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.

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

Functionalized graphene sheets (FGS) are successfully utilized as a novel air electrode for Li-O2 batteries. An extremely high capacity of 15,000 mAh...
High-resolution characterizations of intergranular attack in alloy 600 (Ni-17Cr-9Fe) exposed to 325 °C simulated pressurized water reactor (...
Ensuring data quality and proper instrument functionality is a prerequisite for scientific investigation. Manual validation for quality assurance is...
Single crystalline Na4Mn9O18 nanowires were synthesized via pyrolysis of polyacrylate salt precursors prepared by in-situ polymerization of the metal...
Strain confinement in heterostructured films significantly affects ionic conductivity of the electrolytes for solid oxide fuel cells based on a multi...

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.

Enhanced Quantum Efficiency From Hybrid Cesium Halide/Copper Photocathode.

Abstract: 

The quantum efficiency of Cu is found to increase dramatically when coated by a CsI film and then irradiated by a UV laser. Over three orders of magnitude quantum efficiency enhancement at 266 nm is observed in CsI/Cu(100), indicating potential application in future photocathode devices. Upon laser irradiation, a large work function reduction to a value less than 2 eV is also observed, significantly greater than for similarly treated CsBr/Cu(100). The initial QE enhancement, prior to laser irradiation, is attributed to interface interaction, surface cleanliness and the intrinsic properties of the Cs halide film. Further QE enhancement following activation is attributed to formation of inter-band states and Cs metal accumulation at the interface induced by laser irradiation.

Citation: 
Kong L, AG Joly, TC Droubay, Y Gong, and WP Hess.2014."Enhanced Quantum Efficiency From Hybrid Cesium Halide/Copper Photocathode."Applied Physics Letters 104(17):Article No. 171106. doi:10.1063/1.4874339
Authors: 
L Kong
AG Joly
TC Droubay
Y Gong
WP Hess
Instruments: 
Volume: 
0
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0
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0
Publication year: 
2014

Quantitatively Probing the Al Distribution in Zeolites.

Abstract: 

The degree of substitution of Si4+ by Al3+ in the oxygen-terminated tetrahedra (Al T-sites) of zeolites determines the concentration of ion-exchange and Brønsted acid sites. As the location of the tetrahedra and the associated subtle variations in bond angles influence the acid strength, quantitative information about Al T-sites in the framework is critical to rationalize catalytic properties and to design new catalysts. A quantitative analysis is reported that uses a combination of extended X-ray absorption fine structure (EXAFS) analysis and 27Al MAS NMR spectroscopy supported by DFT-based molecular dynamics simulations. To discriminate individual Al atoms, sets of ab initio EXAFS spectra for various T-sites are generated from DFT-based molecular dynamics simulations allowing quantitative treatment of the EXAFS single- and multiple-photoelectron scattering processes out to 3-4 atom shells surrounding the Al absorption center. It is observed that identical zeolite types show dramatically different Al-distributions. A preference of Al for T-sites that are part of one or more 4-member rings in the framework over those T-sites that are part of only 5- and 6-member rings in the HBEA150 sample has been determined from a combination of these methods. This work was supported by the U. S. Department of Energy (DOE), Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences.

Citation: 
Vjunov A, JL Fulton, T Huthwelker, S Pin, D Mei, GK Schenter, N Govind, DM Camaioni, JZ Hu, and JA Lercher.2014."Quantitatively Probing the Al Distribution in Zeolites."Journal of the American Chemical Society 136(23):8296-8306. doi:10.1021/ja501361v
Authors: 
A Vjunov
JL Fulton
T Huthwelker
S Pin
D Mei
GK Schenter
N Govind
DM Camaioni
JZ Hu
JA Lercher
Instruments: 
Volume: 
136
Issue: 
23
Pages: 
8296-8306
Publication year: 
2014

Molecular Structure and Stability of Dissolved Lithium Polysulfide Species.

Abstract: 

Ability to predict the solubility and stability of lithium polysulfide is vital in realizing longer lasting lithium-sulfur batteries. Herein we report a combined computational and experimental spectroscopic analysis to understand the dissolution mechanism of lithium polysulfide species in an aprotic solvent medium. Multinuclear NMR and sulfur K-edge X-ray absorption (XAS) analysis reveals that the lithium exchange between polysulfide species and solvent molecule constitutes the first step in the dissolution process. Lithium exchange leads to de-lithiated polysulfide ions which subsequently forms highly reactive free radicals through disproportion reaction. The energy required for the disproportion and possible dimer formation reactions of the polysulfide species are analyzed using density functional theory (DFT) calculations. We validate our calculations with variable temperature electron spin resonance (ESR) measurements. Based on these findings, we discuss approaches to optimize the electrolyte in order to control the polysulfide solubility. The energy required for the disproportion and possible dimer formation reactions of the polysulfide species are analyzed using density functional theory (DFT) calculations. We validate our calculations with variable temperature electron spin resonance (ESR) measurements. Based on these findings, we discuss approaches to optimize the electrolyte in order to control the polysulfide solubility.

Citation: 
Vijayakumar M, N Govind, ED Walter, SD Burton, AK Shukla, A Devaraj, J Xiao, J Liu, CM Wang, AM Karim, and S Thevuthasan.2014."Molecular Structure and Stability of Dissolved Lithium Polysulfide Species."Physical Chemistry Chemical Physics. PCCP 16(22):10923-10932. doi:10.1039/c4cp00889H
Authors: 
M Vijayakumar
N Govind
ED Walter
SD Burton
AK Shukla
A Devaraj
J Xiao
J Liu
CM Wang
AM Karim
S Thevuthasan
Instruments: 
Volume: 
16
Issue: 
22
Pages: 
10923-10932
Publication year: 
2014

Electrochemically Stable Cathode Current Collectors for Rechargeable Magnesium Batteries .

Abstract: 

Rechargeable Mg batteries are attractive energy storage systems and could bring cost-effective energy solutions. Currently, however, no practical cathode current collectors that can withstand high voltages in Mg2+ electrolytes has been identified and therefore cathode research is greatly hindered. Here we identified that two metals, Mo and W, are electrochemically stable through formation of surface passive layers. The presented results could have significant impacts on the developments of high voltage Mg batteries.

Citation: 
Cheng Y, TL Liu, Y Shao, MH Engelhard, J Liu, and G Li.2014."Electrochemically Stable Cathode Current Collectors for Rechargeable Magnesium Batteries ."Journal of Materials Chemistry A 2(8):2473 - 2477. doi:10.1039/C3TA15113A
Authors: 
Y Cheng
TL Liu
Y Shao
MH Engelhard
J Liu
G Li
Facility: 
Instruments: 
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0
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0
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0
Publication year: 
2014

Methanol Synthesis from CO2 Hydrogenation over a Pd4/In2O3 Model Catalyst: A Combined DFT and Kinetic Study.

Abstract: 

Methanol synthesis from CO2 hydrogenation on Pd4/In2O3 has been investigated using density functional theory (DFT) and microkinetic modeling. In this study, three possible routes in the reaction network of CO2 + H2 → CH3OH + H2O have been examined. Our DFT results show that the HCOO route competes with the RWGS route whereas a high activation barrier kinetically blocks the HCOOH route. DFT results also suggest that H2COO* + H* ↔ H2CO* +OH* and cis-COOH* + H* ↔CO* + H2O* are the rate limiting steps in the HCOO route and the RWGS route, respectively. Microkinetic modeling results demonstrate that the HCOO route is the dominant reaction route for methanol synthesis from CO2 hydrogenation. We found that the activation of H adatom on the small Pd cluster and the presence of H2O on the In2O3 substrate play important roles in promoting the methanol synthesis. The hydroxyl adsorbed at the interface of Pd4/In2O3 induces the transformation of the supported Pd4 cluster from a butterfly structure into a tetrahedron structure. This important structure change not only indicates the dynamical nature of the supported nanoparticle catalyst structure during the reaction but also shifts the final hydrogenation step from H2COH to CH3O.

Citation: 
Ye J, C Liu, D Mei, and Q Ge.2014."Methanol Synthesis from CO2 Hydrogenation over a Pd4/In2O3 Model Catalyst: A Combined DFT and Kinetic Study."Journal of Catalysis 317:44-53. doi:10.1016/j.jcat.2014.06.002
Authors: 
Ye J
C Liu
D Mei
Q Ge
Instruments: 
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0
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0
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0
Publication year: 
2014

Mineralization of Basalts in the CO2-H2O-SO2-O2 System.

Abstract: 

Sequestering carbon dioxide (CO2) containing minor amounts of co-contaminants in geologic formations was investigated in the laboratory through the use of high pressure static experiments. Five different basalt samples were immersed in water equilibrated with supercritical CO2 containing 1wt% sulfur dioxide (SO2) and 1wt% oxygen (O2) at reservoir conditions (~100 bar, 90°C) for 49 and 98 days. Gypsum (CaSO4) was a common precipitate, occurred early as elongated blades with striations, and served as substrates for other mineral products. Bimodal pulses of water released during dehydroxylation were key indicators along with X-ray diffraction for verifying the presences of jarosite-alunite group minerals. Well-developed pseudocubic jarosite crystals formed surface coatings, and in some instances mixtures of natrojarosite and natroalunite aggregated into spherically shaped structures measuring 100 μm in diameter. Reaction products were also characterized using infrared spectroscopy, which indicated OH and Fe-O stretching modes. The presences of jarosite-alunite group minerals were found in the lower wavenumber region from 700–400 cm-1. A strong preferential incorporation of Fe(III) into natrojarosite was attributed to the oxidation potential of O2. Evidence of CO2 was detected during thermal decomposition of precipitates, suggesting the onset of mineral carbonation.

Citation: 
Schaef HT, JA Horner, AT Owen, CJ Thompson, JS Loring, and BP McGrail.2014."Mineralization of Basalts in the CO2-H2O-SO2-O2 System."Environmental Science & Technology 48(9):5298-5305. doi:10.1021/es404964j
Authors: 
HT Schaef
JA Horner
AT Owen
CJ Thompson
JS Loring
BP McGrail
Volume: 
48
Issue: 
9
Pages: 
5298-5305
Publication year: 
2014

Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates.

Abstract: 

In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of charged peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.

Citation: 
Wang D, S Dasari, MC Chambers, JD Holman, K Chen, D Liebler, DJ Orton, SO Purvine, ME Monroe, CY Chung, KL Rose, and DL Tabb.2013."Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates."Genomics, Proteomics & Bioinformatics 11(2):86-95. doi:10.1016/j.gpb.2012.11.004
Authors: 
D Wang
S Dasari
MC Chambers
JD Holman
K Chen
D Liebler
DJ Orton
SO Purvine
ME Monroe
CY Chung
KL Rose
DL Tabb
Facility: 
Instruments: 
Volume: 
11
Issue: 
2
Pages: 
86-95
Publication year: 
2013

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence

Abstract: 

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Citation: 
Chien MP, AS Carlini, D Hu, CV Barback, AM Rush, DJ Hall, G Orr, and NC Gianneschi.2013."Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence Imaging."Journal of the American Chemical Society 135(50):18710-18713. doi:10.1021/ja408182p
Authors: 
MP Chien
AS Carlini
D Hu
CV Barback
AM Rush
DJ Hall
G Orr
NC Gianneschi
Facility: 
Instruments: 
Volume: 
135
Issue: 
50
Pages: 
18710-18713
Publication year: 
2013

A Facile Approach Using MgCl2 to Formulate High Performance Mg2+ Electrolytes for Rechargeable Mg Batteries.

Abstract: 

Rechargeable Mg batteries have been regarded as a viable battery technology for grid scale energy storage and transportation applications. However, the limited performance of Mg2+ electrolytes has been a primary technical hurdle to develop high energy density rechargeable Mg batteries. In this study, MgCl2 is demonstrated as a non-nucleophilic and cheap Mg2+ source in combining with Al Lewis acids (AlCl3, AlPh3 and AlEtCl2) to formulate a series of Mg2+ electrolytes characteristic of high oxidation stability (up to 3.4 V vs Mg), sulfur compatibility and electrochemical reversibility (up to 100% coulombic efficiency). Three electrolyte systems (MgCl2-AlCl3, MgCl2-AlPh3, and MgCl2-AlEtCl2) were prepared free of purification and fully characterized by multinuclear NMR (27Al{1H} and 25Mg{1H}) spectroscopies, single crystal X-ray diffraction, and electrochemical analysis. The reaction mechanism of MgCl2 and the Al Lewis acids in THF is discussed to highlight the formation of the electrochemically active [(µ-Cl)3Mg2(THF)6]+ monocation in these electrolytes. We are grateful for the financial support from the Pacific Northwest National Laboratory (PNNL)-Laboratory Directed Research and Development (LDRD) program for developing magnesium battery technology. The XRD and SEM data were collected at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at PNNL. PNNL is a multiprogram laboratory operated by Battelle Memorial Institute for the Department of Energy under Contract DE-AC05-76RL01830.

Citation: 
Liu TL, Y Shao, G Li, M Gu, JZ Hu, S Xu, Z Nie, X Chen, CM Wang, and J Liu.2014."A Facile Approach Using MgCl2 to Formulate High Performance Mg2+ Electrolytes for Rechargeable Mg Batteries."Journal of Materials Chemistry A 2(10):3430 - 3438. doi:10.1039/c3ta14825d
Authors: 
TL Liu
Y Shao
G Li
M Gu
JZ Hu
S Xu
Z Nie
X Chen
CM Wang
J Liu
Volume: 
0
Issue: 
0
Pages: 
0
Publication year: 
2014

Metal-Insulator Photocathode Heterojunction for Directed Electron Emission.

Abstract: 

New photocathode materials capable of producing intense and directed electron pulses are needed for development of next generation light sources and dynamic transmission electron microscopy. Ideal photocathodes should have high photoemission quantum efficiency (QE) and be capable of delivering collimated and well-shaped pulses of consistent charge under high-field operating conditions. High-brightness and low-intrinsic emittance electron pulses have been predicted for hybrid metal-insulator photocathode designs constructed from three to four monolayer MgO films on atomically flat silver. Here we use angle-resolved photoelectron spectroscopy to confirm directional photoemission and a large increase in QE under ultraviolet laser excitation of an ultrathin MgO film on Ag(001). We observe new low-binding energy photoemission, not seen for Ag(001), and greater electron emission in the normal direction. Under 4.66 eV laser excitation, the photoemission quantum efficiency of the MgO/Ag(001) hybrid photocathode is a factor of seven greater than that for clean Ag(001).

Citation: 
Droubay TC, SA Chambers, AG Joly, WP Hess, K Nemeth, KC Harkay, and L Spentzouris.2014."Metal-Insulator Photocathode Heterojunction for Directed Electron Emission."Physical Review Letters 112(6):067601(5). doi:10.1103/PhysRevLett.112.067601
Authors: 
TC Droubay
SA Chambers
AG Joly
WP Hess
K Nemeth
KC Harkay
L Spentzouris
Facility: 
Volume: 
0
Issue: 
0
Pages: 
0
Publication year: 
2014

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