Optimal Instrument Settings Research Articles

Optimization of whole slide imaging scan settings for computer vision using human lung cancer tissue.

Digital pathology has become increasingly popular for research and clinical applications. Using high-quality microscopes to produce Whole Slide Images of tumor tissue enables the discovery of insights into biological aspects invisible to the human eye. These are acquired through downstream analyses using spatial statistics and artificial intelligence. Determination of the quality and consistency of these images is needed to ensure accurate outcomes when identifying clinical and subclinical image features. Additionally, the time-intensive process of generating high-volume images results in a trade-off that needs to be carefully balanced. This study aims to determine optimal instrument settings to generate representative images of pathological tissue using digital microscopy. Using various settings, an H&E stained sample was scanned using the ZEISS Axio Scan.Z1. Next, nucleus segmentation was performed on resulting images using StarDist. Subsequently, detections were compared between scans using a matching algorithm. Finally, nucleus-level information was compared between scans. Results indicated that while general matching percentages were high, similarity between information from replicates was relatively low. Additionally, settings resulting in longer scanning times and increased data volume did not increase similarity between replicates. In conclusion, the scan setting ultimately deemed optimal combined consistent and qualitative performance with low throughput time.

Origins and consequences of asymmetric nano-FTIR interferograms

Infrared scattering-type near-field optical microscopy, IR s-SNOM, and its broadband variant, nano-FTIR, are pioneering, flagship techniques for their ability to provide molecular identification and material optical property information at a spatial resolution well below the far-field diffraction limit, typically less than 25 nm. While s-SNOM and nano-FTIR instrumentation and data analysis have been discussed previously, there is a lack of information regarding experimental parameters for the practitioner, especially in the context of previously developed frameworks. Like conventional FTIR spectroscopy, the critical component of a nano-FTIR instrument is an interferometer. However, unlike FTIR spectroscopy, the resulting interference patterns or interferograms are typically asymmetric. Here, we unambiguously describe the origins of asymmetric interferograms recorded with nano-FTIR instruments, give a detailed analysis of potential artifacts, and recommend optimal instrument settings as well as data analysis parameters.

Improved plasma stoichiometry recorded by laser ablation ionization mass spectrometry using a double-pulse femtosecond laser ablation ion source.

Femtosecond (fs) laser ablation ion sources have allowed for improved measurement capabilities and figures of merit of laser ablation based spectroscopic and mass spectrometric measurement techniques. However, in comparison to longer pulse laser systems, the ablation plume from fs lasers is observed to be colder, which favors the formation of polyatomic species. Such species can limit the analytical capabilities of a system due to isobaric interferences. In this contribution, a double-pulse femtosecond (DP-fs) laser ablation ion source is coupled to our miniature Laser Ablation Ionization Mass Spectrometry (LIMS) system and its impact on the recorded stoichiometry of the generated plasma is analyzed in detail. A DP-fs laser ablation ion source (temporal delays of +300 to - 300 ps between pulses) is connected to our miniature LIMS system. The first pulse is used for material removal from the sample surface and the second for post-ionization of the ablation plume. To characterize the performance, parametric double- and single-pulse studies (temporal delays, variation of the pulse energy, voltage applied on detector system) were conducted on three different NIST SRM alloy samples (SRM 661, 664 and 665). At optimal instrument settings for both the double-pulse laser ablation ion source and the detector voltage, relative sensitivity coefficients were observed to be closer (factor of ~2) to 1 compared with single-pulse measurements. Furthermore, the optimized settings worked for all three samples, meaning no further optimization was necessary when changing to another alloy sample material during this study. The application of a double-pulse femtosecond laser ablation ion source resulted in the recording of improved stoichiometry of the generated plasma using our LIMS measurement technique. This is of great importance for the quantitative chemical analysis of more complex solid materials, e.g., geological samples or metal alloys, especially when aiming for standard-free quantification procedures for the determination of the chemical composition.

Bacterial killing efficacy of synthesized rod shaped cuprous oxide nanoparticles using laser ablation technique

Recent interests with copper nanoparticles (Cu-NPs) has arisen because of its low cost and antibacterial properties, as it may be one of the very important alternatives to silver NPs. Pulsed laser ablation of a solid target (copper) in liquid media is used to synthesize colloidal oxide copper (Cu2O and CuO NPs) NPs. These oxides of copper NPs were synthesized using Nd:YAG laser energy to maintain a 40 mJ/pulse wavelength at 1064 nm. During the synthesis of colloidal NPs only two conditions were opted, employing double distilled water (without PEG) and 10% preparation of PEG. Both NPs were prepared under the similar parameters such as optimized instrument settings, laser energy and time of laser exposure (1 h ablation). Further, these copper oxide NPs were characterized by advance technologies including UV–visible, X-ray diffraction, transmission electron microscopy and attenuated total reflection Fourier transform infrared spectroscopy techniques. The significant antibacterial properties of synthesized materials were also observed. The cuprous oxide NPs, showed remarkable antibacterial effect conducted using disc diffusion techniques. The minimum inhibitory concentration and minimum bactericidal concentration of synthesized Cu2O NPs were recorded as 120 and 140 μg/L respectively against Staphylococcus aureus used as positive control. Therefore, based on the findings of present study Cu2O NPs can be exploited as stable antimicrobial agents for multipurpose uses.

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material

ABSTRACTExtracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

Experimental design for optimizing MALDI-TOF-MS analysis of palladium complexes

This paper presents optimization of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer (MS) instrumental parameters for the analysis of chloro(2,2'',2"-terpyridine)palladium(II) chloride dihydrate complex applying design of experiments methodology (DoE). This complex is of interest for potential use in the cancer therapy. DoE methodology was proved to succeed in optimization of many complex analytical problems. However, it has been poorly used for MALDI-TOF-MS optimization up to now. The theoretical mathematical relationships which explain the influence of important experimental factors (laser energy, grid voltage and number of laser shots) on the selected responses (signal to noise ? S/N ratio and the resolution ? R of the leading peak) is established. The optimal instrumental settings providing maximal S/N and R are identified and experimentally verified.

The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility.

Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of the images. Here we focus on the effect of variations in the microscope imaging system and software analysis using fixed preparations of cells and a single cell processing protocol. To determine the effect of the microscope imaging and analysis on the measured percentage of damaged DNA (% DNA in tail), we used preparations of mammalian cells treated with etoposide or electrochemically induced DNA damage conditions and varied the settings of the automated microscope, camera, and commercial image analysis software. Manual image analysis revealed measurement variations in percent DNA in tail as high as 40% due to microscope focus, camera exposure time and the software image intensity threshold level. Automated image analysis reduced these variations as much as three-fold, but only within a narrow range of focus and exposure settings. The magnitude of variation, observed using both analysis methods, was highly dependent on the overall extent of DNA damage in the particular sample. Mitigating these sources of variability with optimal instrument settings facilitates an accurate evaluation of cell biological variability.

In vivo quantitative imaging of photoassimilate transport dynamics and allocation in large plants using a commercial positron emission tomography (PET) scanner.

BackgroundAlthough important aspects of whole-plant carbon allocation in crop plants (e.g., to grain) occur late in development when the plants are large, techniques to study carbon transport and allocation processes have not been adapted for large plants. Positron emission tomography (PET), developed for dynamic imaging in medicine, has been applied in plant studies to measure the transport and allocation patterns of carbohydrates, nutrients, and phytohormones labeled with positron-emitting radioisotopes. However, the cost of PET and its limitation to smaller plants has restricted its use in plant biology. Here we describe the adaptation and optimization of a commercial clinical PET scanner to measure transport dynamics and allocation patterns of 11C-photoassimilates in large crops.ResultsBased on measurements of a phantom, we optimized instrument settings, including use of 3-D mode and attenuation correction to maximize the accuracy of measurements. To demonstrate the utility of PET, we measured 11C-photoassimilate transport and allocation in Sorghum bicolor, an important staple crop, at vegetative and reproductive stages (40 and 70 days after planting; DAP). The 11C-photoassimilate transport speed did not change over the two developmental stages. However, within a stem, transport speeds were reduced across nodes, likely due to higher 11C-photoassimilate unloading in the nodes. Photosynthesis in leaves and the amount of 11C that was exported to the rest of the plant decreased as plants matured. In young plants, exported 11C was allocated mostly (88 %) to the roots and stem, but in flowering plants (70 DAP) the majority of the exported 11C (64 %) was allocated to the apex.ConclusionsOur results show that commercial PET scanners can be used reliably to measure whole-plant C-allocation in large plants nondestructively including, importantly, allocation to roots in soil. This capability revealed extreme changes in carbon allocation in sorghum plants, as they advanced to maturity. Further, our results suggest that nodes may be important control points for photoassimilate distribution in crops of the family Poaceae. Quantifying real-time carbon allocation and photoassimilate transport dynamics, as demonstrated here, will be important for functional genomic studies to unravel the mechanisms controlling phloem transport in large crop plants, which will provide crucial insights for improving yields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0658-3) contains supplementary material, which is available to authorized users.

Design concept of the high-resolution end-station PEAXIS at BESSY II: Wide-Q-range RIXS and XPS measurements on solids, solutions, and interfaces

The design of a soft X-ray end-station for the Berlin Electron Synchrotron BESSY II is presented. It will be used for Resonant Inelastic X-ray Scattering (RIXS) and Angle-dependent X-ray Photoelectron Spectroscopy (AdXPS) studies for energy material science. In RIXS-mode the instrument operates with two spherical Variable Line Space (VLS) gratings for energy resolving measurements in two overlapping energy ranges from 200 to 1200eV. The end-station will allow measurements of solid samples, solutions and interfaces in a wide range of experimental conditions with high energy resolution covering a large Q-range realized by a continuous rotation of the RIXS detector arm by 120°.Besides the description of this end-station, a systematic way is shown for the design of a RIXS instrument assessing the grating parameters based on existing theories and for the calculation of optimal instrument settings as a function of photon energy. Different grating inclinations, line densities, groove shapes and RIXS instrument lengths were investigated. Possible parameter combinations were calculated analytically and the resulting instrument performance was determined by ray-tracing simulations using the simulation package RAY. The performance of the RIXS instrument was evaluated by choosing the product of the intensity at the detector and the square of the resolving power as figure of merit to optimize the spectrometer. The robustness of the optimized parameters has been checked in order to define tolerance parameters for the engineering design of the spectrometer.

Fucoidan Analysis by Tandem MALDI-TOF and ESI Mass Spectrometry.

The application of mass spectrometry towards the structural analysis of the most interesting sulfated biopolymers of the brown algae-fucoidans only developed relatively recently. During method development, many problems, both chemical and instrumental, have to be solved. For example, mass spectrometry has a limitation in the analysis of anionic high molecular weight (HMW) polysaccharides because of the labile nature of sulfate groups which cause the polysaccharide to desulfate rather than ionize. Thus, decomposition methods should be developed taking into account the structural features of such a complex and fragile compound. The selection of optimal instrument settings for the electrospray ionization mass spectrometry (ESIMS) and of matrix media for matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) is also required. When optimal parameters for mass spectrometric analyses are found, the application of these methods to the elucidation of structural features of fucoidans (by studying their fragments) allows researchers to rapidly obtain new and unique data, often impossible to achieve by other techniques. Herein, we describe tandem mass spectrometry of sulfated fucooligosaccharides, obtained by an autohydrolysis technique from structurally different fucoidans.

Effect of scanning and image reconstruction settings in X-ray computed microtomography on quality and segmentation of 3D soil images

Over the last decade, X-ray computed tomography (CT) has been used increasingly to characterise the microscale architecture of soils. As a result significant progress has been made in the acquisition and interpretation of X-ray CT data, as well as in the thresholding of 3D greyscale CT images in order to produce binary (black and white) ones. Nevertheless, sizeable uncertainties persist, in particular concerning optimal instrumental settings used to generate the greyscale images. In this context, the key aim of this study is to investigate in detail the effect of scanning resolution and reconstruction settings such as noise reduction and 32-bit to 8-bit mapping interval on the 3D X-ray CT imaging of soil structure and the impact on the performance of thresholding methods. To assess the quality of the X-ray CT greyscale images, measures of contrast, noise and sharpness are proposed and tested on a series of images of five different soil samples. At the same time, performance of four segmentation algorithms, i.e., three methods recently developed to deal specifically with soil samples and Otsu's method as a benchmark, was evaluated using functional measures of 3D binary images, including Minkowski functionals and surface pore connected fraction. Results of these analyses suggest that the acquisition and reconstruction parameters investigated significantly affect the quality of soil images, and the subsequent thresholding process. In particular, it was found that thresholding by any of the four methods is greatly affected by the quality of image sharpness, which for soil images appears to be mainly controlled by the scanning resolution. As a result, it is concluded that no matter what reconstruction resolution is required in a study, in order to allow an accurate identification of the pore space, the sample should always be scanned at the highest resolution permitted by the scanning instrument and the sample size. Results also suggest that the three segmentation methods recently developed for soil images thresholding are robust to different levels of noise as well as the choice of the 32-bit mapping interval, as long as lower and upper interval limits for mapping are chosen within suitable boundaries.

A detailed study of gold-nanoparticle loaded cells using X-ray based techniques for cell-tracking applications with single-cell sensitivity

In the present study complementary high-resolution imaging techniques on different length scales are applied to elucidate a cellular loading protocol of gold nanoparticles and subsequently its impact on long term and high-resolution cell-tracking utilizing X-ray technology. Although demonstrated for malignant cell lines the results can be applied to non-malignant cell lines as well. In particular the accumulation of the gold marker per cell has been assessed quantitatively by virtue of electron microscopy, two-dimensional X-ray fluorescence imaging techniques and X-ray CT with micrometric and sub-micrometric resolution. Moreover, utilizing these techniques the three dimensional distribution of the incorporated nanoparticles, which are sequestered in lysosomes as a permanent marker, could be determined. The latter allowed elucidation of the gold partition during mitosis and the cell size, which subsequently enabled us to define the optimal instrument settings of a compact microCT system to visualize gold loaded cells. The results obtained demonstrate the feasibility of cell-tracking using X-ray CT with compact sources.

An exclusion list based label‐free proteome quantification approach using an LTQ Orbitrap

Label-based mass spectrometry is a powerful tool for large-scale protein identification and quantification. However, it requires the chemical or metabolic incorporation of the labeled compound(s) which can be difficult to attain, e.g. for non-cultivable organisms or scarce sample, such as biopsies. Therefore, we set out to develop and validate an efficient label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) workflow based on optimized instrument settings and incremental exclusion lists. To increase the number of quantified peptides an incremental exclusion list was incorporated along with optimized instrument settings for the used LTQ Orbitrap. As a proof of concept, label-free quantification data from this optimized approach were compared to the results of control measurements without exclusion lists and of an in vivo metabolic labeling GeLC/MS/MS experiment. The data were drawn from Staphylococcus aureus whole cell lysates of non-stressed and nitric oxide (NO)-stressed cells. Compared to MS analysis without exclusion lists the new approach resulted in an increased number of identified peptides, enabling label-free quantification of more than 990 S. aureus proteins. With respect to the number of quantified proteins and differences in protein levels between the control and NO-treated samples the results of the new method were consistent with those of the GeLC/MS/MS experiment. The application of exclusion lists and optimized instrument settings in LC/MS/MS analysis significantly enhances the sensitivity and resolution of label-free protein identification and quantification. Therefore, the new workflow is a powerful alternative to label-based quantification methods.

An investigation of hydrogen depth profiling using ToF‐SIMS

Hydrogen depth distributions in silicon, zinc oxide, and glass are of great interest in material research and industry. Time‐of‐flight SIMS has been used for hydrogen depth profiling for many years. However, some critical information, such as optimal instrumental settings and detection limits, is not easily available from previous publications. In this work, optimal instrumental settings and detection limits of hydrogen in silicon, zinc oxide, and common glass were investigated. The recommended experimental settings for hydrogen depth profiling using time‐of‐flight SIMS are: (i) keeping pressure in the analysis chamber as low as possible, (ii) using a cesium beam for sputtering and monitoring the H– signal, (iii) employing monatomic ion analysis beams with the highest currents, and (iv) using interlace mode. In addition, monatomic secondary ions from a matrix are recommended as references to normalize the H– signal. Detection limits of hydrogen are limited by the pressure of residual gases in the analysis chamber. The base pressure of the analysis chamber (with samples) is about 7 × 10−10 mbar in this study, and the corresponding detection limits of hydrogen in silicon, zinc oxide, and common glass are 1.3 × 1018 atoms/cm3, 1.8 × 1018 atoms/cm3, and 5.6 × 1018 atoms/cm3, respectively. Copyright © 2011 John Wiley & Sons, Ltd.

Proposal for measurement of earth impedance using variable frequency injection

Measurement of the impedance of earthing systems of high voltage installations at power fundamental frequency has been studied extensively, and standards are available which provide guidance on methods of measurement and types of instrumentation. In the UK, the staged-fault test is seldom performed. Therefore, it is necessary to measure earth impedance without any circuit interruption by injecting current into the earthing system using an auxiliary test circuit. This type of ‘fall-of-potential’ test generally uses low voltage energization and produces test currents of the order of only a few amperes. At this level of current, potential measurements are made difficult due to the presence of relatively high standing voltages in the earthing system. In this paper, a new earth impedance measurement system which is able to overcome the effects of system noise and provide accurate measurements is described. Initial laboratory tests which resulted in optimized instrument settings and subsequent field tests demonstrated successful application at a range of operational electricity installations.

Methods for the design and analysis of sedimentation velocity and sedimentation equilibrium experiments with proteins.

Analytical ultracentrifugation experiments play an integral role in the solution phase characterization of recombinant proteins and other biological macromolecules. This unit discusses the design of sedimentation velocity and sedimentation equilibrium experiments performed with a Beckman Optima XL-A or XL-I analytical ultracentrifuge. Optimal instrument settings and experimental design considerations are explained, and strategies for the analysis of experimental data with the UltraScan data analysis software package are presented. Special attention is paid to the strengths and weaknesses of the available detectors, and guidance is provided on how to extract maximum information from analytical ultracentrifugation experiments.

Quantitative whole spectrum analysis with MALDI-TOF MS, Part II: Determining the concentration of milk in mixtures

The work summarised in this paper presents the second part of a two-paper series on quantitative whole spectrum analysis with MALDI-TOF MS on skimmed milk. In Part I experiments were carried out to search for optimal sample preparation and instrumental settings in terms of signal-to-noise ratios and repeatability. The results were utilised in the present study when trying to predict concentrations of cow, goat and ewe milk in mixed milk samples. Partial least squares regression was combined with suitable pre- and post-processing of spectra and concentration responses. A plotting method was used where predictions are visualised as a mixture design. The objective was to show that MALDI-TOF MS had potential for being used in quantitative analysis without involving peak comparison or other types of expert guided research. Predictions of a validation data set gave promising results with the best RMSEP values ranging from 5.4% (w/w) to 6.5% (w/w), for the different milk types used, and corresponding R 2 pred values ranging from 94.5% to 96.2%. This indicates that MALDI-TOF is sufficiently accurate and repeatable to be used in practical application for quantitative analysis. Three variable selection strategies based on visual inspections and regression modelling were also evaluated. These were all outperformed, with regard to prediction error, by the use of whole spectra and multivariate regression. The results indicate that multivariate regression on whole spectra can be far more effective than using a few selected variables.

Quantitative whole spectrum analysis with MALDI-TOF MS, Part I: Measurement optimisation

The work summarised in this paper presents the first part of a two-paper series on quantitative whole spectrum analysis with MALDI-TOF MS on skimmed milk. To create a reliable foundation for multivariate, quantitative analysis, optimising measurements (Part I) with respect to well chosen criteria is of high importance. This paper describes the whole process from sample acquisition to final mass spectra using skimmed milk from cow, goat and ewe. Experimental design is employed to search for optimal preparation of the milk samples and optimal MALDI-TOF instrumental settings according to well defined quality measures. The results suggest that optimal parameter settings in a quantitative context may differ from the standard settings used to obtain high resolution spectra in traditional mass spectrometry. Improvements from the worst plausible case to optimised factor levels indicate a 6.96 times higher signal-to-noise ratio and 0.63 times lower inter spectra variation (non-repeatability). These results will be the basis for the main experiment with application to quantitative determination of adulterated skimmed milk (Part II).

Laboratory methods to improve SELDI peak detection and quantitation

BackgroundProtein profiling with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry (SELDI-TOF MS) is a promising approach for biomarker discovery. Some candidate biomarkers have been identified using SELDI-TOF, but validation of these can be challenging because of technical parameters that effect reproducibility. Here we describe steps to improve the reproducibility of peak detection.MethodsSELDI-TOF mass spectrometry was performed using a system manufactured by Ciphergen Biosystems along with their ProteinChip System. Serum from 10 donors was pooled and used for all experiments. Serum was fractionated with Expression Difference Mapping kit-Serum Fractionation from the same company and applied to three different ProteinChips. The fractionations were run over a one month period to examine the contribution of sample batch and time to peak detection variability. Spectra were processed and peaks detected using the Ciphergen Express software and variance measured.ResultsExperimental parameters specific to the serum fraction and ProteinChip, including spot protocols (laser intensity and detector sensitivity) were optimized to decrease peak detection variance. Optimal instrument settings, regular calibration along with controlled sample handling and processing nearly doubled the number of peaks detected and decreased intensity variance.ConclusionThis report assesses the variation across fractionated sera processed over a one-month period. The optimizations reported decreased the variance and increased the number of peaks detected.

Degradation of MTBE and Related Gasoline Oxygenates in Aqueous Media by Ultrasound Irradiation

Gasoline oxygenates [methyl \Itert\N-butyl ether (MTBE), di-isopropyl ether, ethyl \Itert\N-butyl ether, and \Itert\N-amyl methyl ether] in aqueous media are readily decompose upon ultrasonic irradiation (665 kHz). The optimal instrumental settings for the production of reactive radical species employing a specific reactor were determined using dosimetry. Since hydroxyl radical is generally believed to be the predominant reactive species leading to ultrasonically induced degradation of organic substrates in aqueous media, a terephthalate dosimeter was employed to selectively determine the yields of hydroxyl radicals. The decomposition of the gasolinie oxygenates and the primary decomposition products of MTBE were compared in the presence of different saturating gases (O\d2, N\d2, and Ar). The observed decomposition rates for the oxygenates were similar under Ar and O\d2 saturated conditions, but significantly slower under nitrogen saturated conditions. The observed decomposition rates for a number of the primary decomposition products of MTBE are significantly slower under N\d2 and O\d2 saturation relative to Ar saturation. All the gasoline oxygenates in this study were degraded by more than 98% within 3 h of ultrasonic irradiation under O\d2 saturationwith half-lives of 21–25 min.