cells. Then, sources of unintentional iron contamination and reaction paths of iron during device manufacturing are discussed. Experimental techniques to measure trace contamination levels of iron in silicon, such as minority carrier lifetime techniques (SPV, μ-PCD, and ELYMAT), deep-level transient spectroscopy (DLTS), total X-ray fluorescence (TXRF) and vapor-phase decomposition TXRF (VPD-TXRF), atomic absorption spectroscopy (AAS), mass spectrometry and its modifications (SIMS, SNMS, ICP-MS), and neutron activation analysis (NAA) are reviewed in the second section of the article. Prospective analytical tools, such as heavy-ion backscattering spectroscopy (HIBS) and synchrotron-based X-ray microprobe techniques (XPS, XANES, XRF) are briefly discussed. The third section includes a discussion of the present achievements and challenges of the electrochemistry and physics of cleaning of silicon wafers, with an emphasis on removal of iron contamination from the wafers. Finally, the techniques for gettering of iron are presented. Received: 16 November 1999 / Accepted: 7 January 2000 / Published online: 5 April 2000     

Comparison of laser-induced and planar laser-induced fluorescence measurements of nitric oxide in a high-pressure, swirl-stabilized, spray flame

We report spatially resolved linear laser-induced fluorescence (LIF) and planar laser-induced fluorescence (PLIF) measurements of nitric oxide (NO) in a pre-heated, high-pressure (4.27 atm), lean direct-injection (LDI) spray flame. The feasibility of using PLIF in lieu of LIF is assessed with respect to measuring NO concentrations in high-pressure LDI spray flames. NO is excited via the resonant Q₂(26.5) transition of the γ(0,0) band while a non-resonant wavelength is employed to subtract background interferences. LIF detection is performed in a 2-nm region centered on the γ(0,1) band. PLIF detection is performed in a 68-nm window that captures fluorescence from several vibrational bands. An in situ NO doping scheme for fluorescence calibration is successfully employed to quantify the LIF signals. However, a similar calibration scheme for the reduction of PLIF images to quantitative field measurements is plagued by the laser-excited background. Excitation scans and calibration comparisons have been performed to assess the background contribution for PLIF detection. Quantitative radial NO profiles measured by LIF are presented and analyzed so as to correct the PLIF measurements to within the accuracy bars of the LIF measurements via a single-point scaling of the PLIF image. Received: 23 November 1999 / Revised version: 17 January 2000 / Published online: 27 April 2000