Applied Spectroscopy

The feasibility of using a two-rank database of reference spectra based on in vivo fluorescence excitation-emission matrix (EEMs) spectra to assess dominant groups of phytoplankton was explored. Twenty-six species belonging to 20 genera of seven divisions were studied. First, fluorescent characteristics of these EEMs were extracted using Daubechies-7 wavelet analysis. Second, the optimal characteristic spectra of scale vectors (SOCS) and time-series vectors (TOCS) were selected; phytoplankton of different genera were classified using Fisher linear discriminant analysis. Third, SOCS and TOCS reference spectra databases were obtained by hierarchical cluster analysis. Using non-negative least squares as the method to assess the phytoplankton, a two-rank reference spectra database was established according to their respective ability to identify the 2818 single-species samples. Using this fluorimetric technique, the correct identification rates (CIRs) for single-species samples were 88.8–100% at the genus level and 98.8–100% at the division level. Dominant species in the 465 laboratory mixtures had corresponding CIRs of 85.6% and 96.1%. Moreover, 15 of the 19 species used as dominants were correctly identified at the genus level. In 27 natural seawater samples, Prorocentrum donghaiense, Thalassiosira nordenskioldi, and Chaetoceros socialis (bloom-forming species with a density of about 10⁷ cell L⁻¹), and Alexandrium sp. (dominant species with abundance of about 10⁶ cell L⁻¹) were qualitatively identified at the genus level; other dominant species, with densities of 10⁵ to 10⁶ cell L⁻¹, were identified at the division level. The quantitative identification was relatively poor in the natural water samples, and several potential resolutions, including trying both new measuring methods and calculating methods, for future study are given.

In production agriculture, savings in herbicides can be achieved if weeds can be discriminated from crop, allowing the targeting of weed control to weed-infested areas only. Previous studies demonstrated the potential of ultraviolet (UV) induced fluorescence to discriminate corn from weeds and recently, robust models have been obtained for the discrimination between monocots (including corn) and dicots. Here, we developed a new approach to achieve robust discrimination of monocot weeds from corn. To this end, four corn hybrids (Elite 60T05, Monsanto DKC 26–78, Pioneer 39Y85 (RR), and Syngenta N2555 (Bt, LL)) and four monocot weeds (Digitaria ischaemum (Schreb.) I, Echinochloa crus-galli (L.) Beauv., Panicum capillare (L.), and Setaria glauca (L.) Beauv.) were grown either in a greenhouse or in a growth cabinet and UV (327 nm) induced fluorescence spectra (400 to 755 nm) were measured under controlled or uncontrolled ambient light intensity and temperature. This resulted in three contrasting data sets suitable for testing the robustness of discrimination models. In the blue-green region (400 to 550 nm), the shape of the spectra did not contain any useful information for discrimination. Therefore, the integral of the blue-green region (415 to 455 nm) was used as a normalizing factor for the red fluorescence intensity (670 to 755 nm). The shape of the normalized red fluorescence spectra did not contribute to the discrimination and in the end, only the integral of the normalized red fluorescence intensity was left as a single discriminant variable. Applying a threshold on this variable minimizing the classification error resulted in calibration errors ranging from 14.2% to 15.8%, but this threshold varied largely between data sets. Therefore, to achieve robustness, a model calibration scheme was developed based on the collection of a calibration data set from 75 corn plants. From this set, a new threshold can be estimated as the 85% quantile on the cumulative frequency curve of the integral of the normalized red fluorescence. With this approach the classification error was nearly constant (16.0% to 18.5%), thereby indicating the potential of UV-induced fluorescence to reliably discriminate corn from monocot weeds.

While the water permeability of the plasma membranes of mammalian cells has been studied extensively, water transport across membranes of subcellular compartments (e.g., lysosomes, macropinosomes) has been difficult to study. Here we demonstrate a new method for measuring water flux in late endosomes and lysosomes of intact living cells using time-lapse fluorescence microscopy. Cells were loaded by fluid-phase uptake with a mixture of the Lucifer Yellow dextran (LY-dex), a D₂O sensitive dye, and a D₂O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D₂O concentration and the LY-dex D₂O sensitivity was not affected by changes in pH, physiological salt, and protein concentrations. The co-loaded control dye, AF546-dex, showed no signal changes as a function of D₂O concentration. To measure membrane water flux, the LY-dex fluorescence in labeled organelles was recorded during rapid superfusion of cells with isotonic buffers prepared in D₂O. The time constant of water exchange across the lysosomal membrane of intact cells was determined by fitting the data to a single exponential function. From these data, together with the measured area of the organelles, observed water permeability for intracellular CHO-K1 lysosomes was calculated to be 5.3 × 10⁻³ ± 0.3 × 10⁻³ cm/s. This work demonstrates the feasibility of measuring water flux into subcellular organelles in live cells using LY-dex.

A new atomization device for direct atomic spectrochemical analysis has been developed that uses the theta-pinch configuration to generate a pulsed, high-energy-density plasma at atmospheric pressure. Energy from a 20-kV, 6.05-μF capacitive electrical discharge was inductively coupled to a sacrificial aluminum thin film to produce a cylindrical plasma. Current waveform analysis indicates an average power dissipation of 0.5 MW in the plasma. Electromagnetic modeling studies were used to identify theta-pinch designs possessing characteristics favorable to both plasma initiation and plasma heating. The discharge was most robust when the induced current and rate of magnetic field change were maximized. Minimizing the ratio of the coil's width to its radius was also critical. Counter to intuition, a larger diameter was found to be more successful. Spectroscopic studies indicate that the discharge forms a heterogeneous plasma with a dense, cylindrical plasma sheet confined by the walls of the discharge tube surrounding a less energetic plasma in the center. Al(II) emission in the outer plasma cylinder was temporally aligned with the induced current whereas in the center it aligns with the magnetic field. Ionization of support gas species (Ar, He, and air) was not observed, although the identity of the gas had a significant influence on the plasma reproducibility. The optimized design utilized a 5.5-turn, 19-mm-diameter theta coil with argon as the support gas. Sb(I) emission from an antimony oxide solid powder sample deposited on the thin film was observed primarily in the outer part of the plasma. Analyte emission shows contributions from magnetic compression early in the discharge and from the induced current late in the discharge. The discharge produced analytically useful signals from solid antimony oxide samples. Using spatially and temporally resolved detection, the line-to-background ratio for Sb(I) was found to be greater than 4 for emission integrated from 55 to 120 μs.

A new setup for spatially resolved laser-induced breakdown spectroscopy (SR-LIBS) is used for the first time to analyze methane–air diffusion flames. Using this configuration, background continuum emission is reduced, signal-to-background noise ratio is increased up to eight times, and spatial resolution is enhanced. The local equivalence ratio is also quantitatively estimated and the width of the secondary combustion region at a specified height above the burner is determined for two different methane flow rates. Furthermore, the threshold energy for spark formation is measured for regions inside and outside the flame. The results show that threshold energy is larger at the secondary combustion region, near the border of the flame, than inside the flame.

Bulk thermal alterations to chlorite schist occurring at temperatures above 450 °C are traditionally studied using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, or thermogravimetric analysis (TGA), but lower temperature changes that lead to structural and chemical alteration, including changes in oxidation state that are not followed by a crystalline modification, are not detectable using these techniques. In this paper we present the results of the first study to examine the application of fiber-optic reflection spectroscopy (FORS) for the detection and quantification of low-temperature thermal alterations in chlorite schist. Such changes have been observed during research on the thermal behavior of medieval (12th–13th centuries AD) chlorite cooking pots from the archaeological site of Merv, Turkmenistan. FORS was used to investigate these changes, testing the archaeological samples against a model data set of experimental reference specimens. The results demonstrate the potential of FORS for tracking low-temperature thermal alterations and offer the opportunity to examine temperatures attained by ancient chlorite vessels during their past use in cooking activities.

In this work, a study concerning the composition of Italian papers from the seventeenth to the twentieth centuries was carried out using energy dispersive X-ray fluorescence spectrometry (EDXRF), X-ray diffraction (XRD), and scanning electron microscopy coupled to energy dispersive spectrometry (SEM-EDS). The analyzed samples consisted of papers employed for drawing, writing, printing, and absorbance. Observations carried out by SEM magnified the typical paper morphology. EDXRF in combination with XRD and SEM-EDS allowed the determination of calcite, gypsum, kaolin, talc, magnesite, and dolomite, used as fillers in the production of the papers studied herein. The inks present in the handwritten and printed papers, investigated by SEM-EDS and μ-EDXRF, were synthetic, Fe based, and iron gall inks.

The competitive adsorption of citrate-capped Ag and Au nanoparticles (∼25 nm in diameter) onto a poly(4-vinyl pyridine) (P4VP) surface has been investigated by means of Raman scattering spectroscopy. The P4VP film prepared on a glass slide was too thin for its normal Raman spectrum to be observed, but the Raman peaks of P4VP could be detected upon the adsorption of Ag and/or Au nanoparticles onto the film, due to the surface-enhanced Raman scattering (SERS) effect associated with the localized surface plasmon of Ag and/or Au nanoparticles. Neither quartz crystal microbalance nor atomic force microscopy (AFM) nor scanning electron microscopy (SEM) methodologies can distinguish between Ag and Au nanoparticles during their adsorption onto P4VP, but it is possible through Raman scattering spectroscopy because Ag (though not Au) nanoaggregates are SERS active at 514.5 nm excitation, while both Ag and Au nanoaggregates are SERS active at 632.8 nm excitation. Coupled with the AFM data, we were thus able to infer that about 120 Ag nanoparticles per 1 μm² were adsorbed, along with 60 Au nanoparticles per 1 μm², onto the P4VP film over a period of 1.5 h from a 1:1 mixture of Ag and Au sols at 1.6 nM each.

Surface contamination of surface-enhanced Raman (SERS)-active metallic substrates has been a limitation to the utility of SERS as an analytical technique, potentially affecting surface coverage, spectral reproducibility, and analytical limits of detection. We have developed a simple and versatile cleaning method for SERS-active Ag nanorod arrays that consists of a short (4 min) exposure of the substrate to an Ar⁺ plasma in a low-pressure environment. The findings presented here demonstrate that this cleaning procedure essentially eliminates organic background contamination. This procedure works equally well for self-assembled monolayers of thiolates that strongly adsorb onto Au and Ag surfaces. For SERS-active surfaces composed of arrays of Ag nanorods prepared by oblique-angle vapor deposition, we investigated the (1) Raman band intensities, (2) nanorod morphology via scanning electron microscopy, and (3) surface hydrophobicity via static contact angle measurements, as a function of exposure time of the Ag nanorods to the Ar⁺ plasma. Short (4 min) exposure to Ar⁺ plasma eliminated background contamination but decreased the observed SERS intensity for re-adsorbed analytes by approximately a factor of 2 while leaving the nanorod morphology essentially unchanged. Prolonged exposure to Ar⁺ plasma (>10 min) resulted in substantial morphological changes of the Ag nanorod lattice and led to a decrease in the observed SERS intensities by a factor of 10. The results presented here suggest that Ar⁺ plasma cleaning is an efficient process for removing carbonaceous and organic contamination as well as thiolate monolayers from SERS-active Ag surfaces, as long as the plasma conditions and exposure times are carefully monitored.

We present here a fully automated spectral baseline-removal procedure. The method uses a large-window moving average to estimate the baseline; thus, it is a model-free approach with a peak-stripping method to remove spectral peaks. After processing, the baseline-corrected spectrum should yield a flat baseline and this endpoint can be verified with the χ²-statistic. The approach provides for multiple passes or iterations, based on a given χ²-statistic for convergence. If the baseline is acceptably flat given the χ²-statistic after the first pass at correction, the problem is solved. If not, the non-flat baseline (i.e., after the first effort or first pass at correction) should provide an indication of where the first pass caused too much or too little baseline to be subtracted. The second pass thus permits one to compensate for the errors incurred on the first pass. Thus, one can use a very large window so as to avoid affecting spectral peaks—even if the window is so large that the baseline is inaccurately removed—because baseline-correction errors can be assessed and compensated for on subsequent passes. We start with the largest possible window and gradually reduce it until acceptable baseline correction based on the χ²-statistic is achieved. Results, obtained on both simulated and measured Raman data, are presented and discussed.

Projective transformation is a mathematical correction (implemented in software) used in the remote imaging field to produce distortion-free images. We present the application of projective transformation to correct minor alignment and astigmatism distortions that are inherent in dispersive spectrographs. Patterned white-light images and neon emission spectra were used to produce registration points for the transformation. Raman transects collected on microscopy and fiber-optic systems were corrected using established methods and compared with the same transects corrected using the projective transformation. Even minor distortions have a significant effect on reproducibility and apparent fluorescence background complexity. Simulated Raman spectra were used to optimize the projective transformation algorithm. We demonstrate that the projective transformation reduced the apparent fluorescent background complexity and improved reproducibility of measured parameters of Raman spectra. Distortion correction using a projective transformation provides a major advantage in reducing the background fluorescence complexity even in instrumentation where slit-image distortions and camera rotation were minimized using manual or mechanical means. We expect these advantages should be readily applicable to other spectroscopic modalities using dispersive imaging spectrographs.

This work presents a theoretical study of a heat transfer effect, taking into account the heat transfer within the heated sample and out to the surrounding medium. The analytical solution is used to model the thermal lens and thermal mirror effects and the results are compared with the finite element analysis (FEA) software solution. The FEA modeling results were found to be in excellent agreement with the analytical solutions. Our results also show that the heat transfer between the sample surface and the air coupling fluid does not introduce an important effect over the induced phase shift in the sample when compared to the solution obtained without considering axial heat flux. On the other hand, the thermal lens created in the air coupling fluid has a significant effect on the predicted time-dependent photothermal signals. When water is used as fluid, the heat coupling leads to a more significant effect in both sample and fluid phase shift. Our results could be used to obtain physical properties of low optical absorption fluids by using a reference solid sample in both thermal lens and thermal mirror experiments.

The ultra-high-vacuum growth of thin chromium and aluminum films on single diamond crystals at various temperatures was monitored by means of infrared spectroscopy, which provides instant information on electronic and morphological properties of the produced metal films. In accordance with atomic force microscopy results, spectra for Al indicate a clear island-like growth in the beginning followed by a transition to a continuous layer. Cr growth results in much smoother films because of a special growth mechanism. Analysis of infrared spectra of both kinds of ultrathin metal layers yields a high DC conductivity well above 10⁶ S/m starting from only a few nanometers thickness for sufficiently high substrate temperatures.

A sensor using a single distributed-feedback (DFB) diode laser at 1.57 μm for the simultaneous measurement of CO₂ and CO concentration at elevated temperatures is developed. A proper line pair near 6361.250 and 6361.344 cm⁻¹ is chosen based on absorption strength, separation of the two lines, and isolation from interference of neighboring transitions of the major combustion gases. The concentrations of CO₂ and CO are inferred from their wavelength modulation spectroscopy (WMS) 1f/-normalized absorption-based WMS-2f signal peak heights. The CO₂ and CO concentration measurements are within 3.3% and 5% of the expected values over the full temperature range.

Cubic indium hydroxide nanomaterials were obtained by a low-temperature soft-chemical method without any surfactants. The transition of nano-cubic indium hydroxide to cubic indium oxide during dehydroxylation has been studied by infrared emission spectroscopy. The spectra are related to the structure of the materials and the changes in the structure upon thermal treatment. The infrared absorption spectrum of In(OH)₃ is characterized by an intense OH deformation band at 1150 cm⁻¹ and two O–H stretching bands at 3107 and 3221 cm⁻¹. In the infrared emission spectra, the hydroxyl-stretching and hydroxyl-bending bands diminish dramatically upon heating, and no intensity remains after 200 °C. However, new low intensity bands are found in the OH deformation region at 915 cm⁻¹ and in the OH stretching region at 3437 cm⁻¹. These bands are attributed to the vibrations of newly formed InOH bonds because of the release and transfer of protons during calcination of the nanomaterial. The use of infrared emission spectroscopy enables the low-temperature phase transition brought about through dehydration of In(OH)₃ nanocubes to be studied.