Thermal-Lens-Induced Anomalous Solvent's effect on Fluorescence Produced by Two-Photon Continuous-Wave Laser Excitation

Measurements of two-photon-excited fluorescence (TPF) of fluorescein and Rhodamine 6G in various solvents were performed with a continuous-wave (cw) laser for excitation and an acousto-optic tunable filter for spectral dispersion. Interestingly, the cw laser excitation produced an unwanted thermal-lens effect when the measurements were performed in solvents that absorb the excitation laser light (e.g., alcohols and water, because these solvents absorb the 780-nm excitation light through the overtone and combination transitions of the O-H group). The defocusing effect of the thermal lens leads to a decrease in the TPF signal. Because the strength of the thermal lens depends on the thermo-optical properties (dn/dT and thermal conductivity) of the solvent, its interference makes the effect of solvents on the TPF much different from those on one-photon-excited fluorescence. However, the thermal-lens interference will not limit the application of this cw laser excited TPF technique because, even when measurements were performed in solvents that absorb cw excitation laser light, the thermal-lens interference was observed only in solvents such as nonpolar organic solvents that have relatively better thermo-optical properties. Interference was not observed in water, which is the most widely used solvent for the TPF technique (because water has poor thermo-optical properties).     

Changes in the luminescence between dried and wet bacillus spores

Fluorescence has been suggested as a method with which to detect and identify bacterial spores. To better understand the nature of the fluorescence signal, we observed the intrinsic steady-state fluorescence and phosphorescence spectra of Bacillus globigii (BG) in both dried and aqueous forms. In vitro, dried, and suspension forms of BG were measured at room temperature in 300-600-nm excitation wavelengths. Also, the phosphorescence of dry BG spores was measured at room temperature at 300-600-nm excitation wavelengths. The wet BG spores exhibited a strong maximum in their fluorescence spectrum, with the peak excitation wavelength near 300 nm and emission wavelength near 400 nm. When the BG was dried, this peak shifted to an approximately 450-nm excitation maximum and an 500-nm emission maximum. The difference between the wet and the dry spore fluorescence spectra cannot be explained by the phosphorescence of the dry spores. Other changes must take place when the spores are wet to account for the large changes observed in the spectrum.     

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.     

Diode-pumped 214.8-nm Nd:YAG /Cr4+:YAG microchip laser system for the detection of NO

A passively Q-switched 214.8-nm Nd:YAG/Cr(4+):YAG microchip laser system for the detection of NO was designed, constructed, and tested. The system uses the fifth harmonic of the 1.074-microm transition in Nd:YAG to detect NO by laser-induced fluorescence. A significant challenge was the development of an environmentally stable coating to provide the necessary discrimination between the 1.074-microm laser line and the stronger transition at 1.064 microm. The exact position of the fifth-harmonic frequency was determined by use of NO fluorescence excitation spectra to be 46556 +/- 1.5 cm(-1). With a pulse energy of approximately 50 nJ of fifth-harmonic light, we observed a detection sensitivity for NO of approximately 15 parts per billion by volume in a simple, compact optical system.     

Saturated patterned excitation microscopy--a concept for optical resolution improvement

The resolution of optical microscopy is limited by the numerical aperture and the wavelength of light. Many strategies for improving resolution such as 4Pi and I5M have focused on an increase of the numerical aperture. Other approaches have based resolution improvement in fluorescence microscopy on the establishment of a nonlinear relationship between local excitation light intensity in the sample and in the emitted light. However, despite their innovative character, current techniques such as stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy require complex optical configurations and instrumentation to narrow the point-spread function. We develop the theory of nonlinear patterned excitation microscopy for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state. The postacquisition manipulation of the acquired data is computationally more complex than in STED or GSD, but the experimental requirements are simple. Simulations comparing saturated patterned excitation microscopy with linear patterned excitation microscopy (also referred to in the literature as structured illumination or harmonic excitation light microscopy) and ordinary widefield microscopy are presented and discussed. The effects of photon noise are included in the simulations.     

Multicolor up-conversion emissions of Tm3+/Er3+/Yb3+ tri-doped YF3 phosphors

Tm3+/Er3+/Yb3+ tri-doped yttrium fluoride (YF3) phosphors were prepared by a facile hydrothermal method. X-ray topographic analysis found that the phosphors were crystallized products. Their sizes and morphologies were characterized by scanning electron microscopy (SEM, Hitachi S-4800), which indicated that most of the YF3 phosphors were hundreds of nanometers in size. Up-conversion (UC) spectra were recorded under 980-nm diode laser excitation at room temperature with a fluorescence spectrometer (Hitachi F-4500). Plenty of UC emissions of Tm3+ and Er3+ were observed from ultraviolet to red. For Tm3+ ions, a five-photon process (approximately 291 nm and approximately 347 nm), a four-photon process (approximately 362 nm and approximately 452 nm), and a three-photon process (approximately 475 nm) were identified in the UC spectra. The UC emissions from the Er3+ were: approximately 380 nm, approximately 408 nm, approximately 521 nm, approximately 537 nm, and approximately 652 nm. Therefore, cyan-white light can be observed by the naked eye at 980-nm excitation, even under low excitation power density. By comparing the UC spectra of the phosphors annealed at different temperatures, we found that the intensity of the UC luminescence increased as annealing temperature increased. Furthermore, the spectral dependencies on Tm3+ doped concentrations were studied. The energy transfer processes and fluorescence dynamics in the tri-doped system are currently being investigated.     

Two-dimensional multiwavelength fluorescence spectra of dipicolinic acid and calcium dipicolinate

Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are major chemical components of bacterial spores. With fluorescence being considered for the detection and identification of spores, it is important to understand the optical properties of the major components of the spores. We report in some detail on the room-temperature fluorescence excitation and emission spectra of DPA and its calcium ion complex and provide a comparison of the excitation-emission spectrum in a dry, wet paste and aqueous form. DPA solutions have weak, if any, fluorescence, with increased fluorescence when the DPA is dry. After exposure to a broad source UV light of the DPA, wet or dry, we observe a large increase in fluorescence with a maximum intensity emission peak at around 440 nm for excitation light with a wavelength of around 360 nm. There is a slight blueshift in the absorption spectra of UV-exposed DPA from the unexposed DPA solution. CaDPA in solution shows a slight fluorescence with increased fluorescence in the dry form, and a substantial increase of fluorescence was observed after UV exposure with an emission peak of around 410 nm for excitation around 305 nm. The detailed excitation-emission spectra are necessary for better interpretation of the fluorescence spectra of bacterial spores where DPA is a major chemical component.     

Two-photon excitation of fluorescence from chicken tissue

We have measured UV fluorescence excited through two-photon absorption from native chicken tissue, using 600-nm, 500-fs pulses from a R6G dye laser. The observed emission signal was found to depend quadratically on the excitation intensity. The two-photon excitation-induced fluorescence spectrum is attributed to tryptophan residues in proteins.     

Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm. The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.     

Temperature measurements from first-negative N(2)(+) spectra produced by laser-induced multiphoton ionization and optical breakdown of nitrogen

A 248-nm excimer laser was used to produce ionized nitrogen by the process of multiphoton excitation in gaseous nitrogen at room temperature. First-negative N(2)(+) emission spectra were analyzed to yield rotational temperatures of typically 600 to 1200 K. Rotational Raman scattering of H(2) in gaseous mixtures of N(2) and H(2) was used to determine if laser heating of the gas produced the observed increase in temperature, but the room temperature value of 295 K was inferred from the H(2) Raman data. Therefore the use of N(2)(+) spectra produced by multiphoton excitation at 248 nm does not appear to be acceptable for air-temperature diagnostics. N(2)(+) emission spectra were also recorded subsequent to optical breakdown in air induced by Nd:YAG 1064-nm radiation, and temperatures were determined to be greater than 5000 K in the decaying plasma.     

Photoluminescence properties of Eu doped Ba2ZnS3 phosphor for white light emitting diodes

The synthesis and photoluminescence properties of novel Eu²⁺ doped Ba₂ZnS₃ phosphors for white light emitting diodes (LEDs) are reported. Diffuse reflection spectra of Ba₂ZnS₃ host and synthesized phosphors have been measured. The excitation spectra of synthesized phosphors consist of three broad bands between 250 nm and 550 nm and are consistent with the diffuse reflectance spectra. The emission spectra show the characteristic 4f⁶5d¹ → 4f⁷ transition of Eu²⁺ ion and there exists efficient energy transfer from host to Eu²⁺ ions when excited by 350-nm light. The dependence of emission spectra on temperature is also measured; the possible reasons applied to explain the experimental results are also discussed. The fluorescence lifetime of Eu²⁺ in Ba_1.995ZnS₃:0.005Eu²⁺ is measured and the values are 1.49 and 23.4 μs.     

Application of 266-nm and 355-nm Nd:YAG laser radiation for the investigation of fuel-rich sooting hydrocarbon flames by raman scattering

We describe the use of linear Raman scattering for the investigation of fuel-rich sooting flames. In comparison, the frequency-tripled and -quadrupled fundamental wavelengths of a Nd:YAG laser have been used as an excitation source for study of the applicability of these laser wavelengths for analysis of sooting flames. The results obtained show that, for the investigation of strongly sooting flames, 266-nm excitation is better than 355-nm excitation. Although the entire fluorescence intensity of polycyclic aromatic hydrocarbons (PAHs) decreases with rising excitation wavelength, there is increased interference with the Raman signals by displacement of the spectral region of the Raman signals toward the fluorescence maximum of the laser-induced fluorescence emissions. Besides the broadband signals of PAHs, narrowband emissions of laser-produced C2 occur in the spectra of sooting flames and affect the Raman signals. These C2 emission bands are completely depolarized and can be separated by polarization-resolved detection. A comparison of the laser-induced fluorescence emissions of an ethylene flame with those of a methane flame shows the same spectral features, but the intensity of the emissions is larger by a factor of 5 for the ethylene fuel. Using 266-nm radiation for Raman signal excitation makes possible measurements in the ethylene flame also.     

Spectroscopy and upconversion processes in YAlO3:Ho crystals

Emission from the high lying excited states, energy transfer, and upconversion processes are investigated in YAlO₃:Ho³⁺. Selectively excited emission spectra in the range from 300 to 800 nm starting from the ³D₃, ³G₅, ⁵F₃, ⁵S₂ and ⁵F₅ multiplets were measured at 15 K. This, together with the detailed absorption and excitation measurements at 15 K allowed determination of the Stark energy levels of Ho³⁺ ions in YAlO₃ up to UV energies. The ⁵S₂ fluorescence decays were recorded as a function of temperature and Ho³⁺ concentration in order to investigate the process of quenching of fluorescence due to cross relaxation among two ions. Conversion of red and infrared laser radiation to green ⁵S₂ and blue ⁵F₃ emission is reported. Under pulsed resonant excitation of the ⁵F₅ or ⁵I₅ levels the upconversion was found to be due to energy transfer process between two excited ions. The photon avalanche effect was observed under cw excitation around 585 nm.     

Low-threshold lasing and broad-band multiphoton-excited light emission from Ag aggregate-adsorbate complexes in microcavity

A novel class of composites for optics, microcavities doped with metal fractal aggregates, is studied. Lasing and broad-band Stokes and anti-Stokes emission from (Ag colloidal aggregates)/(adsorbed molecules)/(micro-cavity) composite at low-intensity cw and pulse laser excitation has been found. At 633 nm cw excitation wavelength the emission spectrum contains many peaks, spanning a range from wavelength 200 nm to 800 nm. Experiments with pulse excitation of Ag/dye/microcavity composite show that the duration of the observed broad-band anti-Stokes emission significantly exceeds the pump pulse duration, dye molecule fluorescence time, and relaxation times in silver particles. It may be interpreted as a luminescence governed by long-living triplet states of dye molecules. These observations were made possible by use of a fractal-microcavity composite, where coupling the localized plasmon modes in fractal aggregates with microcavity resonances is provided. The important role of multiphoton resonant transitions between discrete states of a finite-size metal particle in enhanced local fields is shown. Analysis, based on the model of a spherical potential well, shows that the observed spectra contain fingerprints of the quantum size effect.     

Generation of 224-nm radiation by stimulated Raman scattering of ArF excimer laser radiation in a mixture of H2 and D2

Raman shifting of tunable ArF excimer laser radiation in a mixture of H(2) and D(2) produces tunable radiation in the 224-nm region as a result of Stokes shifting the frequency of the fundamental radiation (193 nm) once in both H(2) and D(2). At a total pressure of 25 bars, a 19% H(2) in D(2) mixture is found to provide a maximum conversion efficiency (2.5%) to the 224-nm range. Both fundamental and 224-nm radiation were used to record laser-induced fluorescence excitation spectra of nitric oxide produced in an oxyacetylene flame. From the excitation spectra, we determined the tuning range of the 224-nm radiation to be 270 cm(-1) with a linewidth of 0.9 cm(-1), which is similar to the fundamental laser radiation. We derived the exact Raman shift of the generated radiation by comparing both excitation spectra which was found to be 7142.3(5) cm(-1).     

Clinical imaging fluorescence apparatus for the endoscopic photodetection of early cancers by use of Photofrin II

A fluorescence imaging device applied to the detection of early cancer is described. The apparatus is based on the imaging of laser-induced fluorescence of a dye that localizes in a tumor with a higher concentration than in the surrounding normal tissue after iv injection. Tests carried out in the upper aerodigestive tract, the tracheobronchial tree, and the esophagus with Photofrin II (1 mg/kg of body weight) as the fluorescent agent are reported as examples. The fluorescence is induced by violet (410-nm) light from a continuous-wave (cw) krypton-ion laser. The fluorescence contrast between tumor and surrounding tissue is enhanced by real-time image processing. This is done by the simultaneous recording of the fluorescence image in two spectral domains (470-600 and 600-720 nm), after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Among the 7 photodetections performed in the tracheobronchial tree, 6 were successful, whereas it was the case for only 5 of the 15 lesions investigated in squamous mucosa (upper aerodigestive tract and esophagus). The sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.     

Deep-ultraviolet Raman microspectroscopy: characterization of wide-gap semiconductors

We have developed a high-throughput deep-ultraviolet (DUV) Raman microspectrometer with excitation from a continuous wave (cw) laser operated at 244 nm that enables us to characterize thin surface layers of wide-gap semiconductors. This spectrometer system consists of a filter spectrometer for the rejection of stray light and a high-dispersion spectrograph combined with a liquid nitrogen cooled charge-coupled device (CCD) detector and extends the low-frequency limit of the observable spectral range down to 170 cm(-1). In the microscope we use a Cassegrain reflective objective for the collection of the scattered light and an off-axis mirror for introduction of the excitation laser light. DUV Raman spectroscopy has been applied for studying wide-gap semiconductors including SiC and AlGaN epitaxial films and shallow implanted layers of these materials. Raman spectra of various crystals have also been measured for examining the performance of this system. Resonance enhancement of Raman bands has been observed for several semiconductors, and the results are discussed.     

Efficient pumping scheme for neodymium-doped materials by direct excitation of the upper lasing level

An efficient pumping scheme that involves direct excitation of the upper lasing level of the Nd(3+) ion is demonstrated experimentally. The results obtained for direct upper laser level pumping of Nd:YAG R2 (869 nm) and Nd:YVO(4) (880 nm) were compared with traditional ~808-nm pump band excitation. A tunable cw Ti:sapphire laser was used as the pump source. In Nd:YAG, the oscillator slope efficiency increased by 10% and the threshold decreased by 11%. In Nd:YVO(4), the slope efficiency increased by 5% and the threshold decreased by 11%. These results agree with theory. The increase in optical efficiency indicates that laser material thermal loading can be substantially reduced.     

UV resonance Raman detection and quantitation of domoic acid in phytoplankton

Cultures of the phytoplankton diatom, Pseudonitzschia multiseries, have been harvested under controlled growth conditions ranging from late logarithmic to late stationary phase (17-58 days). The amount of domoic acid (DA) present in the growth media and in the homogenized cells has been determined by HPLC. Defined samples of media, homogenized cells, whole cells, and whole cells in media have been laser excited at 251 nm for the purpose of selectively exciting intense UV resonance Raman spectra from DA in the samples. Neither media nor cell component spectra from algae seriously interfere with DA spectra. The spectral cross sections for the dominant 1652-cm-1 mode of DA have been determined for 242-, 251-, and 257-nm excitation. Maximum sensitivities are achieved with 251-nm excitation because cross sections for DA are a maximum, and interference from other algal components becomes very small. DA concentrations that have been determined with 251-nm excitation by resonance Raman methods correlate closely with values determined independently with HPLC, especially at higher DA concentrations. The UV resonance Raman analysis of DA in phytoplankton algae is shown to be very sensitive and quantitative as well as rapid and nonintrusive.     

Two-Line Laser-Induced Fluorescence Imaging of Vibrational Temperatures in a NO-Seeded Flame

Two-dimensional temperature fields are measured in lean and sooting flames by means of two-color laser-induced fluorescence (LIF) imaging that uses seeded NO. Vibrational thermometry is performed by the probing of different vibrational ground-state levels. Spectral properties of the excited transitions within the A (2)?(+)-X (2)? system are well known from previous studies. The energy difference of 1974 cm(-1) between the (0, 0)Q(1) + P(21)(33.5) and the (0, 2)O(12)(5.5) lines offers great sensitivity in the temperature range that is relevant for combustion processes. Excitation is possible by use of a tunable KrF excimer laser on its fundamental (248-nm) and Raman shifted (in H(2), 225-nm) wavelengths. An excitation scheme for instantaneous two-line measurements by use of a single laser is developed. The possibility of single-shot measurements is discussed.