The correlation of individual results in laser microanalysis applied to histological slices

Summary This work is a continuation of investigations concerning the applicability of laser analysis in histochemical techniques (Kozik et al., 1970a, b; Kozik et al., 1971a, b).Agar standards containing known amounts of Pb were subjected to laser microanalysis and optical densities of the resulting Pb spectral bands as well as volumes of the resultant craters were determined.—Based on mathematical considerations and equation has been educed by means of which the experimentally obtained photometric readings may be converted into densities corresponding to mean crater volumes. In that way it is possible to eliminate inconveniences resulting from diversity of individual crater volumes and from eventual differences in the degree of atomic excitation between samples. Using this method, the concentrations of the investigated element in histological slices become expressed in percentages.     

Reliability of an electrophoretic and image processing analysis of human skeletal muscle taken from m. vastus lateralis

The relative content of myosin heavy chain (MHC) isoforms IIb, IIa and I in human skeletal muscle taken from the m. vastus lateralis of 30 healthy male subjects was analysed using mini-gel electrophoresis. Repeated electrophoretic gels utilizing the same methods were produced for all subjects and the determination of MHC protein bands was performed using a digital scanner and National Institutes of Health (NIH) Image software and laser densitometry. A comparison between the NIH Image processing technique and laser densitometry revealed differences of 6.47%, 6.35% and 6.84% between these measurement techniques for MHC-IIb, -IIa and -I isoforms, respectively. The percentage technical error of measurement (TEM%) between electrophoretic gels was shown to be 19.1%, 17.8% and 14.2%, with regard to percentage of occurrence of MHC-IIb, -IIa and -I isoforms respectively. The variation in electrophoretic gel analyses was shown to be 5.7%, 7.3% and 5.5%, with regard to the percentage of MHC-IIb, -IIa and -I isoforms respectively. Intra-class correlations comparing NIH Image and laser densitometry produced r values in the range 0.38–0.63. Comparisons between and within gel analyses produced r values in the range 0.59–0.94 and 0.93–0.98, respectively. Analyses of variance revealed no significant differences (P < 0.05) between analysis techniques, between␣gels or within gels for the measurement of MHC-IIb, -IIa and -I isoforms. The inter-gel error between fibre subgroups was moderate for the two type-II MHC populations and less for type-I MHC; the intra-individual error in the measuring technique used for classifying the MHC-IIb, -IIa and -I protein bands was small. The results obtained in this investigation showed consistent trends which may reflect a false classification of the type-II MHC populations for the inter-gel and intra-individual analyses. The NIH Image software and digitizing process was shown to be a valid and reliable method for distinguishing between MHC protein bands of human skeletal tissue as separated by mini-gel electrophoretic techniques. Accepted: 6 January 1997     

Quantum Well Energetics of an n = 2 Ruddlesden–Popper Phase Perovskite

A comprehensive investigation of the electronic energy levels of an n = 2 Ruddlesden–Popper phase perovskite is presented. Ultraviolet and inverse photoemission spectroscopies are used to probe the density of states in the valence and conduction bands, respectively, of the quasi‐2D perovskite, butylammonium cesium lead iodide (BA₂CsPb₂I₇). By comparing experimental spectra with calculated projected density of states, the contributions from Cs, Pb, and I to the quantum well states are identified, and distinguished from those of the organic ligand barrier layer. The ionization energy, electron affinity, and exciton binding energy of this material are derived. The energetics of the quantum well structure are discussed in terms of the number of Pb‐halide layers. The resulting energy diagram suggests that a type‐I heterojunction would be formed with the n = 1 BA₂PbI₄. Finally, surface photovoltage performed via Kelvin probe force microscopy is used to evaluate band bending at the surface of the BA₂CsPb₂I₇ thin films.     

The assay of acid phosphatase activity by means of a laser method

Summary A new method of quantification of the acid phosphatase activity assay by means of laser rays along with spectrography and photometry is described. Calculated S values, representing differences between densities of the spectral bands corresponding to Pb and Ag account for the amount of acP activity.The presented laser method enables a relative evaluation of the acP activity in a given region, yielding results which can be objectively expressed by figures.A special advantage of the presented method is, that it allows to compare the individual acP activities in different regions of the investigated organ, which may be of importance for the quantitative chemoarchitectonic of the brain.     

FTIR Emission Spectra of Bacteriorhodopsin in a Vibrational Excited-State

Vibrational IR-emission spectra of bacteriorhodopsin (bR) were recorded under continuous illumination with visible light at room temperature. They contain selective information about the chromophore, Schiff base, and opsin. The spectral bands were identified by comparing the data with resonance Raman and IR absorption data. The IR-emission spectra were shown to contain a set of bands characteristic for both all-trans (bR₅₆₈) and 13-cis conformations (K₆₁₀-like intermediate) simultaneously. Variation of spectral composition and the intensity of visible light illumination influenced the spectral traces and intensity distribution between them. Greater intensity of deformational vibrations suggests distorted retinal structure in the vibrationally excited ground electronic state. The origin of the emitting species of bR is discussed.     

WAVELENGTH DEPENDENCY OF THE MAXIMUM QUANTUM YIELD OF CARBON FIXATION FOR TWO RED TIDE DINOFLAGELLATES,HETEROCAPSA PYGMAEA AND PROROCENTRUM MINIMUM (PYRROPHYTA): IMPLICATIONS FOR MEASURING PHOTOSYNTHETIC RATES1

The influence of photoadaptive state on the spectral dependency of the maximum quantum yield for carbon fixation was determined for two red tide dinoflagellates, Heterocapsa pygmaea Loeblich, Schmidt, et Sherley and Prorocentrum minimum Pavillard. Cultures were acclimated to green, blue, red, and white light. The spectral dependency in the light-limited slope of the photosynthesis–irradiance curves (α) was measured with carbon action spectra that, when divided by the spectrally weighted absorption coefficient, provided estimates of the maximum quantum yield (φ_max) for carbon fixation. Values of φ_max varied with wavelength within each culture condition as well as between different culture conditions. The degree to which the spectral dependency in φ_max was influenced by the presence of photoprotective carotenoids and/or energy imbalances between photosystems I and II was assessed for both dinoflagellates. The impact of photoprotective pigmentation on the spectral dependency of φ_max was most significant for cells grown under high light conditions reflecting the enrichment of diadinoxanthin. Energy imbalances between the photosystems was assessed by quantifying enhancement effects on spectral φ_max in the presence of background illumination. Under our experimental conditions, enhancement effects on carbon action spectra were evident for H. pygmaea under nearly all growth conditions but were not detectable for P. minimum under any growth condition. We hypothesize that sensitivity to enhancement effects reflected differences in the structure of the photosynthetic machinery of these two peridinin-containing dinoflagellates. While measurements of φ_max are sensitive to the color of the light within an incubator, the relative impact on the spectral dependency of a was less than the wavelength dependency associated with the cellular absorption properties. Finally we used our data to validate an approach proposed by others to aid in the correction of photosynthetic measurements where the in situ spectral light field cannot be easily mimicked. The average error using this approach was 8%, which was significantly less than the error associated with ignoring the spectral dependency in α.     

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI–TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20 kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI–TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI–TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI–TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.     

Model-free analysis for large proteins at high magnetic field strengths

Protein backbone dynamics is often characterized using model-free analysis of three sets of ¹⁵N relaxation data: longitudinal relaxation rate (R ₁), transverse relaxation rate (R ₂), and ¹⁵N–{H} NOE values. Since the experimental data is limited, a simplified model-free spectral density function is often used that contains one Lorentzian describing overall rotational correlation but not one describing internal motion. The simplified spectral density function may be also used in estimating the overall rotational correlation time, by making the R ₂/R ₁ largely insensitive to internal motions, as well as used as one of the choices in the model selection protocol. However, such approximation may not be valid for analysis of relaxation data of large proteins recorded at high magnetic field strengths since the contribution to longitudinal relaxation from the Lorentzian describing the overall rotational diffusion of the molecule is comparably small relative to that describing internal motion. Here, we quantitatively estimate the errors introduced by the use of the simplified spectral density in model-free analysis for large proteins at high magnetic field strength.     

Stable isotope characteristics across narrow savanna/woodland ecotones in Wolfe Creek Meteorite Crater,Western Australia

The stable isotopic composition (δ¹³C) of sediments from lakes are frequently analyzed to reconstruct the proportion of the regional vegetation that used either the C₃ or C₄ photosynthetic pathways, often without conducting a detailed survey of the current local vegetation. We performed a study on the modern vegetation composition within the Wolfe Creek Meteorite Crater to complement our future paleoecological investigation of the crater. A bull’s-eye pattern exists where C₄ grasses dominate an outer ring and salt tolerant species, including shrubs, herbs, chenopods, and halophytic algae, dominate the inner pan of the crater. The ecotone between the inner and outer zones is narrow and occupied by tall (>7 m) Acacia ampliceps, with some C₄ grasses in the understory. Along with the highest water table and most saline soils the center of the crater has C₃ plants present with the highest δ¹³C and δ¹⁵N values. The range of δ¹³C and δ¹⁵N values from the analysis of surface soil organic matter (OM) was much smaller compared with the range of values from plant materials implying that either: (1) the current plant OM has not yet been integrated into the soils, or (2) processes within the soil have acted to homogenize isotopic variability within the crater. The application of a two end member mixing model to calculate %C₄ and %C₃ biomass from the δ¹³C of surface soil OM was complicated by: (1) the crater containing both a dry habitat with C₄ grasses and a central pan with C₄ halophytic plants and, (2) the large variation in the δ¹³C of the plants and soil OM.     

Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Q_y bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 μm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.     

Long-term biomonitoring of lichen and bryophyte biodiversity at Burnham Beeches SAC and global environmental change

Long-term monitoring began 20 years ago at Burnham Beeches Site of Special Interest (SSSI), National Nature Reserve (NNR) and European Special Area of Conservation (SAC) lying 40 km west of London as a consequence of the authorization of an application to extract gravel from an adjacent site lying north of Slough Trading Estate. Dust monitoring (sticky pads) and photographic monitoring, recording and image analysis was instigated in 1992 on Parmelion communities to assess changes in lichen growth, health and community composition. Long-term monitoring identifies that the lichen flora on free-standing trees has undergone rapid expansion from a near dominance by the SO₂-tolerant ‘acidophyte’ species Lecanora conizaeoides and Hypogymnia physodes following reductions in SO₂ concentrations. Long-term influences of low levels of eutrophication, gaseous pollutants (particularly globally rising background ozone concentrations) on lichen and bryophyte communities and succession under changing climatic conditions are unknown. Soil–plant relationships, lichen–invertebrate interactions and a pollution legacy must also be considered.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.     

Energy transfer process in a rare earth (Ce3+, Dy3+, Sm3+)-doped nanocrystalline phosphate phosphor

This work presents the optimized luminescence spectra for the Ce³⁺,Sm³⁺-doped NaSrPO₄ phosphor that was synthesized using a wet chemical method. Ce³⁺ and Sm³⁺ are activator impurities that show spectral splitting bands that corresponds to the d–f and f–f transitions, respectively. These impurity elements shows the characteristics spectral bands when doped with the NaSrPO₄ host lattice. Spectral splitting in the Ce³⁺ excitation band was monitored in the 240–340 nm range, in which the observed bands were located at 269 nm, 292 nm and 321 nm, and emission bands were observed in the broad spectral range 330–430 nm. However, when Sm³⁺ ion was doped in the same host lattice we obtained a characteristic emission band at 590 and 645 nm in the orange–red region, under sharp excitation bands located at 345, 361, 375, and 403 nm respectively. Also, we carried out energy transfer analysis in the Ce³⁺/Dy³⁺-doped NaSrPO₄ phosphor. Further crystalline phase and the nanophase nature of the phosphor compound were confirmed using X-ray diffraction and transmission electron microscopy analyses.     

Growth and Spectral Assessment of Yb3+-Doped KBaGd(MoO4)3 Crystal: A Candidate for Ultrashort Pulse and Tunable Lasers

In order to explore new more powerful ultrashort pulse laser and tunable laser for diode-pumping, this paper reports the growth and spectral assessment of Yb³⁺-doped KBaGd(MoO₄)₃ crystal. An Yb³⁺:KBaGd(MoO₄)₃ crystal with dimensions of 50×40×9 mm³ was grown by the TSSG method from the K₂Mo₂O₇ flux. The investigated spectral properties indicated that Yb³⁺:KBaGd(MoO₄)₃ crystal exhibits broad absorption and emission bands, except the large emission and gain cross-sections. This feature of the broad absorption and emission bands is not only suitable for the diode pumping, but also for the production of ultrashort pulses and tunability. Therefore, Yb³⁺:KBaGd(MoO₄)₃ crystal can be regarded as a candidate for the ultrashort pulse and tunable lasers.     

Quantitative covariance NMR by regularization

The square root of a covariance spectrum, which offers high spectral resolution along both dimensions requiring only few t ₁ increments, yields in good approximation the idealized 2D FT spectrum provided that the amount of magnetization exchanged between spins is relatively small. When this condition is violated, 2D FT and covariance peak volumes may differ. A regularization method is presented that produces a modified covariance spectrum with cross-peak volumes that closely match their 2D FT analogues. The method is demonstrated for TOCSY spectra with variable mixing times.     

New approach to measurement of IR absorption spectra of dissolved oxygen molecules based on photochemical activity of oxygen upon direct laser excitation

Generation of singlet oxygen upon excitation of oxygen molecules with IR diode lasers has been studied in organic media (carbon tetrachloride and acetone) saturated with air under normal pressure and temperature. A new approach to analysis of the experimental data has been developed, which takes into account the degree of overlapping of the spectral bands of oxygen absorption and laser radiation. Optical density, molar absorption coefficient, and the cross section of light absorption were determined for the main absorption maxima of O₂ at 765 and 1273 nm. The results are compared with the data of previous studies. Significance of these results for elucidation of photophysics and photochemistry of oxygen molecules and investigation of biological action of laser radiation is discussed.     

Growth and photosynthesis of soybean (Glycine max (L.) Merr.) in simulated vegetation shade: influence of the ratio of red to far-red radiation*

Abstract. Glycine max (L.) Merr. was grown under several light conditions to determine the role of red and far-red radiation in plant adaptation to vegetation shade. Neutral density,‘neutral’ density with elevated far-red radiation, and green shade treatments were used in a greenhouse, producing calculated phytochrome photostationary state (P_fr/P_r+P_fr) values of 0.68, 0.63 and 0.51, respectively. Cool-white fluorescent lamps either alone or in conjunction with far-red fluorescent lamps were used in a growth chamber, providing P_fr/P_r+P_fr of 0.79 and 0.61, respectively. Daily photo-synthetically active radiation was about 25% of daylight and was approximately equal for both greenhouse (2.15MJ m^?2) and growth chamber (2.57MJ m^?2). Developmental stage 4 weeks after sowing was similar for all treatments, but axillary growth and rates of leaf area and dry matter accretion differed between plants from greenhouse and growth chamber. Light conditions simulating vegetation shade (i.e. a low ratio of red to far-red radiation) significantly promoted petiole elongation and retarded the rate of stem elongation in both greenhouse and growth chamber experiments. Other aspects of growth either were not significantly altered by spectral quality or were not modified consistently in both greenhouse and growth chamber environments. Net photosynthetic rates measured under growth conditions for unifoliate and first trifoliolate (TF₁) leaves of growth chamber plants between 9 and 21 d after sowing were generally unaffected by spectral quality, but supplemental FR enhanced TF₁ leaf area expansion. The latter effect was not correlated with increased dry matter accumulation. The significance of spectral quality for adaptation of soybeans to canopy closure and intercropping is discussed.     

Low-temperature energy transfer in FMO trimers from the green photosynthetic bacterium Chlorobium tepidum

The pump-probe kinetics of the slowest spectral equilibrations between inequivalent BChl a Q_y states in FMO trimers from Chlorobium tepidum are decelerated by nearly two orders of magnitude when the temperature is lowered from 300 K to 19 K. The pump-probe anisotropy decays are also markedly slower at 19 K than at 300 K. Singlet-singlet annihilation in FMO trimers is negligible at the laser powers used here. However, reduced temperatures greatly accentuate the probability of singlet-triplet annihilation, due to accumulation of metastable BChl a states under high laser repetition rates.Abbreviations BChl bacteriochlorophyll - FMO Fenna-Matthews-Olson - fwhm full width at half maximum - PB photobleaching - SE stimulated emission     

Spectral analysis on fluctuation of heat period in paralyzed,vagotomized, and unanesthetized decerebrate cats

Spectral analysis was performed on the heart period fluctuation in vagotomized, paralyzed, and unanesthetized decerebrate cats. The heart period was measured as the time interval between successive R waves of the electrocardiograms. When end-tidal P _{CO 2} was set at the same level as that before immobilization, the power spectral density plot of the heart period fluctuation showed several distinct peaks: one peak corresponded to the frequency of the artificial ventilator and the others to its harmonics. In addition, the spectral density plot had another peak centered at the intrinsic respiratory frequency evaluated by recording efferent phrenic neural discharges. The amplitude of these spectral peaks tended to become greater when the end-tidal P _{CO 2}was increased by adding CO₂ to the input gas. Our results, therefore, provide evidence that the heart period is modulated not only by the artificial ventilation rhythm but also by the centrally generated respiratory rhythm, and suggested that the strength of such central interactions between cardiac and respiratory rhythms varies depending on the end-tidal P _{CO 2} level.     

Colour vision and visual ecology of the blue-spotted maskray, <Emphasis Type="Italic">Dasyatis kuhlii</Emphasis> Müller &; Henle, 1814

Relatively little is known about the physical structure and ecological adaptations of elasmobranch sensory systems. In particular, elasmobranch vision has been poorly studied compared to the other senses. Virtually nothing is known about whether elasmobranchs possess multiple cone types, and therefore the potential for colour vision, or how the spectral tuning of their visual pigments is adapted to their different lifestyles. In this study, we measured the spectral absorption of the rod and cone visual pigments of the blue-spotted maskray, Dasyatis kuhlii, using microspectrophotometry. D. kuhlii possesses a rod visual pigment with a wavelength of maximum absorbance (λ_max) at 497 nm and three spectrally distinct cone types with λ_max values at 476, 498 and 552 nm. Measurements of the spectral transmittance of the ocular media reveal that wavelengths below 380 nm do not reach the retina, indicating that D. kuhlii is relatively insensitive to ultraviolet radiation. Topographic analysis of retinal ganglion cell distribution reveals an area of increased neuronal density in the dorsal retina. Based on peak cell densities and using measurements of lens focal length made using laser ray tracing and sections of frozen eyes, the estimated spatial resolving power of D. kuhlii is 4.10 cycles per degree.