Cell Isolation and Systems Analysis

The function of proteins, cells and cell communities can be investigated using super resolution, quantitative fluorescence microscopy with single molecule sensitivity, cell sorting and transcriptomic analysis, together with proteomics, metabolomics and electron microscopy.

Resources and Techniques

  • High resolution fluorescence, electron and ion microscopy can image living systems for unprecedented molecular- and atomic-level resolution.
  • Transcriptomics generate massive parallel sequencing to support whole-transcriptome analysis, gene expression profiling, small RNA analysis and novel transcript identification.
  • Cell isolation and fractionation detect and isolate distinct cells and organelles for analyses from complex cultures, communities or tissues.
  • High-throughput data interpretation can be employed for intensive data analysis.

Additonal Information:

Description

Capability Details
• CISA offers a microscopy system that integrates nonlinear two-photon excitation, laser scanning confocal microscopy, and FLIM, enabling minimally invasive and deeply penetrating laser excitation for three-dimensional imaging and detection of molecular interactions in living tissues and cells.
• The stochastic optical reconstruction microscope (STORM) is a unique instrument for imaging intact cells at nanoscale resolution using single-molecule fluorescence techniques to construct super-resolution images with software that acquires and analyzes data in real time.
• Two Applied Biosystems SOLiD systems offer a non-biased, highly quantitative and accurate method to sequence hundreds of distinct DNA samples in parallel within a single run, generating up to 700 million, 50-base pair reads with extremely high accuracy; multiplexing capabilities under varied conditions; and epigenetic modifications.
• The influx flow cytometer/cell sorter provides powerful detection capabilities and diverse sorting modes using advanced multiple-parameter sorting technologies based on the presence and content of distinct genes, proteins and other molecules or intracellular structures. Influx technology supports detection and sorting of nanoscale particles, making it highly suited for sorting and analyzing organelles and microorganisms.

Instruments

Two massively parallel next-generation sequencing platforms (SOLiD4) are currently incorporated in users' research for transcriptomics analysis. The...
Custodian(s): Galya Orr
The atomic force microscope (AFM) compound microscope, is designed primarily for fluorescence imaging in the study of nanoscale chemical processes,...
Custodian(s): Dehong Hu
This FEI Tecnai T-12 cryo-transmission electron microscope (TEM) complements EMSL's broader microscopy suite and JEOL 2010 analytical high-...
Custodian(s): Alice Dohnalkova
High-sensitivity total internal reflection fluorescence (FLIM) microscopes support time-lapse single-molecule fluorescence imaging of individual...
Custodian(s): Galya Orr, Dehong Hu
The single-molecule optical microscope is designed to study complex reaction dynamics such as enzymatic reactions, protein-protein interactions, and...
Custodian(s): Dehong Hu

Publications

Electrochemical performance of the existing state-of-the art capacitors is not very high, key scientific barrier is that its charge storage mechanism...
The metallic compound MnBi is a promising rare-earth-free permanent magnet material. Compare to other rare-earth-free candidates, MnBi stands out for...
We report on ab initio molecular dynamics simulations of Ca-rich montmorillonite systems, in different hydration states in the presence of...
A thermophilic, filamentous, heterotrophic bacterium designated strain JAD2T was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells...
Porous carbon nanofiber (CNF)-supported tin-antimony (SnSb) alloys is synthesized and applied as sodium ion battery anode. The chemistry and...

Science Highlights

Posted: June 17, 2014
The Science Many bacterial species have genes called mraZ and mraW, which are located in a cluster of genes that regulate cell division and cell...
Posted: May 13, 2014
EMSL and Pacific Northwest National Laboratory scientists isolated two bacterial consortia from a microbial mat in Hot Lake, in north-central...
Posted: September 16, 2013
A new transcriptomics-based model accurately predicts how much isoprene the bacterium Bacillus subtilis will produce when stressed or nourished....
Posted: March 19, 2013
One of the most noteworthy concerns for the U.S. Department of Energy is controlling atmospheric carbon dioxide to mitigate its effects on global...
Posted: May 02, 2012
Researchers at Pacific Northwest National Laboratory revealed new knowledge about the effects ionizing radiation has on human skin by using mass...

The function of proteins, cells and cell communities can be investigated using super resolution, quantitative fluorescence microscopy with single molecule sensitivity, cell sorting and transcriptomic analysis, together with proteomics, metabolomics and electron microscopy.

Resources and Techniques

  • High resolution fluorescence, electron and ion microscopy can image living systems for unprecedented molecular- and atomic-level resolution.
  • Transcriptomics generate massive parallel sequencing to support whole-transcriptome analysis, gene expression profiling, small RNA analysis and novel transcript identification.
  • Cell isolation and fractionation detect and isolate distinct cells and organelles for analyses from complex cultures, communities or tissues.
  • High-throughput data interpretation can be employed for intensive data analysis.

Additonal Information:

Attachments: 

Effect of Composition and Heat Treatment on MnBi Magnetic Materials.

Abstract: 

The metallic compound MnBi is a promising rare-earth-free permanent magnet material. Compare to other rare-earth-free candidates, MnBi stands out for its high intrinsic coercivity (Hci) and its large positive temperature coefficient. Several groups have demonstrated that the Hci of MnBi compound in thin film or in powder form can exceed 12 kOe and 26 kOe at 300 K and 523 K, respectively. Such steep increase in Hci with increasing temperature is unique to MnBi. Consequently, MnBi is a highly sought-after hard phase for exchange coupling nanocomposite magnets. The reaction between Mn and Bi is peritectic, so Mn tends to precipitate out of the MnBi liquid during the solidification process. As result, the composition of the Mn-Bi alloy with the largest amount of the desired LTP (low temperature phase) MnBi and highest saturation magnetization will be over-stoichiometric and rich in Mn. The amount of additional Mn required to compensate the Mn precipitation depends on solidification rate: the faster the quench speed, the less Mn precipitates. Here we report a systematic study of the effect of composition and heat treatments on the phase contents and magnetic properties of Mn-Bi alloys. In this study, Mn-Bi alloys with 14 compositions were prepared using conventional metallurgical methods such as arc melting and vacuum heat treatment, and the obtained alloys were analyzed for compositions, crystal structures, phase content, and magnetic properties. The results show that the composition with 55 at.% Mn exhibits the highest LTP MnBi content and the highest magnetization. The sample with this composition shows >90 wt.% LTP MnBi content. Its measured saturation magnetization is 68 emu/g with 2.3 T applied field at 300 K; its coercivity is 13 kOe and its energy product is 12 MGOe at 300 K. A bulk magnet fabricated using this powder exhibits an energy product of 8.2 MGOe.

Citation: 
Cui J, JP Choi, E Polikarpov, ME Bowden, W Xie, G Li, Z Nie, N Zarkevich, MJ Kramer, and DD Johnson.2014."Effect of Composition and Heat Treatment on MnBi Magnetic Materials."Acta Materialia 79:374-381. doi:10.1016/j.actamat.2014.07.034
Authors: 
J Cui
JP Choi
E Polikarpov
ME Bowden
W Xie
G Li
Z Nie
N Zarkevich
MJ Kramer
DD Johnson
Volume: 
Issue: 
Pages: 
Publication year: 
2014

In Situ One-Step Synthesis of Hierarchical Nitrogen-Doped Porous Carbon for High Performance Supercapacitors.

Abstract: 

Electrochemical performance of the existing state-of-the art capacitors is not very high, key scientific barrier is that its charge storage mechanism wholly depends on adsorption of electrolyte on electrode. We present a novel method for the synthesis of nitrogen -doped porous carbons and address the drawback by precisely controlling composition and surface area. Nitrogen-doped porous carbon was synthesized using a self-sacrificial template technique without any additional nitrogen and carbon sources. They exhibited exceptionally high capacitance (239 Fg-1) due to additional pseudocapacitance originating from doped nitrogen. Cycling tests showed no obvious capacitance decay even after 10,000 cycles, which meets the requirement of commercial supercapacitors. Our method is simple and highly efficient for the production of large quantities of nitrogen-doped porous carbons.

Citation: 
Jeon JW, R Sharma, P Meduri, BW Arey, HT Schaef, J Lutkenhaus, JP Lemmon, PK Thallapally, MI Nandasiri, BP McGrail, and SK Nune.2014."In Situ One-Step Synthesis of Hierarchical Nitrogen-Doped Porous Carbon for High Performance Supercapacitors."ACS Applied Materials & Interfaces 6(10):7214-7222. doi:10.1021/am500339x
Authors: 
JW Jeon
R Sharma
P Meduri
BW Arey
HT Schaef
J Lutkenhaus
JP Lemmon
PK Thallapally
MI Nasiri
BP McGrail
SK Nune
Instruments: 
Volume: 
6
Issue: 
10
Pages: 
7214-7222
Publication year: 
2014

Microstructural Response of Variably Hydrated Ca-Rich Montmorillonite to Supercritical CO2.

Abstract: 

We report on ab initio molecular dynamics simulations of Ca-rich montmorillonite systems, in different hydration states in the presence of supercritical CO2. Analysis of the molecular trajectories provides estimates of the relative H2O:CO2 ratio per interspatial cation. The vibrational density of states in direct comparison with dipole moment derived IR spectra for these systems provide unique signatures that can used to follow molecular transformation. In a co-sequestration scenario, these signatures could be used to identify the chemical state and fate of Sulfur compounds. Interpretation of CO2 asymmetric stretch shift is given based on a detailed analysis of scCO2 structure and intermolecular interactions of the intercalated species. Based on our simulations, smectites with higher charge interlayer cations at sub-single to single hydration states should be more efficient in capturing CO2, while maintaining caprock integrity. This research would not have been possible without the support of the office of Fossil Energy, Department of Energy. The computational resources were made available through a user proposal of the EMSL User facility, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.

Citation: 
Lee MS, BP McGrail, and VA Glezakou.2014."Microstructural Response of Variably Hydrated Ca-Rich Montmorillonite to Supercritical CO2."Environmental Science & Technology 48(15):8612-8619. doi:10.1021/es5005889
Authors: 
MS Lee
BP McGrail
VA Glezakou
Instruments: 
Volume: 
48
Issue: 
15
Pages: 
8612-8619
Publication year: 
2014

Production and Early Preservation of Lipid Biomarkers in Iron Hot Springs.

Abstract: 

The bicarbonate-buffered anoxic vent waters at Chocolate Pots hot springs in Yellowstone National Park are 51–54°C, pH 5.5–6.0, and are very high in dissolved Fe(II) at 5.8–5.9 mg/L. The aqueous Fe(II) is oxidized by a combination of biotic and abiotic mechanisms and precipitated as primary siliceous nanophase iron oxyhydroxides (ferrihydrite). Four distinct prokaryotic photosynthetic microbial mat types grow on top of these iron deposits. Lipids were used to characterize the community composition of the microbial mats, link source organisms to geologically significant biomarkers, and investigate how iron mineralization degrades the lipid signature of the community. The phospholipid and glycolipid fatty acid profiles of the highest-temperature mats indicate that they are dominated by cyanobacteria and green nonsulfur filamentous anoxygenic phototrophs (FAPs). Diagnostic lipid biomarkers of the cyanobacteria include midchain branched mono- and dimethylalkanes and, most notably, 2-methylbacteriohopanepolyol. Diagnostic lipid biomarkers of the FAPs (Chloroflexus and Roseiflexus spp.) include wax esters and a long-chain tri-unsaturated alkene. Surprisingly, the lipid biomarkers resisted the earliest stages of microbial degradation and diagenesis to survive in the iron oxides beneath the mats. Understanding the potential of particular sedimentary environments to capture and preserve fossil biosignatures is of vital importance in the selection of the best landing sites for future astrobiological missions to Mars. This study explores the nature of organic degradation processes in moderately thermal Fe(II)-rich groundwater springs—environmental conditions that have been previously identified as highly relevant for Mars exploration. Key Words: Lipid biomarkers—Photosynthesis—Iron—Hot springs—Mars. Astrobiology 14, 502–521.

Citation: 
Parenteau MN, LL Jahnke, JD Farmer, and SL Cady.2014."Production and Early Preservation of Lipid Biomarkers in Iron Hot Springs."Astrobiology 14(6):, doi:10.1089/ast.2013.1122
Authors: 
MN Parenteau
LL Jahnke
JD Farmer
SL Cady
Instruments: 
Volume: 
Issue: 
Pages: 
Publication year: 
2014

Structures and Stabilities of (MgO)n Nanoclusters.

Abstract: 

Global minima for (MgO)n structures were optimized using a tree growth−hybrid genetic algorithm in conjunction with MNDO/MNDO/d semiempirical molecular orbital calculations followed by density functional theory geometry optimizations with the B3LYP functional. New lowest energy isomers were found for a number of (MgO)n clusters. The most stable isomers for (MgO)n (n > 3) are 3-dimensional. For n < 20, hexagonal tubular (MgO)n structures are more favored in energy than the cubic structures. The cubic structures and their variations dominate after n = 20. For the cubic isomers, increasing the size of the cluster in any dimension improves the stability. The effectiveness of increasing the size of the cluster in a specific dimension to improve stability diminishes as the size in that dimension increases. For cubic structures of the same size, the most compact cubic structure is expected to be the more stable cubic structure. The average Mg−O bond distance and coordination number both increase as n increases. The calculated average Mg−O bond distance is 2.055 Å at n = 40, slightly smaller than the bulk value of 2.104 Å. The average coordination number is predicted to be 4.6 for the lowest energy (MgO)40 as compared to the bulk value of 6. As n increases, the normalized clustering energy ΔE(n) for the (MgO)n increases and the slope of the ΔE(n)vs n curve decreases. The value of ΔE(40) is predicted to be 150 kcal/mol, as compared to the bulk value ΔE(∞) = 176 kcal/mol. The electronic properties of the clusters are presented and the reactive sites are predicted to be at the corners.

Citation: 
Chen M, AR Felmy, and DA Dixon.2014."Structures and Stabilities of (MgO)n Nanoclusters."Journal of Physical Chemistry A 118(17):3136-3146. doi:10.1021/jp412820z
Authors: 
M Chen
AR Felmy
DA Dixon
Instruments: 
Volume: 
118
Issue: 
17
Pages: 
3136-3146
Publication year: 
2014

Oxidative Remobilization of Technetium Sequestered by Sulfide-Transformed Nano Zerovalent Iron.

Abstract: 

The dissolution of Tc(IV) sulfide and concurrent transformation of sulfidated ZVI during 2 oxidation were examined. Kinetic data obtained with 10 mL batch reactors showed that Tc(VII) 3 reduced by sulfidated nZVI has significantly slower reoxidation rates than Tc(VII) reduced by 4 nZVI only. In a 50 mL batch reactor, initial inhibition of Tc(IV) dissolution was apparent and 5 lasted until 120 hours at S/Fe = 0.112, presumably due to the redox buffer capacity of FeS. This 6 is evidenced by the parallel trends in oxidation-reduction potentials (ORP) and Tc dissolution 7 kinetics. Mӧssbauer spectra and micro X-ray diffraction of S/Fe = 0.112 suggested the 8 persistence of FeS after 24-h oxidation although X-ray photoelectron spectroscopy indicated 9 substantial surface oxidation. After 120-h oxidation, all characterizations showed complete 10 oxidation of FeS, which further indicates that FeS inhibits Tc oxidation. X-ray absorption 11 spectroscopy for S/Fe = 0.011 showed significantly increasing percentage of TcS2 in the solid 12 phase after 24-h oxidation, indicating TcS2 is more resistant to oxidation than TcO2. At S/Fe = 13 0.112, the XAS results revealed significant transformation of Tc speciation from TcS2 to TcO2 14 after 120-h oxidation at S/Fe = 0.112. Given that no apparent Tc dissolution occurred during this 15 period, the speciation transformation might play a secondary role in hindering Tc oxidation, 16 especially as redox buffer capacity approached depletion.

Citation: 
Fan D, R Anitori, BM Tebo, PG Tratnyek, JS Lezama Pacheco, RK Kukkadapu, L Kovarik, MH Engelhard, and ME Bowden.2014."Oxidative Remobilization of Technetium Sequestered by Sulfide-Transformed Nano Zerovalent Iron."Environmental Science & Technology 48(13):7409-7417. doi:10.1021/es501607s
Authors: 
D Fan
R Anitori
BM Tebo
PG Tratnyek
JS Lezama Pacheco
RK Kukkadapu
L Kovarik
MH Engelhard
ME Bowden
Instruments: 
Volume: 
48
Issue: 
13
Pages: 
7409-7417
Publication year: 
2014

Secondary Ion Mass Spectrometry Imaging of Dictyostelium discoideum Aggregation Streams.

Abstract: 

High resolution imaging mass spectrometry could become a valuable tool for cell and developmental biology, but both, high spatial and mass spectral resolution are needed to enable this. In this report, we employed Bi3 bombardment time-of-flight (Bi3 ToF-SIMS) and C60 bombardment Fourier transform ion cyclotron resonance secondary ion mass spectrometry (C60 FTICR-SIMS) to image Dictyostelium discoideum aggregation streams. Nearly 300 lipid species were identified from the aggregation streams. High resolution mass spectrometry imaging (FTICR-SIMS) enabled the generation of multiple molecular ion maps at the nominal mass level and provided good coverage for fatty acyls, prenol lipids, and sterol lipids. The comparison of Bi3 ToF-SIMS and C60 FTICR-SIMS suggested that while the first provides fast, high spatial resolution molecular ion images, the chemical complexity of biological samples warrants the use of high resolution analyzers for accurate ion identification.

Citation: 
Debord JD, DF Smith, CR Anderton, RM Heeren, L Pasa-Tolic, RH Gomer, and FA Fernandez-Lima.2014."Secondary Ion Mass Spectrometry Imaging of Dictyostelium discoideum Aggregation Streams."PLoS One 9(6):e99319. doi:10.1371/journal.pone.0099319
Authors: 
JD Debord
DF Smith
CR Anderton
RM Heeren
L Pasa-Tolic
RH Gomer
FA Fernez-Lima
Instruments: 
Volume: 
Issue: 
Pages: 
Publication year: 
2014

Automated High-Pressure Titration System with In Situ Infrared Spectroscopic Detection.

Abstract: 

A fully automated titration system with infrared detection was developed for investigating interfacial chemistry at high pressures. The apparatus consists of a high-pressure fluid generation and delivery system coupled to a high-pressure cell with infrared optics. A manifold of electronically actuated valves is used to direct pressurized fluids into the cell. Precise reagent additions to the pressurized cell are made with calibrated tubing loops that are filled with reagent and placed in-line with the cell and a syringe pump. The cell’s infrared optics facilitate both transmission and attenuated total reflection (ATR) measurements to monitor bulk-fluid composition and solid-surface phenomena such as adsorption, desorption, complexation, dissolution, and precipitation. Switching between the two measurement modes is accomplished with moveable mirrors that direct radiation from a Fourier transform infrared spectrometer into the cell along transmission or ATR light paths. The versatility of the high-pressure IR titration system is demonstrated with three case studies. First, we titrated water into supercritical CO2 (scCO2) to generate an infrared calibration curve and determine the solubility of water in CO2 at 50 °C and 90 bar. Next, we characterized the partitioning of water between a montmorillonite clay and scCO2 at 50 °C and 90 bar. Transmission-mode spectra were used to quantify changes in the clay’s sorbed water concentration as a function of scCO2 hydration, and ATR measurements provided insights into competitive residency of water and CO2 on the clay surface and in the interlayer. Finally, we demonstrated how time-dependent studies can be conducted with the system by monitoring the carbonation reaction of forsterite (Mg2SiO4) in water-bearing scCO2 at 50 °C and 90 bar. Immediately after water dissolved in the scCO2, a thin film of adsorbed water formed on the mineral surface, and the film thickness increased with time as the forsterite began to dissolve. However, after approximately 2.5 hours, the trend reversed, and a carbonate precipitate began to form on the forsterite surface, exposing dramatic chemical changes in the thin-water film. Collectively, these applications illustrate how the high-pressure IR titration system can provide molecular-level information about the interactions between variably wet scCO2 and minerals relevant to underground storage of CO2 (geologic carbon sequestration). The apparatus could also be utilized to study high-pressure interfacial chemistry in other areas such as catalysis, polymerization, food processing, and oil and gas recovery.

Citation: 
Thompson CJ, PF Martin, J Chen, P Benezeth, HT Schaef, KM Rosso, AR Felmy, and JS Loring.2014."Automated High-Pressure Titration System with In Situ Infrared Spectroscopic Detection."Review of Scientific Instruments 85(4):Article No. 044102. doi:10.1063/1.4870411
Authors: 
CJ Thompson
PF Martin
J Chen
P Benezeth
HT Schaef
KM Rosso
AR Felmy
JS Loring
Instruments: 
Volume: 
Issue: 
Pages: 
Publication year: 
2014

Molecular Hydrogen Formation from Proximal Glycol Pairs on TiO2(110).

Abstract: 

Understanding hydrogen formation on TiO2 surfaces is of great importance as it could provide fundamental insight into water splitting for hydrogen production using solar energy. In this work, hydrogen formation from glycols having different numbers of methyl end-groups have been studied using temperature pro-grammed desorption on reduced, hydroxylated, and oxidized TiO2(110) surfaces. The results from OD-labeled glycols demon-strate that gas-phase molecular hydrogen originates exclusively from glycol hydroxyl groups. The yield is controlled by a combi-nation of glycol coverage, steric hindrance, TiO2(110) order and the amount of subsurface charge. Combined, these results show that proximal pairs of hydroxyl aligned glycol molecules and subsurface charge are required to maximize the yield of this redox reaction. These findings highlight the importance of geometric and electronic effects in hydrogen formation from adsorbates on TiO2(110).

Citation: 
Chen L, Z Li, RS Smith, BD Kay, and Z Dohnalek.2014."Molecular Hydrogen Formation from Proximal Glycol Pairs on TiO2(110)."Journal of the American Chemical Society 136(15):5559-5562. doi:10.1021/ja500992b
Authors: 
L Chen
Z Li
RS Smith
BD Kay
Z Dohnalek
Facility: 
Volume: 
136
Issue: 
15
Pages: 
5559-5562
Publication year: 
2014

Arginine-Containing Ligands Enhance H-2 Oxidation Catalyst Performance.

Abstract: 

In H2 fuel cells, performance depends on factors controlling turnover frequency and energy efficiency in the electrocatalytic oxidation of H2. Nature uses the hydrogenase enzymes to oxidize H2 at high turnover frequencies (up to 20,000 s-1) and low overpotentials (<100 mV), while the fastest synthetic catalyst reported to date only oxidizes H2 at 50 s-1 under 1 atm H2. Here we report a water-soluble complex incorporating the amino acid arginine, [NiII(PCy2NArg2)2]6+, that operates at 210 s-1 (180 mV overpotential) under 1 atm H2 and 144,000 s-1 (460 mV overpotential) under 133 atm H2. The complex functions from pH 0-14 with rates increasing at lower pH values. The arginine groups impart water solubility and play a critical role in enhancing turnover frequency, most consistent with an intramolecular Arg-Arg interaction that controls the structure of the catalyst active site. This work was funded by the Office of Science Early Career Research Program through the US DOE, BES (AD, WJS), and the Center for Molecular Electrocatalysis, an Energy Frontier Research Center funded by the US DOE, BES (JASR). PNNL is operated by Battelle for the US DOE.

Citation: 
Dutta A, JA Roberts, and WJ Shaw.2014."Arginine-Containing Ligands Enhance H-2 Oxidation Catalyst Performance."Angewandte Chemie International Edition 53(25):6487-6491. doi:10.1002/anie.201402304
Authors: 
A Dutta
JA Roberts
WJ Shaw
Facility: 
Volume: 
53
Issue: 
25
Pages: 
6487-6491
Publication year: 
2014

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