Infrared Laser Excitation Research Articles

Two-Photon Multicolor FISH: A Versatile Technique to Detect Specific Sequences with in Single DNA Molecules in Cells and Tissues

Two-photon multicolor fluorescence in situ hybridisation (FISH) is presented as an advanced single molecule detection technique. Based on a two-photon excitation process it enables pinhole-free 3D laser scanning microscopy. Nonresonant two-photon absorption in a sub-femtoliter excitation volume was realized with a femtosecond laser scanning microscope equipped with a high numerical aperture objective. Nonlinear near infrared laser excitation and multicolor FISH has been used to image a variety of specific DNA regions. This innovative single molecule technique was applied to visualize genomic regions in fiber-DNA, human metaphase chromosomes, interphase nuclei and histological sections. Intense 170 fs laser pulses at 800 nm and GW/cm2 intensities have been employed to induce visible fluorescence of a variety of FISH fluorophores coupled to DNA probes. In particular, two-photon excited FITC-labeled 40 kb probes and multicolor labeled centromeric probes that bind to repetitive sequences of 0.340-2.000 kb have been used to visualize subtelomeric and different centromeric regions in metaphase chromosome spreads. Spectrum Orange and Spectrum Green labeled bcr and abl gene probes that target a stretch of 300 kb and 650 kb, respectively, have been imaged in a single fiber-DNA molecule with an estimated number of fluorescent molecules of 75 - 225 μm-1. Using the advantages of pinhole-free optical sectioning with submicron spatial resolution and multi-fluorophore single-wavelength-excitation, 3D images of multiple labeled centromeric regions of amniotic fluid cells in interphase have been obtained and used in diagnosis of trisomy 18. In addition, first 3D two-photon studies on centromer distribution in human kidney biopsies by labeling chromosomes X, Y and 4 with the FISH-fluorophores Spectrum Blue, Spectrum Green and Spectrum Orange have been performed. As demonstrated, two-photon Multicolor FISH has the potential to perform Multi-Gene-Imaging with high spatial resolution also in turbid tissue layers.

Laser spectroscopy ofNd3+ions inGeO2−PbO−Bi2O3glasses

The optical properties of ${\mathrm{Nd}}^{3+}$ ions in six different compositions of glasses based on ${\mathrm{GeO}}_{2},$ PbO, and ${\mathrm{Bi}}_{2}{\mathrm{O}}_{3}$ have been investigated by using steady-state and time-resolved laser spectroscopy. Judd-Ofelt parameters were derived from the absorption spectra and used to calculate the ${}^{4}{\stackrel{\ensuremath{\rightarrow}}{{F}_{3/2}}}^{4}{I}_{11/2}$ stimulated emission cross section and the ${}^{4}{F}_{3/2}$ radiative lifetime. Site-selective spectroscopy and time resolved fluorescence line narrowing experiments were performed in the ${}^{4}{\stackrel{\ensuremath{\rightarrow}}{{F}_{3/2}}}^{4}{I}_{9/2}{,}^{4}{I}_{11/2}$ transitions. For all samples studied a line narrowing of the ${}^{4}{\stackrel{\ensuremath{\rightarrow}}{{F}_{3/2}}}^{4}{I}_{11/2}$ emission has been observed when exciting at the long wave side of the ${}^{4}{\stackrel{\ensuremath{\rightarrow}}{{I}_{9/2}}}^{4}{F}_{3/2}$ transition. The lifetimes of the ${}^{4}{F}_{3/2}$ state do not show a monotonic variation with the excitation wavelength indicating large site-to-site variations in the local crystal field. Spectral migration of excitation among the ${\mathrm{Nd}}^{3+}$ ions has been studied from the time evolution of the ${}^{4}{\stackrel{\ensuremath{\rightarrow}}{{F}_{3/2}}}^{4}{I}_{9/2}$ spectra under resonant excitation. The results can be interpreted in terms of a dipole-dipole energy transfer mechanism. Visible upconversion has been observed in these glasses under infrared laser excitation.

Vibrational relaxation of NO(υ=1) by oxygen atoms

The rate constant kO(υ=1) for NO(υ=1) vibrational relaxation by O has been measured at room temperature using a laser photolysis-laser probe technique. Vibrationally excited NO and relaxer O atoms were formed using 355 nm laser photolysis of a dilute mixture of NO2 in argon bath gas. The time evolution of both the NO(υ=1) and the O atoms was monitored using laser-induced fluorescence (LIF). The required absolute O-atom densities were obtained through a comparison of O-atom LIF signals from the photolysis source and from a titrated cw microwave source. At early times the O atoms constitute the most important loss mechanism for the nascently produced NO(υ=1). Possible effects from NO(υ=1) vibrational ladder-climbing and from thermal expansion have been shown to be minimal. The rate constant kO(υ=1)=(2.4±0.5)×10−11 cm3 s−1 determined herein is a factor of 2 to 3 lower than the generally accepted value of kO(υ=1) used in thermospheric modeling. The present value for kO(υ=1) is the same, within the error bars, as the kO(υ=2,3) previously measured in this laboratory using an entirely different technique, resonant infrared laser excitation of NO(υ=0). This result suggests that the collisional relaxation rates are independent of υ. A recent quasiclassical trajectory calculation, in which both allowed NO–O surfaces have been explicitly considered, predicts a collisional relaxation rate which is in good agreement with the present result. The kO(υ=1) value, along with previously measured rate constants for NO–O high-pressure recombination (krec∞) and isotope exchange (kiso), can serve as a proxy for the rate coefficient kC describing the formation of a long-lived NO2* intermediate from O+NO collisions. The present value for kO(υ=1) is significantly lower, however, than a recent determination of krec∞ and also the value of kC derived from kiso. In the latter case the comparison is not as straightforward.

Eigenstate-Resolved Studies of Gas-Surface Reactivity:CH4(ν3) Dissociation on Ni(100)

A new experimental technique uses state-resolved infrared laser excitation to probe a polyatomic molecule's dissociative chemisorption dynamics with previously unattainable detail. Methane molecules excited to $v\phantom{\rule{0ex}{0ex}}=\phantom{\rule{0ex}{0ex}}1$ of the ${\ensuremath{\nu}}_{3}$ C-H stretching vibration are up to 1600 times more reactive on a clean Ni(100) surface than are molecules in the ground vibrational state. Over a translational energy range of 27 to 54 kJ/mol, their absolute reaction probability increases from $3\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}4}$ to $6\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}3}$, which indicates that ${\ensuremath{\nu}}_{3}$ is responsible only in part for the vibrational activation reported in previous studies.

Infrared Depletion Spectroscopy of the Aniline Dimer

The infrared depletion spectrum of the aniline dimer, formed in a supersonic jet, has been recorded in the N−H stretch region by combining infrared laser excitation and resonant two-photon ionization/time-of-flight mass spectrometry. Only two bands have been found at 3394.0 and 3465.9 cm-1 (±0.5 cm-1) in the region 3130−3530 cm-1. These are red-shifted by 27.8 and 42.3 cm-1 from the symmetric and asymmetric NH stretching vibrations of the aniline monomer, respectively. A configuration with mutual NH2···π bonds and phenyl groups stacked in parallel is suggested for the ground-state aniline dimer.

Reaction of Cl with vibrationally excited CH4 and CHD3: State-to-state differential cross sections and steric effects for the HCl product

The mechanism for the reaction of atomic chlorine with vibrationally excited methane is investigated by measurement of correlated state and scattering distributions using the method of core extraction (see preceding paper). Laser photolysis of molecular chlorine creates monoenergetic chlorine atoms (≳98% Cl 2P3/2) that react with vibrationally excited methane molecules prepared by linearly polarized infrared laser excitation. The resulting HCl product population distributions are determined by (2+1) resonance-enhanced multiphoton ionization (REMPI), and the differential cross section for each product rovibrational state is measured by core extraction. Approximately 30% of the product is formed in HCl(υ=1,J) with a cold rotational distribution; the remaining population is formed in HCl(υ=0,J) and is more rotationally excited. We observe a rich variation of the scattered flux that is dependent on the internal-energy state of the product. The HCl(υ=1) product is sharply forward scattered for low J and becomes nearly equally forward–backward scattered for high J; the HCl(υ=0,J) product is back and side scattered. The reactions of Cl with C–H stretch-excited methane (CH4) and C–H stretch-excited CHD3 are found to have similar angular and internal-state distributions. Observation of the spatial anisotropy of the HCl(υ=0, J=3) product shows that significant vibrational excitation of the methyl fragment does not occur. The measured spatial anisotropy is most consistent with a model in which backscattered HCl(υ=0, J=3) is formed in coincidence with slight methyl vibrational excitation and the forward-scattered HCl(υ=0, J=3) is formed in coincidence with no methyl excitation. The approach of the attacking chlorine atom with respect to the C–H stretch direction can be varied by rotating the plane of polarization of the infrared excitation. A marked steric effect is observed in which Cl atoms approaching perpendicular to the C–H stretch preferentially yield forward-scattered HCl(υ=1) product. On the other hand, the reaction is weakly dependent on the rotational quantum state of CH4(υ3=1,J), and on the rotational polarization. The data are consistent with a model that has a widely open ‘‘cone of acceptance’’ in which the impact parameter controls the internal-state and scattering distributions of the HCl product.

Infrared laser excitation of gas phase molecules prior to field ionization

Infrared multiphoton absorption is introduced as a method of direct heating of gas phase molecules prior to field ionization. This approach appears to be a very effective way to increase fragmentation following field ionization of the vibrationally excited molecules. Comparison is made with the method of electrical heating of the emitter which results in an indirect heating of the molecules prior to field ionization.

Non linear effects in monomode ytterbium doped silica fibers : cooperative luminescence

The visible cooperative luminescence in Ytterbium-doped optical fibres was observed using a titanium-sapphire infrared laser excitation (≡900nm). The evolution of this luminescence as a function of absorbed power is discussed. The optical confinement therefore enables us to observe new saturation effects which are studied for two different ytterbium fibres

Rotational relaxation in a free expansion of HCl

The rotational relaxation of HCl in a free jet expansion was studied in a pump-and-probe experiment using infrared laser excitation and resonant multiphoton ionization detection. Rate constants were determined for various J to J′ energy transfer processes in the vibrationally excited molecule. There is a strong indication that the rotational energy transfer occurs via a near-resonant dipole–dipole interaction between vibrationally excited and vibrationally cold HCl molecules.

Fourier Transform Raman spectroscopy — a tool for inorganic, organometallic and solid state chemists?

The use of Fourier Transform Raman spectroscopy with near infrared laser excitation for studying inorganic and organometallic compounds is reviewed with the aim of encouraging the use of this developing technique. A range of compounds to which the method has been applied are considered. These are: (1) oxoanions containing transition metal atoms; (2) co-ordination compounds; (3) compounds of bio-inorganic interest; (4) σ- and π-bonded organometallic species; (5) inorganic materials; and (6) matrix-isolated species. Shortcomings of the technique, and possible ways in which these may be overcome, and a selection of potential systems to which the method could be extended are included.

Chemisorption on the SiO 2 and silicon surfaces and the influence of infrared laser excitation

Binding energies and vibrational frequencies of adsorbate-substrate clusters modelling the chemisorption of F, CF 3, and CF 2 on SiO 2 and silicon surfaces have been calculated using the quantum chemical MNDO method. The calculated data are discussed with respect to the mechanism of infrared laser-induced etching and with respect to the choice of the etch gas molecules and the laser frequencies for selective etching of SiO 2 and Si.

Vibrational predesorption of carbon monoxide from sodium chloride at 20 K induced by resonant infrared laser excitation

The linear vibrational absorption spectrum, the laser infrared absorption spectrum and the laser-induced desorption spectrum of carbon monoxide 13C 16O adsorbed on sodium chloride at 20 K were observed and found to coincide, the linewidth being 11±3 cm -1, the peak wavenumber 2107±2 cm -1. The energy of the fundamental internal vibration of adsorbed CO is greater than the adsorption energy. The resonant laser-induced process detected is described as a vibrational predesorption.

Vibrational predesorption of carbon monoxide from sodium chloride at 20 K induced by resonant infrared laser excitation

Abstract The linear vibrational absorption spectrum, the laser infrared absorption spectrum and the laser-induced desorption spectrum of carbon monoxide 13 C 16 O adsorbed on sodium chloride at 20 K were observed and found to coincide, the linewidth being 11±3 cm -1 , the peak wavenumber 2107±2 cm -1 . The energy of the fundamental internal vibration of adsorbed CO is greater than the adsorption energy. The resonant laser-induced process detected is described as a vibrational predesorption.

Infrared-laser excitation of the internal vibrational mode of a diatomic molecule adsorbed on a metal surface.

The infrared-laser excitation of the internal vibrational mode of a diatomic molecule adsorbed on a metal surface is analyzed theoretically. This vibrational energy is damped into the metal by electron-hole excitations. Simple expressions for the populations of the vibrational levels, the mean number of vibrational quanta, and the rate of energy transfer between the infrared laser and the metal surface in the steady state are derived. An equation of evolution can readily be solved numerically to determine the time necessary to reach this steady state. The criteria of applicability of the Markov approximation (which leads to the golden rule) is clearly established, where it is seen that this approximation may not be used to compute the evolution of the populations of the vibrational levels. The random-phase approximation is shown to give the correct kinetic equation for the populations of the vibrational levels. The excitation of carbon monoxide adsorbed on a copper surface is analyzed quantitatively.

Nearly thermalized systems under continuous infrared laser excitation: Expected and unexpected effects

Nearly thermalized systems under continuous infrared laser excitation: Expected and unexpected effects

Angular, velocity, rotational, and electronic distributions of vibrationally elastically scattered NO(v=1) from LiF(100)

Infrared laser excitation has been utilized to excite part of a molecular beam of NO to a single well-defined quantum state, NO(v=1, J=3/2, Ω=1/2), which is scattered from a cleaved LiF(100) surface. Laser spectroscopic detection techniques then allow the determination of rotational and electronic distributions as well as state-specific angular and velocity distributions for scattering from a single initial vibrational-rotational state.

Photoacoustic Detection of Phase Transitions in 4-Octyl-4′-Cyanobiphenyl

Abstract Phase transitions in a Ihermotropic liquid crystal are investigated by means of photoacoustic detection with an infrared laser excitation. With samples as small as 0.2 mg the technique is shown to be sensitive for the analysis of second order or weak first order phase transitions.

Spectral characteristics of the excitation of SF6molecules by an intense infrared laser field under strong cooling conditions

A study was made of the fine structure of the excitation of the lower vibrational levels in SF6 molecules gasdynamically cooled to temperatures of the order of 30 K. A two-frequency infrared–infrared laser excitation method was employed. A continuously tunable CO2 laser was used for the first stage. At a frequency of 944.39 cm−1 a contrast absorption peak was detected, having a width of less than 0.1 cm−1 and corresponding to the A1g two-photon resonance.

Resonance, Rate, and Quantum Yield of Infrared-Laser-Induced Desorption by Multiquantum Vibrational Excitation of the Adsorbate CH3F on NaCl

The linear infrared absorption spectra of C${\mathrm{H}}_{3}$F gas, multilayer film, and C${\mathrm{H}}_{3}$F adsorbate on NaCl crystal surfaces have been found to be distinctly different and significantly coverage dependent. On resonant infrared-laser excitation of the ${\ensuremath{\nu}}_{3}$ internal vibration of the adsorbate C${\mathrm{H}}_{3}$F on NaCl, molecular desorption of C${\mathrm{H}}_{3}$F occurs. Measurement of the resonance, rate coefficient, and quantum yield has revealed high spectral selectivity and efficiency of the desorption due to multiphoton ($n>2$) adsorbate vibrational excitation.

Time of flight spectroscopy of particles in electronically excited state, produced via infrared laser excitation

A method for determination of the translational energy of the fragments in long living electronic states obtained under ir multiple-photon dissociation of the molecules is proposed. The measurement of the translational distribution function and of the average translational energy of the particles appeared under MPD of OsO4 has been carried out by this method. It has been shown that the proposed technique may be usefull for identification of visible radiation as belonging to the fragment or to the parent molecule itself.