100% efficient sub-nanoliter sample introduction in laser-induced breakdown spectroscopy and inductively coupled plasma spectrometry: implications for ultralow sample volumes.

Recently, nanoflow nebulizers with low-volume drain-free spray chambers became available for inductively coupled plasma-mass spectrometry application for analysis of very small sampling volumes. The present technical note reports on a different approach for 100% efficient subnanoliter sample introduction, the application of monodisperse piezoelectric microdroplet dispensers which generate 40-50 microm droplets with high reproducibility if nozzles of 30 microm diameter are applied. The droplets having volumes below 0.1 nL can be introduced loss-free and without plasma loading, with frequencies up to approximately 100 Hz into analytical plasmas. In this technical note, the analytical figures of merit of laser-induced breakdown spectroscopy and inductively coupled plasma-optical emission spectrometry with single droplet introduction are reported using Ca and Au standard solutions as examples. Future engineering is required to reduce the total sample volumes from the relatively large sample reservoir of the current study, thereby reducing potential issues of washout while enabling analysis of ultralow total sample volumes.

Alternative statistical methods for spectral data processing: applications to laser-induced breakdown spectroscopy of gaseous and aerosol systems.

A new spectral data processing scheme based on the standard deviation of collected spectra is compared with the traditional ensemble-averaging of laser-induced breakdown spectroscopy (LIBS)-based spectral data for homogenous (i.e., pure gas phase) systems and with a LIBS-based traditional conditional spectral analysis scheme for non-homogenous (e.g., aerosol system) analyte systems under discrete particle loadings. The range of conditions enables quantitative assessment of the analytical approaches under carefully controlled experimental conditions. In the homogeneous system with gaseous carbon dioxide producing the carbon atomic emission signal, the standard deviation method provided a suitable metric that is directly proportional to the analyte signal and compares favorably with a traditional ensemble averaging scheme. In contrast, the applicability of the standard deviation method for analysis of non-homogenous analyte systems (e.g., aerosol systems) must be carefully considered. It was shown both experimentally and via Monte Carlo simulations that the standard deviation approach can produce an analyte response that is monotonic with analyte concentration up to a point at which the analyte signal starts to transition from a non-homogeneous system to a homogeneous systems (i.e., around a 50% sampling point for aerosol particles). In addition, the standard deviation spectrum is capable of revealing spectral locations of non-homogeneously dispersed analyte species without a priori knowledge.

Excimer laser photofragmentation/fragment detection for analysis of the oxygenated hydrocarbon ethyl-3-ethyoxypropionate: implications for atmospheric monitoring.

In the present study, the use of ArF excimer laser photofragmentation/fragment detection (PF/FD) is considered for the characterization of gaseous ethyl-3-ethyoxypropionate (EEP), a representative solvent within the class of oxygenated volatile organic compounds (VOCs). PF/FD measurements of EEP resulted in identification of the resulting photofragments as C2 and CH, allowing photofragmentation pathways to be proposed for the parent molecule for the first time. In addition, PF/PD measurements of EEP recorded in the presence of sodium-based aerosol particles resulted in a reduced signal from the EEP, indicating adsorption onto the particulates, thereby demonstrating proof-of-concept that PF/FD can be used to study heterogeneous chemical reactions relevant to atmospheric chemistry. Finally, both time-integrated and time-resolved measurements of the EEP photofragmentation signal in varying concentrations of oxygen revealed that oxygen effectively acts as a dynamic quencher of the EEP signal. In consideration of oxygen quenching, care must be taken in calibrating/analyzing data in selected applications of PF/FD for VOC characterization (such as combustion emission monitoring) in order to account for the potential variability of oxygen concentration.

Laser-induced breakdown spectroscopy for ambient air particulate monitoring: correlation of total and speciated aerosol particle counts.

A statistical analysis of ambient air particle monitoring, namely PM2.5, is presented to elucidate the correlations between laser-induced breakdown spectroscopy (LIBS)-based speciated aerosol monitoring and non-speciated aerosol monitoring (i.e., total particle counts). LIBS was used in a real-time, conditional-processing mode to identify individual aerosol particles containing detectable quantities of either calcium or sodium, as based on the resulting atomic emission signals. Using this technique, real-time measurements of speciated aerosol particle concentrations and analyte mass concentrations were evaluated for a total of 60 1-hour sampling periods spread over a 5-week period. For each 1-hour sampling period, total aerosol counts were simultaneously monitored using a commercial light scattering-based instrument. Over the 30 sampling periods, aerosol counts (both total and LIBS-based) were found to vary by more than one order of magnitude. For aerosol particles in the 500 nm to 2.5 microm size range, significant correlations were found between the two sampling methods, resulting in correlation coefficients (r2) ranging from 0.22 to 0.93. In addition, transient fluctuations in aerosol counts on a timescale of 5 to 10 minutes were successfully observed simultaneously with the two monitoring techniques, thereby demonstrating the temporal resolution of LIBS.

Plasma-particle interactions in a laser-induced plasma: implications for laser-induced breakdown spectroscopy.

The interaction between laser-induced plasmas and individual particles controls the rate of particle dissociation and subsequent atomic diffusion and emission processes, with implications for single-particle spectroscopy, as well as materials synthesis and other plasma sources. It is demonstrated through quantitative plasma imaging studies that discrete particles dissociate on a time scale of tens of microseconds within plasmas formed by 300-mJ Nd:YAG laser pulses. Significant spatial nonhomogeneity, as measured by localized atomic emission from particle-derived calcium atoms, persists on a comparable time scale, providing a measure of their average atomic diffusion rate of 0.04 m(2)/s. In addition, the resulting calcium atomic emission is explored using image analysis as well as traditional spectroscopic analysis.

Analysis of dense-medium light scattering with applications to corneal tissue: experiments and Monte Carlo simulations.

Dense-medium scattering is explored in the context of providing a quantitative measurement of turbidity, with specific application to corneal haze. A multiple-wavelength scattering technique is proposed to make use of two-color scattering response ratios, thereby providing a means for data normalization. A combination of measurements and simulations are reported to assess this technique, including light-scattering experiments for a range of polystyrene suspensions. Monte Carlo (MC) simulations were performed using a multiple-scattering algorithm based on full Mie scattering theory. The simulations were in excellent agreement with the polystyrene suspension experiments, thereby validating the MC model. The MC model was then used to simulate multiwavelength scattering in a corneal tissue model. Overall, the proposed multiwavelength scattering technique appears to be a feasible approach to quantify dense-medium scattering such as the manifestation of corneal haze, although more complex modeling of keratocyte scattering, and animal studies, are necessary.

Hydrogen leak detection using laser-induced breakdown spectroscopy.

Laser-induced breakdown spectroscopy (LIBS) is investigated as a technique for real-time monitoring of hydrogen gas. Two methodologies were examined: The use of a 100 mJ laser pulse to create a laser-induced breakdown directly in a sample gas stream, and the use of a 55 mJ laser pulse to create a laser-induced plasma on a solid substrate surface, with the expanding plasma sampling the gas stream. Various metals were analyzed as candidate substrate surfaces, including aluminum, copper, molybdenum, stainless steel, titanium, and tungsten. Stainless steel was selected, and a detailed analysis of hydrogen detection in binary mixtures of nitrogen and hydrogen at atmospheric pressure was performed. Both the gaseous plasma and the plasma initiated on the stainless steel surface generated comparable hydrogen emission signals, using the 656.28 Halpha emission line, and exhibited excellent signal linearity. The limit of detection is about 20 ppm (mass) as determined for both methodologies, with the solid-initiated plasma yielding a slightly better value. Overall, LIBS is concluded to be a viable candidate for hydrogen sensing, offering a combination of high sensitivity with a technique that is well suited to implementation in field environments.

Calibration effects for laser-induced breakdown spectroscopy of gaseous sample streams: analyte response of gas-phase species versus solid-phase species.

The effects of analyte phase on the calibration response for laser-induced breakdown spectroscopy is investigated for a range of carbon species. Significant differences in the atomic emission signal from carbon were observed when comparing calibration streams of gas-phase and submicrometer-sized solid-phase carbon species. The resulting calibration curve slopes varied by a factor of 8 over a comparable range of atomic carbon concentrations for five different analyte sources, while the plasma electron density and temperature remained essentially constant. The current findings challenge a widely held assumption that complete dissociation of constituent species within a highly energetic laser-induced plasma results in independence of the analyte atomic emission signal on the analyte source. A physical model of the plasma-analyte interaction is proposed that provides a framework to account for the observed dependence on the physical state of the analyte.

Feasibility of detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy.

The detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy (LIBS) is investigated using aerosolized Bacillus spores. Spores of Bacillus atrophaeous, Bacillus pumilus, and Bacillus stearothemophilus were introduced into an aerosol flow stream in a prescribed manner such that single-particle LIBS detection was realized. Bacillus spores were successfully detected based on the presence of the 393.4- and 396.9-nm calcium atomic emission lines. Statistical analyses based on the aerosol number density, the LIBS-based spore sampling frequency, and the distribution of the resulting calcium mass loadings support the conclusion of individual spore detection within single-shot laser-induced plasmas. The average mass loadings were in the range of 2-3 fg of calcium/Bacillus spore, which corresponds to a calcium mass percentage of approximately 0.5%. While individual spores were detected based on calcium emission, the resulting Bacillus spectra were free from CN emission bands, which has implications for the detection of elemental carbon, and LIBS-based detection of single spores based on the presence of magnesium or sodium atomic emission was unsuccessful. Based on the current instrumental setup and analyses, real-time LIBS-based detection and identification of single Bacillus spores in ambient (i.e., real life) conditions appears unfeasible.

Analysis of polyethylene wear debris using micro-Raman spectroscopy: a report on the presence of beta-carotene.

This paper describes micro-Raman spectroscopy of ultra-high molecular weight polyethylene wear debris isolated from revised knee replacements. The novel application of micro-Raman spectroscopy to the analysis of in vivo-generated wear debris was used to evaluate the chemical nature of individual, retrieved polyethylene particles. The analysis revealed the presence of beta-carotene on particles from both synovial fluid and tissue samples. Raman analysis of retrieved polyethylene tibial inserts also revealed localized beta-carotene signals within the primary wear region. In this paper, a mechanism is suggested that may account for the coupling of beta-carotene and polyethylene wear debris. We also discuss the origin of beta-carotene within the implanted joint and the implications that beta-carotene, an anti-oxidant, has for the overall host response to polyethylene orthopedic components.

Characterization of submicron polyethylene wear debris from synovial-fluid samples of revised knee replacements using a light-scattering technique.

The objectives of this study were to determine whether submicron-sized ultra-high-molecular-weight polyethylene (UHMWPE) wear debris was present in synovial fluid surrounding knee implants, and to report on the utility of a light-scattering technique for the in situ analysis of submicron-sized wear debris. The measured light-scattering coefficients of the implant synovial fluid samples were significantly larger than the coefficients of the control samples (p < 0.0001). The enhanced light scattering was attributed to the presence of submicron UHMWPE particles. This is consistent with light-scattering considerations and a Raman spectroscopy survey of synovial fluid. The mean particle volume fraction of UHMWPE was 1.11 x 10(-5) cm3/mL for the six implant samples, with mean particle diameters in the range of 200-300 nm. The UHMWPE volume fractions were found to differ by a factor of 2 between the osteolytic and nonosteolytic cases. The current findings warrant further work to determine the role of submicron polyethylene debris in the wear mechanisms of biomaterials and in the development of osteolysis following total knee replacement.

The effect of specific hormones on fibrinolysis in pregnancy.

OBJECTIVE: Plasminogen activator inhibitor-1, the major serum protease inhibitor of fibrinolysis, increases steadily during pregnancy. The study objective was to examine four hormones, namely, estradiol-17 beta, progesterone, prolactin, and hydrocortisone to determine their individual contributions in the production of tissue plasminogen activator antigen, plasminogen activator inhibitor-1 antigen, and plasminogen activator inhibitor-1 activity. STUDY DESIGN: Human umbilical vein endothelial cells were grown with physiologic third-trimester concentrations of the above hormones, and fibrinolytic parameters were measured. RESULTS: Of the four hormones evaluated, only hydrocortisone significantly increased plasminogen activator inhibitor-1 antigen and activity at both concentrations tested (p < 0.001). Estradiol-17 beta significantly increased tissue plasminogen activator antigen and progesterone significantly decreased tissue plasminogen activator antigen, but neither affected the overall fibrinolytic balance. CONCLUSION: Hydrocortisone demonstrated antifibrinolytic properties at physiologic concentrations in pregnancy, suggesting that there may be a role for hydrocortisone in the prothrombotic tendency associated with pregnancy. The overall process of fibrinolysis was unaffected by estradiol-17 beta, progesterone, or prolactin.

Induction of lambda prophage by 213 nm laser radiation: a quantitative comparison with 193 nm excimer radiation using image analysis.

We compared the DNA damage produced by radiation from two UV laser wavelengths, 213 nm and 193 nm, with that produced by noncoherent 254 nm radiation. Following irradiation of Escherichia coli BR339, a bacteriophage lambda lysogen containing the lacZ gene, pro-phage induction was measured by assaying for beta-galactosidase. Because of the limited penetration by UV laser wavelengths an agar overlay of the lysogen was used as the irradiation target. Irradiation of 254 nm was performed in buffer suspension followed by transfer of 5 microL spots onto assay plants. Computer image analysis was used to monitor the rate of product formation, observed as an increase in optical density of the irradiated zones on assay plates. We found that the rate of product formation was a more reproducible unit of comparison than the optical density present at the end of the reaction. Although the rate of product formation was not linearly related to enzyme concentration, the data could be fit to a simple logarithmic function. Using this method, we concluded that the DNA damaging ability of 213 nm radiation was 10 times more efficient than 193 nm radiation and about 100 times less efficient than 254 nm noncoherent radiation.

Electron paramagnetic resonance spectroscopy of free radicals in corneal tissue following excimer laser irradiation.

BACKGROUND AND OBJECTIVES: Free radicals, detected previously in corneal tissue following 193 nm laser irradiation, may be important agents in the laser/tissue interaction. Electron paramagnetic resonance spectroscopy (EPR) has been used to examine such radical formation in detail. STUDY DESIGN/MATERIALS AND METHODS: Bovine corneal strips were frozen in liquid nitrogen, irradiated with excimer laser pulses, and assayed by EPR. Exposure conditions were varied to study radical formation dependence on laser intensity and repetition. Results were measured against a quantifiable standard to calculate radical quantum yield. RESULTS: Either weak or intense laser fluences produced comparable tissue EPR signals. Radicals accumulated in frozen tissue for at least 10 initial ablation pulses. Radical quantum yield in cornea was 0.15%. CONCLUSION: Corneal radical formation is largely a photochemical process driven by the 193 nm laser radiation. Reactive radical species are produced in substantial numbers and likely have a significant clinical role.

Potent pituitary-gonadal axis suppression and extremely low anaphylactoid activity of a new gonadotropin releasing hormone (GnRH) receptor antagonist "azaline B".

We report here the biological characterization of azaline B, a new gonadotropin releasing hormone (GnRH) receptor antagonist, with the following amino acid sequence: [Ac-D-Nal1, D-Cpa2, D-Pal3, Aph5(atz), D-Aph6(atz), Ilys8, D-Ala10]-GnRH. Azaline B was shown to suppress several reproductive processes in rats including ovulation, and had very low anaphylactoid activity compared with other GnRH antagonists. Azaline B inhibited histrelin (a GnRH agonist)-mediated follicle stimulating hormone (FSH) and luteinizing hormone (LH) release from cultured rat pituitary cells. Three antagonists ([Nal-Glu]-GnRH, [Nal-Lys]-GnRH ("antide"), and azaline B) inhibited 0.1 nM histrelin-mediated gonadotropin release to baseline levels with EC50 values of approximately 0.6 nM. Azaline B, when injected s.c. into rats on the afternoon of proestrus, was more potent at inhibiting ovulation than either [Nal-Glu]-GnRH or [Nal-Lys]-GnRH. The relative order of antiovulatory potencies of the three antagonists was azaline B > [Nal-Glu]-GnRH > [Nal-Lys]-GnRH. Similar azaline B potency was shown by its ability to suppress gonadotropin levels in castrated rats. The improved selectivity of azaline B was demonstrated when it was compared with other GnRH antagonists in the cutaneous anaphylactoid assay (local wheal response) in rats. Results with azaline B were not significantly different from results with vehicle in this assay. [Nal-Glu]-GnRH was more than twice as potent as [Nal-Lys]-GnRH in stimulating a wheal response. Furthermore, the maximal wheal response produced by azaline B was only 0.6 times that of [Nal-Lys]-GnRH, currently one of the most selective antagonists identified. Finally, both azaline B and [Nal-Lys]-GnRH were much less potent than [Nal-Glu]-GnRH in the guinea pig cardiopulmonary anaphylactoid assay after i.v. administration. These data show that azaline B is a potent and selective GnRH receptor antagonist with little or no anaphylactoid activity in animal models, and therefore has potential for use in the treatment of many reproductive endocrine disorders, as well as for use as a contraceptive.

Gap junction regulation in the uterus and ovaries of immature rats by estrogen and progesterone.

The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (alpha 1 = Cx43, beta 1 = Cx32, beta 2 = Cx26) in the uterus and ovaries of immature rats by immunohistochemistry, electron microscopy and northern blot analysis. E2 induced the expression of alpha 1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2-induced alpha 1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of beta 1 and beta 2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical-lateral cell borders of the secretory epithelium following P-treatment. The co-administration of E2 + P had a synergistic effect on beta 1 and beta 2 expression in the luminal epithelium, but an inhibitory effect on beta 2 expression in glandular epithelium. Myometrial GJs were detected in freeze-fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated beta 1 and beta 2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.

Dynamics of ablation plume particles generated during excimer laser corneal ablation.

BACKGROUND AND OBJECTIVE: Although the empirical characteristics of ArF excimer laser corneal ablation have been well documented, the exact ablation mechanisms are not well understood. The present paper reports a quantitative analysis of corneal ablation plumes using in situ time resolved laser light scattering and Raman spectroscopy. STUDY DESIGN/MATERIALS AND METHODS: Bovine corneas were used as the ArF excimer laser ablation targets. Light scattering data were recorded from the ablation plume as a function of height above the tissue surface and as function of delay time with respect to the ablative ArF laser pulse. RESULTS: Raman spectra of the ablation plume allow identification of the particles as water. Mean plume particle diameters are found to decrease with height, while the particle volume fractions are relatively constant. The total volume of plume particles correlates well with the total volume of water in the ablated corneal tissue. CONCLUSION: The finding of a non-evolving plume composed of water spherules, combined with the excellent agreement between total volume of water in the plume and the content of water in the ablated corneal tissue, support the concept of photodecomposition or "cold ablation" for corneal tissue during ArF excimer laser ablation.

Enhanced ArF laser absorption in a collagen target under ablative conditions.

The time-resolved transmission of collagen targets during ArF excimer laser ablation has been measured. The transmitted excimer pulse measurements demonstrate enhanced media attenuation that is a nonlinear function of incident laser fluence. Forward scattering of the transmitted pulse has been assessed to be a negligible contributor to the observed phenomena. Results of pump/probe interrogation of the ablation site indicate that the onset of enhanced attenuation occurs on the time course of the ablating laser pulse and persists for times on the order of hundreds of microseconds.

Preclinical evaluation of norgestimate, a progestin with minimal androgenic activity.

Norgestimate is a novel progestin with highly selective progestational activity and minimal androgenicity. In rabbits, norgestimate binds to uterine progestin receptors, stimulates the endometrium, and inhibits ovulation. Norgestimate acts directly on target organs, stimulating rabbit endometrium when injected into the uterine cavity and inhibiting luteinizing hormone-releasing hormone-stimulated luteinizing hormone release in dispersed rat pituitary cells in culture. Norgestimate has no estrogenic activity, and like other progestins, it suppresses the action of estrogen. Unlike some other progestins, it is relatively free of androgenic activity. Norgestimate and its 17-deacetylated metabolite demonstrate very poor affinity for androgen receptors compared with levonorgestrel and gestodene and do not exhibit androgenic activity when measured as the stimulation of prostatic growth in immature rats. Norgestimate's lack of affinity for human sex hormone-binding globulin is further evidence of its minimal androgenicity.

PIP: The androgenic activity of progestins used in oral contraceptives (OCs) may be responsible for the adverse changes in lipid and lipoprotein metabolism, so researchers are continuing to work on developing progestins that reduce androgenicity. They have developed norgestimate (NGM) which is a progestin with improved selectivity. i.e., extent of maximization of progestational potency and minimization of androgenic potency. The receptor binding affinities of ngm and its major metabolite, 17-deacetylated norgestimate for the progestin receptor are on par with progesterone. Their binding affinities are only 0.003 and 0.013 times that of dihydrotestosterone, respectively, while the affinities for levonorgestrel and gestodene are 0.220 and 0.154 times that of dihydrotestosterone, respectively. They have almost no affinity for human sex hormone binding globulin (SHBG) in vitro. clinical research of 40 women using combined OCs with NGM and ethinyl estradiol over 4 cycles shows that NGM does not prevent estrogen-induced rises in SHBG levels indicating its minimal androgenicity. In addition, NGM successfully suppresses ovulation in rabbits, rats, hamsters, and mice by preventing the preovulatory rise of luteinizing hormone and, in rats, by targeting the hypothalamic/pituitary axis. NGM also allows rats and rabbits to effectively maintain pregnancy by stimulating the endometrium. It also has no estrogenic activity in vivo and does not bind to estrogen receptors in vitro. Specifically NGM prevents estrogen-induced vaginal cornification. These findings from various preclinical research and 1 clinical study demonstrate NGM to be a selective progestin which reduces androgenicity.

Role of metal additives in light scattering from flame particulates.

The scattering and extinction behavior of multicomponent particles formed in flame systems is significant in a number of research areas and practical applications, such as the production of superconducting and other ceramic materials by means of the aerosol route as well as pollutant suppression through the use of metal additives. The objective of this study is to assess the role of iron pentacarbonyl vapor addition on the light scattering and extinction behavior of flame soot. Calculations were carried out by using the scattering models for homogeneous and coated spheres and comparisons were made between the particle diameters and volume fractions. In addition, scattering, absorption, and dynamic light-scattering measurements at the wavelength of 488 nm in a premixed propane-oxygen flame with a fuel-equivalence ratio of ? = 2.4 unseeded and seeded with iron pentacarbonyl vapor 0.32% by weight iron to fuel were performed. The refractive index and number densities of the soot particles in the unseeded flame we determined as functions of position above the burner by combining the scattering and absorption measurements with the particle size-distribution parameters determined from photocorrelation. In the seeded flames the soot particles were found to contain iron oxide throughout the flame. Thus the data were analyzed by using both the scattering-absorption model for coated spheres and the Maxwell-Garnett relation for the effective refractive index. Differences up to 131% in particle-volume fractions were found from the data analysis by using the constant and variable effective index of the mixture (soot plus iron oxide). The results of the coated-sphere analysis are discussed and the effects of particle agglomeration on the inference of particle-volume fractions are assessed. It is concluded that the effects of particle optical inhomogeneity in the analysis of scattering and absorption data from multicomponent particles cannot be neglected.