Laser Excitation Source Research Articles

Two-photon microscopy with wavelength switchable fiber laser excitation

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

The application of Raman spectrometry to investigate and characterize cement, Part I: A review

Raman spectrometry has received attention for the past 3 decades in its application to the characterisation of pure cement phases, white and grey cements. Various configurations of instrumentation and laser excitation sources have been used, as reported in a limited number of papers. The first demonstrated investigation in 1976 by Bensted, illustrated the characterisation of various pure mineral cement phases with the use of a visible excitation source and a standard dispersive configuration. This was soon followed by an investigation of pure phases, as well as white and grey cements in 1980 by Conjeaud and co-workers using a microprobe configuration and visible excitation. Considerable difficulties with excessive fluorescence phenomena, especially with grey cements, have however been reported from the initial to the most recent publication. This review aimed to report on Raman studies of pure synthesized cement phases, cement phases as found in white and grey cements, various forms of gypsum and related compounds as found in cement and concrete, hydration of pure cement phases, as well as those of white and grey cement and lastly carbonation effects. Results reported are discussed and tables of summarized observed Raman shifts are given.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 13. The Raman characteristics of phthalocyanine in unsubstituted and peripherally octa(octyloxy)-substituted homoleptic bis(phthalocyaninato) rare earth complexes

The Raman spectroscopic data in the range of 500–1800 cm −1 for two series of thirty homoleptic bis(phthalocyaninato) rare earth complexes M(Pc) 2 and M[Pc(OC 8H 17) 8] 2 [M = Y, La–Lu except Pm; H 2Pc = unsubstituted phthalocyanine, H 2Pc(OC 8H 17) 8 = 2,3,9,10,16,17,23,24-octakis(octyloxy)phthalocyanine] have been collected and comparatively studied using laser excitation sources emitting at 633 and 785 nm. Under both laser excitations, the marker Raman band of Pc 2− and [Pc(OC 8H 17) 8] 2− in the Raman spectra of Ce(Pc) 2 and Ce[Pc(OC 8H 17) 8] 2 appears as a strong scattering at 1498–1501 cm −1 with contributions from both pyrrole C C and aza C N stretches together with isoindole stretchings. This band has been found to upshift to 1502–1528 cm −1 in the Raman spectra of M III(Pc) 2 and M III[Pc(OC 8H 17) 8] 2 as the marker Raman band of phthalocyanine monoanion radicals, Pc − or [Pc(OC 8H 17) 8] − . With laser excitation at 633 nm, Raman vibrations derived from isoindole ring and aza stretchings in the range of 1300–1600 cm −1 for both series are selectively intensified. In contrast, when excited with laser radiation of 785 nm, the ring radial vibrations of isoindole moieties and dihedral plane deformations between 500 and 1000 cm −1 for M(Pc) 2 and M[Pc(OC 8H 17) 8] 2 intensify to become the strongest scatterings. The present Raman results also reveal that the frequencies of Pc breathing, pyrrole stretching, isoindole stretchings, aza stretchings and coupling of pyrrole and aza stretchings depend on the rare earth ionic size, shifting to higher wavenumbers along with the lanthanide contraction due to the increased ring–ring interaction across the series. Moreover, under these laser excitations, in particular under 785 nm laser line, the Raman spectrum appearance, i.e., the pattern of relative intensities, also changes systematically depending on the rare earth ionic size.

Electrophoresis with Polarized Fluorescence Detection. Application to Capillary Fluorescence Rejection

In this paper, we integrate the apparatus of electrophoresis with polarized fluorescence detection to suppress the background fluorescence arising from illuminating the coating layer of channel containers with a laser excitation source. It has demonstrated moderate background suppression efficiency, close to the ideal 3fold reduction on the surface doped with randomly distributed static fluorophores. In addition, when the fluorescence coverage is coated along the same orientation more uniformly, the background reduction is 10-fold in our experiments. This detection scheme is applicable to acquire the electrophoregrams of separating small organicmoleculesandbiopolymers,suchasnucleicacids,whentheloadingofstainingdyesisnotheavy.This apparatus is simple. Only adding a pair of polarizer optics is needed. This detection scheme should work equally well with an incoherent light source.

Pulsed laser Raman spectroscopy in the laser-heated diamond anvil cell

We describe the design and operation of a spatially-filtered Raman/fluorescence spectrometer that incorporates a pulsed 532nm laser excitation source and a synchronized and electronically gated CCD detector. This system permits the suppression of undesired continuous radiation from various sources by a factor of up to 50 000 providing the possibility of acquiring Raman signals at temperatures exceeding 5000K. We present performance comparisons of this system with that of a state-of-the-art conventional CW system using a 458nm excitation source. We also demonstrate that the pulsed system is capable of suppressing an impurity-induced (single nitrogen defects) fluorescence in diamond, and further suggest that this capability can be used to suppress the stress-induced fluorescence in diamond that may appear at pressures near or above 150GPa. This work suggests that Raman spectroscopy under conditions of very high temperatures (to 5000K) and/or ultrahigh pressures (to 300GPa) is entirely viable.

Synthesis of Two-Photon Absorbing Unsymmetrical Branched Chromophores through Direct Tris(bromomethylation) of Fluorene

Branched fluorene-based chromophores bearing electron-donating diphenylamino or electron-withdrawing nitro groups were synthesized as well as their linear analogues. An efficient synthetic method was developed via a novel 2,4,7-tris(bromomethyl)-9,9-diethylfluorene intermediate. The bromomethyl groups in this key intermediate were converted to either phosphonate or carboxaldehyde moieties, facilitating preparation of a high functionality branched structure. It was found that the reactivity at position 4 is attenuated in the bromomethyl and phosphorylated derivates, facilitating the selective and systematic functionalization of the fluorenyl system. All compounds were stable up to ca. 350 degrees C, except for a sterically crowded branched derivative. The linear optical properties of the compounds were investigated by UV-visible, steady-state fluorescence, and excitation anisotropy spectroscopic measurements. Fluorescence quantum yields were greater than or equal to 0.84 for symmetric linear and unsymmetric branched derivatives. Very high two-photon absorption (2PA) cross-sections were achieved (5765 GM at 520 nm and 4194 GM at 570 nm), as determined with use of picosecond and femtosecond laser excitation sources, respectively.

Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control

We report a novel Fourier transform method for measuring two-photon excitation spectra. We demonstrate this method using simple dye molecules and discuss its applications in two-photon fluorescence microscopy and optimal control. This method facilitates an intuitive interpretation of recent control experiments in terms of tuning the nonlinear spectrum of the exciting laser source.

Optical defect centers and surface morphology of isotopically enriched diamond layers in designer diamond anvils

We have studied optical defect centers and surface morphology of isotopically enriched layers grown on diamond anvils by microwave plasma chemical vapor deposition for applications as designer diamond anvils in high-pressure diamond anvil cell devices. Various mixtures of methane isotopes were used to grow homoepitaxial diamond with C13 molar fractions of 0.01, 0.41, 0.83, and 0.99 as determined from Raman spectroscopy. Defect centers were studied at temperatures between 80 and 320K using micro-photoluminescence (PL) spectroscopy with an argon ion and krypton laser excitation source. The defect spectra were dominated by zero phonon lines (ZPL) from nitrogen-related defect centers at nominal energies of 1.945eV (640nm defect) and 2.156eV (575nm defect), especially for the non-(100) surfaces. Polished (100) surfaces fluoresced weakly. ZPL’s at 1.77 and 1.68eV are observed, but not for all isotopically mixed samples. The 1.77eV ZPL appears to be associated from the original diamonds, while the 1.68eV ZPL is known to originate with silicon-based defects. Atomic force microscopy of as-grown isotopically enriched layers show rough growth steps in areas with surface roughness of hundred nanometers and smooth areas with surface roughness of few nanometers. Our studies indicate that (100) polished surfaces of isotopically enriched designer diamonds with low concentration of nitrogen defect centers can be fabricated for a variety of applications in high pressure research.

Formulation and implementation of a phase-resolved fluorescence technique for latent-fingerprint imaging: theoretical and experimental analysis

A theoretical and experimental study of the imaging of latent fingerprints by a phase-resolved fluorescence technique along with associated signal-processing analysis is described. The system configuration is optimized by incorporation of a novel approach of homodyne-assisted even-step phase shifting in a signal-processing concept. The excitation laser source and gain of the detection device, which are modulated at megahertz frequency followed by sensitive signal-processing concepts, are employed to separate the fingerprint fluorescence from background fluorescence. Experiments are carried out with fingerprints deposited upon different types of substrate surfaces. Later, a quantitative image-quality assessment is carried out, which confirms the improvement in the quality of the phase-resolved fingerprint image. Imaging of older fingerprints with better contrast is also carried out with the proposed novel technique.

The autofluorescence of plastic materials and chips measured under laser irradiation

Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.

Vibrational spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich‐type phthalocyaninato and porphyrinato rare earth complexes. Part 11—Raman spectroscopic characteristics of phthalocyanine in mixed [tetrakis(4‐chlorophenyl)porphyrinato](phthalocyaninato) rare earth double‐deckers

AbstractRaman spectroscopic data in the range 500–1800 cm−1 for a series of 15 mixed [tetrakis(4‐chlorophenyl)porphyrinato](phthalocyaninato) double‐decker complexes with tervalent rare earths MIII(TClPP)(Pc) (M = Y, LaLu except Ce and Pm) and intermediate‐valent cerium Ce(TClPP)(Pc) were collected using laser excitation sources emitting at 632.8 and 785 nm. Comparison with the Raman spectra of corresponding bis(phthalocyaninato) rare earths reveals that the Raman characteristics of mixed‐ring double‐deckers are dominated by the phthalocyaninato‐metal fragment M(Pc) and phthalocyanine exists as the monoradical anion Pc•− in MIII(TClPP)(Pc) and the dianion Pc2− in Ce(TClPP)(Pc), respectively. With excitation at 632.8 nm, which is in close resonance with the main Q absorption band of the phthalocyanine ligand, a typical Raman marker band of the monoanion radical Pc•− was observed at 1512–1519 cm−1 as a weak band resulting from the coupling of pyrrole CC and aza CN stretchings. For Ce(TClPP)(Pc), a strong band at 1492 cm−1 with contributions from both pyrrole CC and aza CN stretchings and also isoindole stretching was the marker Raman band of Pc2−. When excited with laser radiation of 785 nm, which is far away from the main Q absorption band of the phthalocyanine ligand, the marker Raman band of Pc•− in MIII(TClPP)(Pc) is observed at 1495–1514 cm−1 and of Pc2− in Ce(TClPP)(Pc) at 1511 cm−1 as a strong band. Copyright © 2004 John Wiley & Sons, Ltd.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: (Part 10) The infrared and Raman characteristics of phthalocyanine in heteroleptic bis(phthalocyaninato) rare earth complexes with decreased molecular symmetry

The infrared (IR) spectroscopic data for a series of eleven heteroleptic bis(phthalocyaninato) rare earth complexes MIII(Pc)[Pc(α-OC5H11)4] (M = Sm–Lu, Y) [H2Pc = unsubstituted phthalocyanine, H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected with 2cm−1 resolution. Raman spectroscopic properties in the range of 500–1800cm−1 for these double-decker molecules have also been comparatively studied using laser excitation sources emitting at 632.8 and 785nm. Both the IR and Raman spectra for M(Pc)[Pc(α-OC5H11)4] are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues due to the decreased molecular symmetry of these double-decker compounds, namely C4. For this series, the IR Pc− marker band appears as an intense absorption at 1309–1317cm−1, attributed to the pyrrole stretching. With laser excitation at 632.8nm, Raman vibrations derived from isoindole ring and aza stretchings in the range of 1300–1600cm−1 are selectively intensified. In contrast, when excited with laser radiation of 785nm, the ring radial vibrations of isoindole moieties and dihedral plane deformations between 500 and 1000cm−1 for M(Pc)[Pc(α-OC5H11)4] intensify to become the strongest scatterings. Both techniques reveal that the frequencies of pyrrole stretching, isoindole breathing, isoindole stretchings, aza stretchings and coupling of pyrrole and aza stretchings depend on the rare earth ionic size, shifting to higher energy along with the lanthanide contraction due to the increased ring-ring interaction across the series. The assignments of the vibrational bands for these compounds have been made and discussed in relation to other unsubstituted and substituted bis(phthalocyaninato) rare earth analogues, such as M(Pc)2 and M(OOPc)2 [H2OOPc = 2,3,9,10,16,17,23,24-octakis(octyloxy)phthalocyanine].

Review of the development of laser fluorosensors for oil spill application

As laser fluorosensors provide their own source of excitation, they are known as active sensors. Being active sensors, laser fluorosensors can be employed around the clock, in daylight or in total darkness. Certain compounds, such as aromatic hydrocarbons, present in petroleum oils absorb ultraviolet laser light and become electronically excited. This excitation is quickly removed by the process of fluorescence emission, primarily in the visible region of the spectrum. By careful choice of the excitation laser wavelength and range-gated detection at selected emission wavelengths, petroleum oils can be detected and classified into three broad categories: light refined, crude or heavy refined. This paper will review the development of laser fluorosensors for oil spill application, with emphasis on system components such as excitation laser source, and detection schemes that allow these unique sensors to be employed for the detection and classification of petroleum oils. There have been a number of laser fluorosensors developed in recent years, many of which are strictly research and development tools. Certain of these fluorosensors have been ship-borne instruments that have been mounted in aircraft for the occasional airborne mission. Other systems are mounted permanently on aircraft for use in either surveillance or spill response roles.

Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.

Surface-Enhanced Raman Scattering Studies on Aggregated Gold Nanorods

Surface-enhanced Raman scattering (SERS) of adsorbed molecules on gold nanorods (NRs) with dimensions of 10 nm × 27 nm was studied on silica surface with low to high surface coverage of NRs. The study was carried out to investigate both the dependence of the SERS intensity on the number of NRs and the NRs spacing on the silica surface. SERS of adsorbed molecules such as 2-aminothiophenol (2-ATP) and the capping molecules (hexadecyltrimethylammonium bromide) was studied on these surfaces using a near-IR laser excitation source (1064 nm). To produce silica surfaces covered with NRs, two approaches were used. In the first approach, monodispersed NRs gradually deposited from solution to silica surface and their number was increased by increasing the deposition time. In the second one, the NRs were first aggregated in solution and then deposited on the surface. Although using the first approach it was possible to prepare surfaces with high NR surface coverage, SERS intensity was found to be stronger for adsorb...

Two-dimensional photoacoustic imaging by use of Fourier-transform image reconstruction and a detector with an anisotropic response.

Theoretical and experimental aspects of two-dimensional (2D) biomedical photoacoustic imaging have been investigated. A 2D Fourier-transform-based reconstruction algorithm that is significantly faster and produces fewer artifacts than simple radial backprojection methods is described. The image-reconstruction time for a 208 x 482 pixel image is approximately 1 s. For the practical implementation of 2D photoacoustic imaging, a rectangular detector geometry was used to obtain an anisotropic detection sensitivity in order to reject out-of-plane signals, thereby permitting a tomographic image slice to be reconstructed. This approach was investigated by the numerical modeling of the broadband directional response of a rectangular detector and imaging of various spatially calibrated absorbing targets immersed in a turbid phantom. The experimental setup was based on a Q-switched Nd:YAG excitation laser source and a mechanically line-scanned Fabry-Perot polymer-film ultrasound sensor. For a 800 microm x 200 microm rectangular detector, the reconstructed image slice thickness was 0.8 mm up to a vertical distance of z = 3.5 mm from the detector, increasing thereafter to 2 mm at z = 10 mm. Horizontal and vertical spatial resolutions within the reconstructed slice were approximately 200 and 60 microm, respectively.

Normal Raman and surface enhanced Raman spectroscopic experiments with thin layer chromatography spots of essential amino acids using different laser excitation sources

A comparative study of the feasibility and efficiency of Raman spectroscopic detection of thin layer chromatography (TLC) spots of some weak Raman scatterers (essential amino acids, namely, glycine and l-forms of alanine, serine, valine, proline, hydroxyproline, and phenylalanine) was carried out using four different visible and near-infrared (NIR) laser radiations with wavelengths of 532, 633, 785, and 1064 nm. Three types of commercial TLC plates were tested and the possibility of inducing surface enhanced Raman scattering (SERS) by means of Ag-sol was also investigated. The spectra obtained from spotted analytes adsorbed on TLC plates were of very different quality strongly depending on the excitation wavelength, the wetness of the samples, and the compounds examined. The best results were obtained with the simple silica TLC plate, and it has been established that the longest wavelength (lowest energy) NIR excitation of a Nd:YAG laser is definitely more suitable for generating normal Raman scattering of analyte spots than any of the visible radiations. Concerning SERS with application of Ag-sol to the TLC spots, 1–3 orders of magnitude enhancement was observed with wet samples, the greatest with the 532 nm radiation and gradually smaller with the longer wavelength excitations. It is shown, however, that due to severe adsorption-induced spectral distortions and increased sensitivity to microscopic inhomogeneity of the sample, none of the SERS spectra obtained with the dispersive Raman microscope operating in the visible region were superior to the best NIR normal FT-Raman spectra, as far as sample identification is concerned.

An all fiber optic system modeling for the gastrointestinal endoscopy: design concepts and fluorescent analysis

A theoretical model, which describes both the spatial distributions of photons in fluorescence endoscopic images for the detection of abnormalities/defects in the intracavities of human body, specifically gastrointestinal path is presented. The design concept of the image probe, which was developed for collecting the low fluorescent emission using an excitation laser source and the imaging done through an image fiber bundle, are discussed here. A band-pass/neutral density filter combination was used to separate the fluorescence emission from the normal coherent background induced by the laser. Finally, a quantitative analysis done by varying the different parameters affecting the tissue fluorescence is discussed in this paper.

Resonance Raman effects in TS-1: the structure of Ti(iv) species and reactivity towards H2O, NH3 and H2O2: an in situ studyPresented at the International Congress on Operando Spectroscopy, Lunteren, The Netherlands, March 2-6, 2003.

The isomorphous insertion of 1–2 wt% of Ti into the MFI framework leads to a Titanium silicalite-1 (TS-1) material, which is an active and highly selective catalyst in a remarkable number of low-temperature oxidation reactions with aqueous H2O2 as oxidant. Such Ti(IV) species exhibit a local Td-like symmetry, forming [TiO4] units, and induces to the hosting MFI matrix two Ti-specific vibrational modes at 960 and 1125 cm−1. We report a Raman study on the perturbation caused by interaction with H2O, NH3 and H2O/H2O2 on the vibrational modes of the [TiO4] unit embedded in the MFI framework. The selective use of different excitation laser sources in the near-IR (1064 nm; 9398 cm−1), visible (442 nm; 22 625 cm−1), near-UV (325 nm; 30 770 cm−1) and far-UV (244 nm; 40 985 cm−1) allowed us to progressively enter into the oxygen to titanium charge transfer transition and thus to switch on the resonance effects on the 1125 cm−1 mode, which is the only Ti-specific mode exhibiting the same symmetry of the charge transfer transition. Interaction with both water and ammonia causes the formation of [Ti(H2O)2O4] or [Ti(NH3)2O4] complexes which destroy the Td-like symmetry and thus the Raman enhancement of the 1125 cm−1 mode. Upon dosing a H2O/H2O2 to TS-1, the powders turn yellow as a consequence of the appearance a new charge transfer transition around 385 nm (26 000 cm−1). In order to single out the vibrational mode of the active peroxo complex formed on Ti, we have performed Raman experiments using a visible laser source (442 nm; 22 625 cm−1). In these conditions we have observed the strong enhancement of a mode at 618 cm−1, which has been attributed to the symmetric breathing mode of the Ti(O)2 ring.

Optical spectroscopy and imaging of the dentin-enamel junction in human third molars.

A 351-nm laser excitation source was used to perform autofluorescence microscopy of dentin, enamel, and the dentin-enamel junction (DEJ) to obtain information regarding their morphology and spectral characteristics. The emission spectra of these calcified dental tissues were different from one another, and this enabled the DEJ to be imaged and dimensionalized. The DEJ displayed sharp and clearly delineated borders at both its enamel and dentin margins. The dentinal tubules and the enamel prisms appeared to terminate abruptly at the DEJ. The median DEJ width was 10 microm, ranging from 7 to 15 microm, and it did not appear to depend on intratooth position.