Laser Excitation Source Research Articles

Raman spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich-type phthalocyaninato and porphyrinato rare earth complexes: Part 4. Raman spectroscopic characteristics of naphthalocyanine in mixed (octaethylporphyrinato)(naphthalocyaninato) rare earth double-deckers

Raman spectroscopic data in the range of 400–1800 cm −1 for a series of 15 mixed (octaethylporphyrinato)(naphthalocyaninato) double-decker complexes with tervalent rare earths M III(OEP)(2,3-Nc) (M=Y, La, …, Lu except Ce and Pm) and intermediate valent cerium Ce(OEP)(2,3-Nc) have been collected using laser excitation sources emitting at 632.8 and 785 nm. Comparison with the Raman spectra of the corresponding bis(naphthalocyaninato) rare earths reveals that the Raman characteristics of mixed ring double-deckers M(OEP)(2,3-Nc) are dominated by the metallonaphthalocyanine M(2,3-Nc) fragment. Under excitation at 632.8 nm, which is far away from the main Q absorption band of the naphthalocyanine ligand, the most intense Raman band appears at 1598 cm −1 for M III(OEP)(2,3-Nc) (M≠Ce), and is assigned to the naphthalene stretching. On the other hand, for Ce(OEP)(2,3-Nc), this vibration appears as a strongly enhanced peak at 1550 cm −1 together with a medium intense band at 1613 cm −1. Typical Raman marker bands of the monoanion radical Nc − were observed at 1499–1512 cm −1 as a medium band and at 1521−1538 cm −1 as a band whose intensity increases along with the rare earth contraction, resulting from the coupling of pyrrole CC and aza CN stretchings and aza CN-only stretchings, respectively. For Ce(OEP)(2,3-Nc), a weak and broad band centered at 1507 cm −1 with contribution from both pyrrole CC and aza CN stretches together with isoindole stretches was the marker Raman band of Nc 2−. When excited with laser radiation of 785 nm which is in close resonance with the main Q absorption band of naphthalocyanine ligand, the ring radial vibrations of the isoindole moieties at ca. 680 and 732 cm −1 in the Raman spectra of both M III(OEP)(2,3-Nc) and Ce(OEP)(2,3-Nc) are selectively intensified and appear as the most intense vibrations. The marker Raman bands of Nc − in M III(OEP)(2,3-Nc) and Nc 2− in Ce(OEP)(2,3-Nc) are observed in similar regions to those found for excitation at 632.8 nm. The scatterings with contribution from the Nc breathings, ring radial vibration, naphthalene stretchings, benzoisoindole stretchings, aza group stretchings, and the coupling of pyrrole CC and aza CN stretchings in M III(OEP)(2,3-Nc) all show dependence on the rare earth radius, shifting to higher energy along with the rare earth contraction and showing the rare earth ionic size effect on the Raman characteristics of naphthalocyanine in the mixed ring double-decker compounds.

Raman spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich-type phthalocyaninato and porphyrinato rare earth complexes.: Part 3. The effect of rare earth ionic size on the Raman characteristics of naphthalocyanine in bis(naphthalocyaninato) rare earth complexes

Raman spectroscopic data of a series of 13 sandwich double-decker bis(naphthalocyaninato) rare earth complexes M(2,3-Nc*) 2 [M=Ce⋯Lu except Nd and Pm, Y; Nc*=Nc( tBu) 4] were collected using laser excitation sources emitting at 632.8 and 785 nm and systematically compared. The Raman characteristics for substituted naphthalocyaninato monoradical anion Nc* − under these two laser excitations are thus comparatively described. The strongly enhanced Raman band at approximately 1592 cm −1, which is assigned to the naphthalene stretches of the Nc ring, is a typical feature of the monoanion radical, Nc* − under excitation with the 632.8 nm laser line. With laser excitation at 785 nm, which is nearly coincident with the Q band absorption of bis(naphthalocyaninato) rare earth complexes, the band at 1512–1530 cm −1 due to the CN (aza group) stretch is a good Raman marker band for the naphthalocyanine anion Nc* −. This band and the Nc breathings, naphthalene stretchings, isoindole stretchings, and coupling of pyrrole CC ring and aza CN stretches all shift to higher frequency with decreasing ionic radius (the rare earth contraction).

Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera.

CCD cameras coupled optically to gated image intensifiers have been used for fast time-resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the 'charge drain' function on an interline-transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.

Influence of light intensity on the photoluminescence of silicon nanostructures

The strong visible photoluminescence (PL) of nanostructured silicon, such as porous Silicon and silicon nanocrystals, is studied as a function of the power and the wavelength of the excitation laser source. The position of the PL peak is a function of the fluence: when the incident fluence is increased, the PL peak is blueshifted, and it is redshifted to its initial position when the fluence is decreased back. The PL yield is strongly attenuated with the increasing fluence and this decrease is partially irreversible. The behavior is also found to be a function of the wavelength of excitation: the shorter the excitation wavelength, the stronger the fluence effect. The PL temporal behavior has also been studied and appears to be weakly sensitive to the fluence. Fluence effects are compared to temperature effects and both are noticeably different, proving the absence of heating effects in our experiment for a wide range of incident power. Auger effect and state filling are discussed in order to understand the experimental results in the framework of the quantum confinement process.

Raman spectroscopy of polymer latexes

AbstractThe influence of elastic light scattering on the Raman signal of polymer latexes was investigated by varying the sample position relative to the confocal Raman probe. Measurements on polystyrene latexes revealed that the absolute Raman signal is very sensitive to the alignment of the sample. Furthermore, it was shown that for particle sizes below 140 nm and solid weight contents below 20%, changes in the absolute Raman signal can be described fairly well by turbidity laws. For larger particle sizes and higher solid weight contents deviations occur, but a correlation between these variables and the Raman signal still exists. In contrast, the normalized intensities are not influenced by varying the sample alignment in measurements on polymer latexes when using an excitation laser source at 532 nm. The important consequence of this is that the determination of chemical composition is not influenced by the elastic light scattering effects. Copyright © 2002 John Wiley & Sons, Ltd.

Coherent thermal wave imaging of subsurface chromophores in biological materials

Thermal wave imaging of discrete subsurface chromophores in biological materials is reported using a phase sensitive coherent detection technique applied to recorded infrared (IR) images. We demonstrate that utilization of a periodically modulated laser source for thermal wave excitation and coherent detection applied to each pixel may be used to compute images of thermal wave amplitude and phase at the laser modulation frequency. In comparison to recorded IR images, the narrow-band detection technique significantly improves the quality of thermal wave amplitude images of subsurface chromophores in biological materials. Additionally, the technique provides phase information, which may be used to estimate chromophore depth in tissue. Application of the technique is demonstrated using tissue phantoms and in vivo biological models. We present a theoretical analysis and computer simulations that demonstrate the effect of tissue optical and thermal properties on thermal wave amplitude and phase. In comparison to the pulsed photothermal technique, coherent thermal wave imaging of subsurface chromophores in tissue for diagnostic applications allows reduction of peak incident laser fluence by as much as four orders of magnitude and is safer and more amenable to in vivo imaging.

Fibre-Optic Hand Held Probe For Opto-Thermal In-vivo Skin Measurements

The potential of using opto-thermal radiometry for skin measurements is well known, but the current bench-top apparatus we use is not suitable for routine use and restricts in-vivo measurements to fingers and forearms. To overcome these shortcomings, we are developing a fibre optic opto-thermal hand held probe, designed for non-invasive, in vivo measurements on any skin site of human body. It uses one fibre (hollow waveguide for Q-switched Er:YAG and glass fibre for Q-switched Nd:YAG laser respectively) to deliver the excitation radiation to the sample surface and another fibre (silver halide) to collect the infrared radiation and send it to the infrared detector. We present details of the design of this probe and the experimental results using it on various skin sites, with different laser excitation sources.

Intracellular coupling of adhesion receptors: Molecular proximity measurements

Publisher Summary This chapter describes frequency-domain fluorescent lifetime imaging microscopy (FLIM), which utilizes a sinusoidally modulated laser excitation source to excite the donor fluorophore. A frequency domain FLIM has a number of components. There are two main methods for measuring fluorescence lifetime, namely—time-domain and frequency-domain-based fluorescence lifetime sensing. The principles of frequency-domain-based fluorescence lifetime sensing, which is based on monitoring the extent of demodulation and phase shift between a sinusoidally modulated excitation light and the donor emission consequent on its excitation, are explained in this chapter. In time-domain FLIM, the sample is excited with a short pulse, and the subsequent fluorescence is integrated into separate time windows. The fluorescence lifetime is then calculated by fitting an exponential model to the measured intensities. This chapter focuses on the frequency-domain-based method, using a conventional inverted microscope configuration that employs phase-sensitive detection of fluorescence emission.

Raman spectroscopy studies on M 2RuH 6 where M=Ca, Sr and Eu

Characterization of ternary and quaternary metal hydrides by Raman spectroscopy appears to be rather scarce due primarily to the decomposition of the metal hydrides by the energy of the laser excitation source. We report the results of some recent room temperature Raman measurements collected with a 2–10 mW 488 nm laser source for M 2RuH 6 where M=Ca, Sr and Eu. The assignments from this study are combined with existing vibrational data for other metal hydrides.

Fiber-Optic System for 77 K Phosphorescence Lifetime Measurements of Polycylic Aromatic Compounds in Shpol'skii Matrices

A fiber-optic system is reported for rapid, accurate, and precise 77 K measurements of relatively long phosphorescence lifetimes ( τp = 200 ms). The instrument employs stimulated Raman scattering in connection with a pulsed laser source for sample excitation. Phosphorescence decay waveforms are collected at maximum emission wavelengths with a single channel detection system and the aid of a LabVIEW based in-house program acting as a data acquisition instrument, data storage device, and data interpreter. By means of program looping, up to 20 decays are recorded from a frozen sample in less than 5 min. A cryogenic fiber-optic probe directly frozen into the sample matrix removes the random errors associated with the classic sample freezing procedure and provides excellent reproducibility of measurements. The relative standard deviations of τp measured from several polycyclic aromatic compounds varied between 0 and 4.30%. The simplicity of the experimental procedure, the rather large τ p differences observed from compounds within the same pollutant class, and the analytical figures of merit provide a solid foundation for pursuing low-temperature phosphorescence, time-resolved low-temperature phosphorescence, and low-temperature multidimensional luminescence analysis.

Development of a Mechanism-Based, DNA Staining Protocol Using SYTOX Orange Nucleic Acid Stain and DNA Fragment Sizing Flow Cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA (∼450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.

Ultraviolet Resonance Raman Spectroscopy of Bulk and Microscopic Human Colon Tissue

A sensitive ultraviolet resonance Raman (UVRR) system, which integrates bulk and microscopic capabilities, is described for use with human tissue. The system incorporates a quasi-continuous tunable laser excitation source in the range 200–300 nm, a liquid nitrogen-cooled charge-coupled device (CCD), a single grating spectrometer, dielectric longpass laser line rejection filters, and cooled specimen holders to minimize bio- and photo-degradation. The system was used on normal human colon tissue, and was compared to our previously used Nd:YAG-based system. Spectra with higher signal-to-noise ratio (S/N) than has previously been achieved, and with less than 5% photobleaching, are presented. The present system outperforms our previous system by improving the S/N in bulk normal colon spectra by a factor of greater than 10. We show for the first time UVRR spectra collected from the various microscopic regions of human colon in a thin section preparation, and the line shapes from the different microscopic morphological components are related to the bulk colon spectra. At 251 nm the epithelial cells and the lamina propria are the major contributors to the UVRR spectrum of bulk colon tissue. Our data suggest that combined bulk and microscopic UVRR spectroscopy of human tissue could provide a valuable tool for bioassay and histochemistry.

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge-coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single-stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

Time-Resolved Resonance Raman Spectroscopy at Low Temperature. The Excited-State Metal-Metal Stretching Frequency of Rh(2)(TMB)(4)(2+) (TMB = 2,5-Dimethyl-2,5-diisocyanohexane).

The time-resolved resonance Raman spectrum of the short-lived triplet (dsigmapsigma) excited state of Rh(2)(TMB)(4)(2+) (TMB = 2,5-dimethyl-2,5-diisocyanohexane) was obtained by lowering the temperature of a 3:1 ethanol/methanol solution until the excited-state lifetime became much greater than the width of the pulsed laser excitation source. The metal-metal stretching frequency is 151 cm(-)(1) in the excited triplet state, as compared to 50 cm(-)(1) in the ground state. The diatomic harmonic force constants derived from these frequencies are in a 9.12:1 ratio (excited state/ground state), consistent with the simple molecular orbital description that predicts that the Rh-Rh bond order is greater in the excited state than in the ground state. A comparison of Rh(2)(TMB)(4)(2+) and Rh(2)b(4)(2+) (b = 1,3-diisocyanopropane) Raman data indicates that the nature of the bridging ligand considerably affects the ground- and excited-state metal-metal stretching frequencies and that the population of the psigma orbital may have very little effect on the bonding in the excited triplet state.

Below-Gap Spectroscopy of Undoped GaAs/AlGaAs Quantum Wells by Two-Wavelength Excited Photoluminescence

By using an improved method of two-wavelength excited photoluminescence, we observed a considerable decrease in the band-to-band photoluminescence intensity of an undoped GaAs/AlGaAs quantum well structure when a below-gap excitation was superposed on an above-gap excitation. This intensity quenching was attributed to the enhanced nonradiative recombination through below-gap states which were activated by the below-gap excitation. By changing the energy of above-gap excitation, it was shown that these below-gap states were formed neither inside GaAs well layers nor at the GaAs/AlGaAs heterointerface, but inside AlGaAs barrier layers. Tuning the photon energy of below-gap excitation revealed that the activation energy of the nonradiative recombination process via these below-gap states is around 1.2 eV. Lowering the above-gap excitation density to a single-photon-counting region enabled us to characterize below-gap states spectroscopically without the need of a high-power tunable laser source for the below-gap excitation.

Role of Intramolecular Torsion and Solvent Dynamics in the Charge-Transfer Kinetics in Triphenylphosphine Oxide Derivatives and DMABN

The photoinduced processes in three dimethylamino derivatives of the triphenylphosphine oxide (OMAP, ODAP, and OTAP) are studied in solution at room temperature by time-resolved fluorescence spectroscopy with a streak camera and a 500 fs UV laser excitation source. These compounds exhibit a dual fluorescence in polar solvents explained by the fast formation of an emissive charge-transfer state as in the model compound (dimethylamino)benzonitrile (DMABN). Fluorescence decays are also measured for solutions of DMABN under the same conditions. For both compounds, the intramolecular charge-transfer time is shown to vary from a few picoseconds to a few tens of picoseconds depending on the polarity of the solvent and, for the triphenylphosphine derivatives, on the number of dimethylamino substituents. The charge-transfer process is described as a barrier-activated process with a solvent polarity dependent height. The solvent dynamics and solvent viscosity effects on the charge-transfer rate are examined for bot...

Diffraction effects in single- and two-laser photothermal lens spectroscopy

A simple method for calculating the effects of optical geometry on photothermal lens signals is shown. This method is based on calculating cumulative electric-field phase shifts produced by a series of Gaussian refractive-index perturbations produced by the photothermal effect. Theoretical results are found for both pulsed-laser and continuous Gaussian laser excitation sources and both single- and two-laser apparatuses commonly employed in photothermal lens spectroscopy. The effects of apparatus geometry on the resulting signal are shown. Analytical time-dependent signal results are found for small signals. Analytical pump-probe focus geometry results allow direct optimization for certain conditions. The calculations indicate that the photothermal lens signal is, in general, optimized for near-field detection-plane geometries.

Simultaneous two-photon activation of type-I photodynamic therapy agents.

The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote nonresonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4'-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamaging near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage.

Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a slow-scan CCD camera. The phase and modulation homogeneity of the MCP image intensifier were determined at frequencies of 40, 100, 160, and 240 MHz. The detected modulation depths were 65, 52, 32, and 23%, respectively, and were highly homogeneously distributed. The phasedistribution image revealed iris effects at frequencies of 160 and 240 MHz but was homogeneous at lower frequencies. Lifetime imaging of a solution of the fluorescent flavoprotein lipoamide dehydrogenase demonstrated (i) the accuracy of the determined lifetimes (< 60 ps), (ii) the time resolution of the instrument (< 50 ps), and (iii) the average precision for single pixel fluorescence lifetimes (50 ps is feasible). The imaging of tiny fluorescent microspheres revealed that even in a volume of 0.3 x 10-15 L, the standard error in the lifetimes can be as low as 79 ps. The spatial resolution of the instrument is estimated to be < 400 nm in the object plane at a 100 x magnification.

Field demonstration of a multichannel fiber-optic laser-induced fluorescence system in a cone penetrometer vehicle

A laser-induced fluorescence system for in situ detection of soil contamination with cone penetrometer technology, capable of collecting three-dimensional fluorescence fingerprints (excitation–emission matrices, or EEMs) as the probe is advanced vertically, has been developed. In this report, we describe a recent field test at Hanscom Air Force Base, Massachusetts, in which we demonstrated successful operation of the system. The system uses a pair of silica-clad-silica optical fibers for each of its channels (eight of which were used in the work described), one to deliver excitation light from the multiwavelength laser excitation source to the sample, and the other to conduct contaminant fluorescence to a grating spectrograph that utilizes a charge-coupled-device detector to record fluorescence intensity as a function of both excitation and emission wavelength. The matrix-formatted field data are displayed as three-dimensional fingerprints of contamination at given push locations and depths; they are also reduced in dimension by summing over one or both wavelength axes and plotting summed fluorescence versus depth to facilitate visualization of the approximate extent of the contaminant plume. Selected EEMs from each push location are examined for characteristic patterns and compared to standard EEMs of pure compounds and fuels. © 1997 John Wiley & Sons, Inc. Field Analyt Chem Technol 1: 343–355, 1997