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At least 973 records · Page 54

OpenMSI Arrayed Analysis Toolkit: Analyzing Spatially Defined Samples Using Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) has primarily been applied in localizing biomolecules within biological matrices. Although well-suited, the application of MSI for comparing thousands of spatially defined spotted samples has been limited. One reason for this is a lack of suitable and accessible data processing tools for the analysis of large arrayed MSI sample sets. In this paper, the OpenMSI Arrayed Analysis Toolkit (OMAAT) is a software package that addresses the challenges of analyzing spatially defined samples in MSI data sets. OMAAT is written in Python and is integrated with OpenMSI (http://openmsi.nersc.gov), a platform for storing, sharing, and analyzing MSI data. By using a web-based python notebook (Jupyter), OMAAT is accessible to anyone without programming experience yet allows experienced users to leverage all features. OMAAT was evaluated by analyzing an MSI data set of a high-throughput glycoside hydrolase activity screen comprising 384 samples arrayed onto a NIMS surface at a 450 μm spacing, decreasing analysis time >100-fold while maintaining robust spot-finding. The utility of OMAAT was demonstrated for screening metabolic activities of different sized soil particles, including hydrolysis of sugars, revealing a pattern of size dependent activities. Finally, these results introduce OMAAT as an effective toolkit for analyzing spatially defined samples in MSI. OMAAT runs on all major operating systems, and the source code can be obtained from the following GitHub repository: https://github.com/biorack/omaat.

47 OTHER INSTRUMENTATION↗

Single-Particle Isotopic Analysis Using the Liquid Sampling-Atmospheric Pressure Glow Discharge Microplasma Coupled to an Orbitrap Mass Spectrometer

Isotope ratio (IR) determinations on individual particles can provide valuable information relative to the sample, including processing and dating, which could be valuable to geological and nuclear material analysis communities. In comparison to “bulk” measurements, in which all particle information is lost and homogenized within a sample, “particle” measurements allow for fingerprinting and can provide unique insights related to the sample’s nano- and micro- compositions. Furthermore, the complex nature of particles, with potential isobaric interferences from either the matrix or other elements within the same particle, poses a significant analytical challenge for conventional mass spectrometry platforms employed for elemental analysis due to their limited mass resolution. Presented here is the first demonstration of an ultrahigh resolution method for single particle (SP) isotopic analysis using the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma ionization source coupled to an Orbitrap mass spectrometer and FTMS Booster X2T acquisition/processing system. For proof of concept, well-characterized CeO 2 microparticles with a nominal diameter of 1.0 μm were analyzed at a mass resolution of ∼330,000. Important instrumental parameters were optimized to enable the detection of single particles. The 142 Ce/ 140 Ce ratio of individual particles and the population average were determined and compared to an SP inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) analysis. The isotopic accuracy and precision were determined by two methods and found to be in good agreement with the SP-ICP-TOF-MS method, with the linear regression slope method providing a more accurate ratio, showing a ∼1.4% relative difference from the ratio determined from a particle digest. The encouraging performance of the method was further supported by a determined detection limit of 2.9 fg ( 142 Ce). The developed method is anticipated to overcome many of the challenges posed by isobaric interferences encountered in conventional mass spectrometry techniques when analyzing real-world samples for particle isotopic composition.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Ambient Metabolic Profiling and Imaging of Biological Samples with Ultrahigh Molecular Resolution using Laser Ablation Electrospray Ionization 21 Tesla FTICR Mass Spectrometry

Mass spectrometry (MS) has been an indispensable analytical tool in capturing the complexity and array of biomolecules within biological systems. However, conventional MS-based analytical workflows are limited in both throughput and measuring the native molecular state these systems, due in large part to sample handling and preparation. The development of ambient ionization methods capable of sampling directly from tissues circumvent many of these issues but requires the use of high-performance MS to resolve the molecular complexity within these samples. Herein, we demonstrate the unique combination of laser ablation electrospray ionization (LAESI) coupled with a 21 Tesla Fourier transform ion cyclotron resonance (21T-FTICR-MS) for MS imaging applications. This analytical platform provides isotopic fine structure information directly from native tissues, delivering a higher degree of confidence for molecular identification. The ability to capture and visualize the complex metabolic behavior of living organisms in situ is a bioanalytical grand challenge that has yet to be fully realized. Due to their excellent sensitivity and selectivity, mass spectrometry (MS)-based platforms offer a unique opportunity to characterize an array of biomolecules from a variety of biological samples.[1] One method in particular, MS imaging (MSI), has demonstrated the ability to simultaneously determine the spatial distributions for hundreds of biomolecules, e. g., metabolites, lipids, peptides, and xenobiotics, within complex biological systems simultaneously.[2-3] Matrix-assisted laser desorption/ionization (MALDI)-MSI remains the most common of these methods.[4] However, the application of an exogenous matrix, and the limited availability of commercial sources that operate under atmospheric pressure,[5] has significantly prohibited the ability of MALDI to be useful for biomolecular mapping of living systems. Ambient ionization methods that require minimal to no sample preparation, like desorption electrospray ionization (DESI and nano-DESI) and laser ablation electrospray ionization (LAESI), demonstrated the ability to spatially map biomolecule distributions within live organisms.

Mass Spectometry, fourier transform ion cyclostron↗

Sample Preparation Methods for Targeted Single-Cell Proteomics

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540–660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390–540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5–25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

59 BASIC BIOLOGICAL SCIENCES↗

Sparge Sampling of Molten Salts for Online Monitoring via Laser-Induced Breakdown Spectroscopy

A method was developed to sample molten salts by sparging to generate and transport aerosols to an isolated instrument for compositional analysis by laser-induced breakdown spectroscopy (LIBS). Real-time monitoring of molten salt composition is critical to developing molten salt nuclear reactors, which offer enhanced safety and efficiency. In this article, the sparge sampling method is described and compared with sampling using a Collison nebulizer. The size distribution and transport of aerosols produced from molten eutectic NaNO 3 –KNO 3 salt were compared for multiple gas flow rates (75–1200 mL min –1 ) and transport distances (0.68–2.61 m). Both methods produced aerosols ranging from 0.5 to 5.0 μm determined using a cascade impactor. Aerosols were effectively transported without pre- or trace-heating of gas lines, but transport efficiency was reduced by the formation of agglomerates. Sparge sampling was found to use less sample and less gas than a Collison nebulizer while producing a more concentrated aerosol stream (up to 5 μg L –1 ). The effects of laser energy and delay time on the signal quality of LIBS measurements of these aerosols were also studied. High energy and short delay times were found to enhance signal and repeatability, whereas signal-to-background and signal-to-noise ratios were highest at low energy and longer delay times. The capabilities of this system for online monitoring of molten salts were demonstrated with calibrations for Sr and Li with relative standard deviations of 2.6% and 1.5% and limits of detection of 380 and 180 μg g –1 , respectively.

Aerosols↗

Microbial Diversity in Bushmeat Samples Recovered from the Serengeti Ecosystem in Tanzania

Bushmeat, the meat and organs derived from wildlife species, is a common source of animal protein in the diets of those living in sub-Saharan Africa and is frequently associated with zoonotic spillover of dangerous pathogens. Given the frequent consumption of bushmeat in this region and the lack of knowledge about the microbial communities associated with this meat, the microbiome of 56 fresh and processed bushmeat samples ascertained from three districts in the Western Serengeti ecosystem in Tanzania was characterized using 16S rRNA metagenomic sequencing. Here, the results show that the most abundant phyla present in bushmeat samples include Firmicutes (67.8%), Proteobacteria (18.4%), Cyanobacteria (8.9%), and Bacteroidetes (3.1%). Regardless of wildlife species, sample condition, season, or region, the microbiome is diverse across all samples, with no significant difference in alpha or beta diversity. The findings also suggest the presence of DNA signatures of potentially dangerous zoonotic pathogens, including those from the genus Bacillus, Brucella, Coxiella, and others, in bushmeat. Together, this investigation provides a better understanding of the microbiome associated with this major food source in samples collected from the Western Serengeti in Tanzania and highlights a need for future investigations on the potential health risks associated with the harvesting, trade, and consumption of bushmeat in Sub-Saharan Africa.

59 BASIC BIOLOGICAL SCIENCES↗

Non-Destructive Study of Bulk Crystallinity and Elemental Composition of Natural Gold Single Crystal Samples by Energy-Resolved Neutron Imaging

Energy-resolved neutron imaging enables non-destructive analyses of bulk structure and elemental composition, which can be resolved with high spatial resolution at bright pulsed spallation neutron sources due to recent developments and improvements of neutron counting detectors. This technique, suitable for many applications, is demonstrated here with a specific study of ~5–10mm thick natural gold samples. Through the analysis of neutron absorption resonances the spatial distribution of palladium (with average elemental concentration of ~0.4 atom% and ~5 atom%) is mapped within the gold samples. At the same time, the analysis of coherent neutron scattering in the thermal and cold energy regimes reveals which samples have a single-crystalline bulk structure through the entire sample volume. A spatially resolved analysis is possible because neutron transmission spectra are measured simultaneously on each detector pixel in the epithermal, thermal and cold energy ranges. With a pixel size of 55μm and a detector-area of 512 by 512 pixels, a total of 262,144 neutron transmission spectra are measured concurrently. The results of our experiments indicate that high resolution energy-resolved neutron imaging is a very attractive analytical technique in cases where other conventional nondestructive methods are ineffective due to sample opacity.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

Sample preparation with sucrose cryoprotection dramatically alters Zn distribution in the rodent hippocampus, as revealed by elemental mapping

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. Furthermore, the choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.

59 BASIC BIOLOGICAL SCIENCES↗

Hanging drop sample preparation improves sensitivity of spatial proteomics

Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm 2 , respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.

47 OTHER INSTRUMENTATION↗

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Further, our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

59 BASIC BIOLOGICAL SCIENCES↗

1WHSP: An IR-based sample of ~1000 VHE γ -ray blazar candidates

Context. Blazars are the dominant type of extragalactic sources at microwave and at γ-ray energies. In the most energetic part of the electromagnetic spectrum (E > ≳ 100 GeV) a large fraction of high Galactic latitude sources are blazars of the High Synchrotron Peaked (HSP) type, that is BL Lac objects with synchrotron power peaking in the UV or in the X-ray band. Building new large samples of HSP blazars is key to understand the properties of jets under extreme conditions, and to study the demographics and the peculiar cosmological evolution of these sources. Aims. HSP blazars are remarkably rare, with only a few hundreds of them expected to be above the sensitivity limits of currently available surveys, some of which include hundreds of millions of sources. To find these very uncommon objects, we have devised a method that combines ALLWISE survey data with multi-frequency selection criteria. Methods. The sample was defined starting from a primary list of infrared colour-colour selected sources from the ALLWISE all sky survey database, and applying further restrictions on IR-radio and IR-X-ray flux ratios. Using a polynomial fit to the multi-frequency data (radio to X-ray) we estimated synchrotron peak frequencies and fluxes of each object. Results. We assembled a sample including 992 sources, which is currently the largest existing list of confirmed and candidates HSP blazars. All objects are expected to radiate up to the highest γ-ray photon energies. In fact, 299 of these are confirmed emitters of GeV γ-ray photons (based on Fermi-LAT catalogues), and 36 have already been detected in the TeV band. The majority of sources in the sample are within reach of the upcoming Cherenkov Telescope Array (CTA), and many may be detectable even by the current generation of Cherenkov telescopes during flaring episodes. The sample includes 425 previously known blazars, 151 new identifications, and 416 HSP candidates (mostly faint sources) for which no optical spectra is available yet. The full 1WHSP catalogue is on-line at http://www.asdc.asi.it/1whsp/ providing a direct link to the SED building tool where multifrequency data for each source can be easily visualised.

79 ASTRONOMY AND ASTROPHYSICS↗

BEYONDPLANCK II. CMB mapmaking through Gibbs sampling

We present a Gibbs sampling solution to the mapmaking problem for cosmic microwave background (CMB) measurements that builds on existing destriping methodology. Gibbs sampling breaks the computationally heavy destriping problem into two separate steps: noise filtering and map binning. Considered as two separate steps, both are computationally much cheaper than solving the combined problem. This provides a huge performance benefit as compared to traditional methods and it allows us, for the first time, to bring the destriping baseline length to a single sample. Here, we applied the Gibbs procedure to simulated Planck 30 GHz data. We find that gaps in the time-ordered data are handled efficiently by filling them in with simulated noise as part of the Gibbs process. The Gibbs procedure yields a chain of map samples, from which we are able to compute the posterior mean as a best-estimate map. The variation in the chain provides information on the correlated residual noise, without the need to construct a full noise covariance matrix. However, if only a single maximum-likelihood frequency map estimate is required, we find that traditional conjugate gradient solvers converge much faster than a Gibbs sampler in terms of the total number of iterations. The conceptual advantages of the Gibbs sampling approach lies in statistically well-defined error propagation and systematic error correction. This methodology thus forms the conceptual basis for the mapmaking algorithm employed in the BEYONDPLANCK framework, which implements the first end-to-end Bayesian analysis pipeline for CMB observations.

79 ASTRONOMY AND ASTROPHYSICS↗

Quantifying Hydraulic and Water Quality Uncertainty to Inform Sampling of Drinking Water Distribution Systems

Sampling of drinking water distribution systems is performed to ensure good water quality and protect public health. Sampling also satisfies regulatory requirements and is done to respond to customer complaints or emergency situations. Water distribution system modeling techniques can be used to plan and inform sampling strategies. However, a high degree of accuracy and confidence in the hydraulic and water quality models is required to support real-time response. One source of error in these models is related to uncertainty in model input parameters. Effective characterization of these uncertainties and their effect on contaminant transport during a contamination incident is critical for providing confidence estimates in model-based design and evaluation of different sampling strategies. In this paper, the effects of uncertainty in customer demand, isolation valve status, bulk reaction rate coefficient, contaminant injection location, start time, duration, and rate on the size and location of the contaminant plume are quantified for two example water distribution systems. Results show that the most important parameter was the injection location. The size of the plume was also affected by the reaction rate coefficient, injection rate, and injection duration, whereas the exact location of the plume was additionally affected by the isolation valve status. As a result, uncertainty quantification provides a more complete picture of how contaminants move within a water distribution system and more information when using modeling results to select sampling locations.

54 ENVIRONMENTAL SCIENCES↗

Calorimeters for Precision Power Dissipation Measurements on Controlled-Temperature Superconducting Radiofrequency Samples

Two calorimeters, with stainless steel and Cu as the thermal path material for high precision and high power versions, respectively, have been designed and commissioned for the surface impedance characterization (SIC) system at Jefferson Lab to provide low temperature control and measurement for CW power up to 22 W on a 5 cm dia. disk sample which is thermally isolated from the RF portion of the system. A power compensation method has been developed to measure the RF induced power on the sample. Simulation and experimental results show that with these two calorimeters, the whole thermal range of interest for superconducting radiofrequency (SRF) materials has been covered. The power measurement error in the interested power range is within 1.2% and 2.7% for the high precision and high power versions, respectively. Temperature distributions on the sample surface for both versions have been simulated and the accuracy of sample temperature measurements have been analysed. Both versions have the ability to accept bulk superconductors and thin film superconducting samples with a variety of substrate materials such as Al, Al{sub 2}O{sub 3}, Cu, MgO, Nb and Si.

43 PARTICLE ACCELERATORS↗

Automated laboratory based X-ray beamline with multi-capillary sample chamber

An automated laboratory based X-ray beamline with a multi-capillary sample chamber capable of undertaking small angle X-ray scattering measurements on a maximum of 104 samples at a time as a function of temperature between 5 and 85 °C has been developed. The modular format of the system enables the user to simultaneously equilibrate samples at eight different temperatures with an accuracy of ±0.005 °C. This system couples a rotating anode generator and 2D optoelectronic detector with Franks X-ray optics, leading to typical exposure times of less than 5 min for lyotropic liquid crystalline samples. Beamline control including sample exchange and data acquisition has been fully automated via a custom designed LabVIEW framework.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Compact ultrahigh vacuum sample environments for x-ray nanobeam diffraction and imaging

X-ray nanobeams present the opportunity to obtain structural insight in materials with small volumes or nanoscale heterogeneity. The effective spatial resolution of the information derived from nanobeam techniques depends on the stability and precision with which the relative position of the x-ray optics and sample can be controlled. Nanobeam techniques include diffraction, imaging, and coherent scattering, with applications throughout materials science and condensed matter physics. Sample positioning is a significant mechanical challenge for x-ray instrumentation providing vacuum or controlled gas environments at elevated temperatures. Such environments often have masses that are too large for nanopositioners capable of the required positional accuracy of the order of a small fraction of the x-ray spot size. Similarly, the need to place x-ray optics as close as 1 cm to the sample places a constraint on the overall size of the sample environment. We illustrate a solution to the mechanical challenge in which compact ion-pumped ultrahigh vacuum chambers with masses of 1–2 kg are integrated with nanopositioners. The overall size of the environment is sufficiently small to allow their use with zone-plate focusing optics. We describe the design of sample environments for elevated-temperature nanobeam diffraction experiments demonstrate in situ diffraction, reflectivity, and scanning nanobeam imaging of the ripening of Au crystallites on Si substrates.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Note: A fast pneumatic sample-shuttle with attenuated shocks

We describe a home-built pneumatic shuttle suitable for the fast displacement of samples in the vicinity of a highly sensitive atomic magnetometer. The samples are magnetized at 1 T using a Halbach assembly of magnets. The device enables the remote detection of free-induction-decay in ultra-low-field and zero-field nuclear magnetic resonance (NMR) experiments, in relaxometric measurements and in other applications involving the displacement of magnetized samples within time intervals as short as a few tens of milliseconds. Other possible applications of fast sample shuttling exist in radiological studies, where samples have to be irradiated and then analyzed in a cold environment.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

An experimental setup for the simultaneous measurement of thermoelectric power of two samples from 77 K to 500 K

We report on an experimental setup for the simultaneous measurement of the thermoelectric power (TEP) of two samples in the temperature range from 77 K to 500 K using optimum electronic instruments. The setup consists of two rectangular copper bars in a bridge arrangement for sample mounting, two surface mount (SM) chip resistors for creating alternate temperature gradient, and a type E thermocouple in differential geometry for gradient temperature (ΔT) measurement across the samples. In addition, a diode arrangement has been made for the alternate heating of SM resistors using only one DC current source. The measurement accuracy of ΔT increases with the differential thermocouple arrangement. For the calibration of the setup, measurements of TEP on a high purity (99.99%) platinum wire and type K thermocouple wires Chromel and Alumel have been performed from 77 K to 500 K with respect to copper lead wires. Additionally, this setup can be utilized to calibrate an unknown sample against a sample of known absolute TEP.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗