Cell Isolation and Systems Analysis

The Cell Isolation and Systems Analysis (CISA) capability provides technologies and expertise to study individual cells and cell communities or tissues at the molecular level. Live cells or organelles can be isolated from complex populations, including environmental microbial communities or plant tissues for analyses spanning quantitative live cell fluorescence imaging with single molecule sensitivity, super resolution fluorescence and atomic force microscopy, and transcriptomic analyses using next generation sequencing technologies.

Together with proteomics, metabolomics, and electron and ion microscopy, these capabilities provide the foundation for attaining a molecular-level understanding of individual cells and cell community dynamics and function to support biofuel research, understand the role of biological systems in carbon cycling and enable research in biodefense and other national needs.

High-resolution quantitative fluorescence microscopy in living cells – use multi-photon, confocal, single-molecule and super resolution fluorescence microscopy, including STORM/PALM coupled with atomic force microscopy and structured illumination microscopy (SIM), fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) in intact cells.

Cell and organelle isolation – use the Influx™ flowcytometer cell sorter, and a high resolution laser capture microdissection microscope for isolating distinct cell populations or single cells and organelles from complex microbial communities or tissues for further analyses.

Transcriptomics – use the SOLiD® 5500 series next-generation sequencing systems together with the Ion Proton™ system for massively parallel unbiased sequencing to enable metatranscriptome analysis of complex microbial communities, whole transcriptome and novel transcript identification, as well as RNA-Seq of single eukaryotic cells.

Cell growth – use bioreactors, plant growth chamber, and cell culture facility to support the controlled growth of microbial cells, plants or cell lines under defined conditions for further analyses.

Data interpretation – use computational and modeling tools for data intensive omics data analyses, assimilation and visualization, and quantitative image analysis and statistics tools to achieve meaningful conclusions.

 

Description

Capability Details

  • Super-resolution fluorescence structured illumination microscopy (SIM) enables 3D imaging of intact hydrated cells with 120 nm lateral resolution - Resolves protein complexes and subcellular structures using any fluorescent protein or dye
  • Stochastic optical reconstruction microscopy (STORM), also known as photo-activated localization microscopy (PALM)  enables imaging intact hydrated cells with 20-30 nm resolution - Reconstructs super-resolution fluorescence images of protein complexes and subcellular structures in intact hydrated cells with unprecedented resolution
  • Combined atomic force microscopy (AFM) and STORM/PALM for 3D mapping of cellular structures with 1-10 nm resolution coupled  with single-molecule and super resolution fluorescence imaging to identify distinct proteins and molecular complexes in the membrane of intact cells or organelles
  • Multi-photon fluorescence microscopy system that seamlessly integrates nonlinear excitation, laser scanning confocal microscopy, fluorescence lifetime imaging (FLIM), and differential interference contrast (DIC) imaging – enables minimally invasive and deeply penetrating laser excitation for high resolution 3D imaging and detection of molecular interactions in single cells, cell communities or tissues
  • Time-lapse single molecule fluorescence imaging uses total internal reflection fluorescence (TIRF) techniques to track individual molecules or organelles in live cells – enables the study of subcellular processes over time, and molecular interaction dynamics using fluorescence resonance energy transfer (FRET) in live cells
  • Two SOLiD® systems together with the Ion Proton™ provide unbiased global transcriptome analyses with high accuracy and throughput using  multiplexing capability for RNA sequencing (RNA-Seq) of multiple samples in parallel - enables global gene expression analyses, novel gene or isoform identification, and regulation of gene expression studies, such as ChIP-Seq or microRNA analyses in complex microbial communities or individual organisms
  • The Influx™ flow cytometer/cell sorter has multiple laser lines and powerful detection capabilities for high throughput analysis and sorting of distinct cells or organelles using advanced multi-parameter sorting technologies based on the presence and content of distinct genes and proteins or intracellular structures - supports detection and sorting of nanoscale particles, making it highly suited for sorting and analyzing organelles and single cells
  • High resolution laser capture microdissection microscope  equipped with a 100x magnification objective lens and multiple fluorescence lines – enables the enrichment of distinct organelles or isolation of single cells from cell populations or tissue sections for further analyses
  • The CyTOF® mass cytometer – uses Time-of-Fight mass spectrometry in single cells to quantify the expression of multiple proteins or mRNA species, each tagged with a different stable heavy metal isotope – enables high throughput single cell analysis of the expression of multiple proteins using antibodies, or multiple genes using in situ hybridization probes
  • Bioreactors for controlled growth and monitoring of diverse microbial cells in volumes ranging from 100 µl wells to multi-liter reactors. These include the Bioscreen-C™ for automated real-time analysis of growth rate in up to 200 independent 100 µl wells; the Micro-24 MicroReactor system for 24 independently controlled 5 ml reactors; and the BioFlo® 310 benchtop fermentor-bioreactor system for larger volume

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

The stability of sub-nanometer size gold clusters ligated with organic molecules is of paramount importance to the scalable synthesis of monodisperse...
The scalable synthesis of subnanometer metal clusters containing an exact number of atoms is of interest due to the highly size-dependent catalytic,...
The oxygen reduction/evolution reaction (ORR/OER) mechanisms in nonaqueous Li-O2 batteries have been investigated by using electron paramagnetic...
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...

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: October 28, 2010
Nanoparticles show promise in solving a host of problems, from pinpointing medical diagnoses to developing alternative forms of energy and creating...

The Cell Isolation and Systems Analysis (CISA) capability provides technologies and expertise to study individual cells and cell communities or tissues at the molecular level. Live cells or organelles can be isolated from complex populations, including environmental microbial communities or plant tissues for analyses spanning quantitative live cell fluorescence imaging with single molecule sensitivity, super resolution fluorescence and atomic force microscopy, and transcriptomic analyses using next generation sequencing technologies.

Together with proteomics, metabolomics, and electron and ion microscopy, these capabilities provide the foundation for attaining a molecular-level understanding of individual cells and cell community dynamics and function to support biofuel research, understand the role of biological systems in carbon cycling and enable research in biodefense and other national needs.

High-resolution quantitative fluorescence microscopy in living cells – use multi-photon, confocal, single-molecule and super resolution fluorescence microscopy, including STORM/PALM coupled with atomic force microscopy and structured illumination microscopy (SIM), fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) in intact cells.

Cell and organelle isolation – use the Influx™ flowcytometer cell sorter, and a high resolution laser capture microdissection microscope for isolating distinct cell populations or single cells and organelles from complex microbial communities or tissues for further analyses.

Transcriptomics – use the SOLiD® 5500 series next-generation sequencing systems together with the Ion Proton™ system for massively parallel unbiased sequencing to enable metatranscriptome analysis of complex microbial communities, whole transcriptome and novel transcript identification, as well as RNA-Seq of single eukaryotic cells.

Cell growth – use bioreactors, plant growth chamber, and cell culture facility to support the controlled growth of microbial cells, plants or cell lines under defined conditions for further analyses.

Data interpretation – use computational and modeling tools for data intensive omics data analyses, assimilation and visualization, and quantitative image analysis and statistics tools to achieve meaningful conclusions.

 

Investigating the Synthesis of Ligated Metal Clusters in Solution Using a Flow Reactor and Electrospray Ionization Mass

Abstract: 

The scalable synthesis of subnanometer metal clusters containing an exact number of atoms is of interest due to the highly size-dependent catalytic, electronic and optical properties of these species. While significant research has been conducted on the batch preparation of clusters through reduction synthesis in solution, the processes of metal complex reduction as well as cluster nucleation, growth and post-reduction etching are still not well understood. Herein, we demonstrate a temperature-controlled flow reactor for studying cluster formation in solution at well-defined conditions. Employing this technique methanol solutions of a chloro(triphenylphosphine)gold precursor, 1,4-bis(diphenylphosphino)butane capping ligand and borane-tert-butylamine reducing agent were combined in a mixing tee and introduced into a heated capillary with an adjustable length. In this manner, the temperature dependence of the relative abundance of different ionic reactants, intermediates and products synthesized in real time was characterized using online mass spectrometry. A wide distribution of doubly and triply charged cationic gold clusters was observed as well as smaller singly charged metal-ligand complexes. The results demonstrate that temperature plays a crucial role in determining the relative population of cationic gold clusters and, in general, that higher temperature promotes the formation of doubly charged clusters and singly charged metal-ligand complexes while hindering the growth of triply charged clusters. Moreover, the distribution of clusters observed at elevated temperatures is found to be consistent with that obtained at longer reaction times at room temperature, thereby demonstrating that heating may be used to access cluster distributions characteristic of different stages of reduction synthesis in solution.

Citation: 
Olivares AM, J Laskin, and GE Johnson.2014."Investigating the Synthesis of Ligated Metal Clusters in Solution Using a Flow Reactor and Electrospray Ionization Mass Spectrometry."Journal of Physical Chemistry A 118(37):8464-8470. doi:10.1021/jp501809r
Authors: 
AM Olivares
J Laskin
GE Johnson
Facility: 
Volume: 
118
Issue: 
37
Pages: 
8464-8470
Publication year: 
2014

Size-dependent stability toward dissociation and ligand binding energies of phosphine-ligated gold cluster ions.

Abstract: 

The stability of sub-nanometer size gold clusters ligated with organic molecules is of paramount importance to the scalable synthesis of monodisperse size-selected metal clusters with highly tunable chemical and physical properties. For the first time, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) equipped with surface induced dissociation (SID) has been employed to investigate the time and collision energy resolved fragmentation behavior of cationic doubly charged gold clusters containing 7-9 gold atoms and 6-7 triphenylphosphine (TPP) ligands prepared by reduction synthesis in solution. The TPP ligated gold clusters are demonstrated to fragment through three primary dissociation pathways: (1) Loss of a neutral TPP ligand from the precursor gold cluster, (2) asymmetric fission and (3) symmetric fission and charge separation of the gold core resulting in formation of complementary pairs of singly charged fragment ions. Threshold energies and activation entropies of these fragmentation pathways have been determined employing Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental SID data. It is demonstrated that the doubly charged cluster ion containing eight gold atoms and six TPP ligands, (8,6)2+, exhibits exceptional stability compared to the other cationic gold clusters examined in this study due to its large ligand binding energy of 1.76 eV. Our findings demonstrate the dramatic effect of the size and extent of ligation on the gas-phase stability and preferred fragmentation pathways of small TPP-ligated gold clusters.

Citation: 
Johnson GE, TA Priest, and J Laskin.2014."Size-dependent stability toward dissociation and ligand binding energies of phosphine-ligated gold cluster ions."Chemical Science 5:3275-3286. doi:10.1039/c4sc00849a
Authors: 
GE Johnson
TA Priest
J Laskin
Facility: 
Volume: 
Issue: 
Pages: 
Publication year: 
2014

The Mechanisms of Oxygen Reduction and Evolution Reactions in Nonaqueous Lithium-Oxygen Batteries.

Abstract: 

The oxygen reduction/evolution reaction (ORR/OER) mechanisms in nonaqueous Li-O2 batteries have been investigated by using electron paramagnetic resonance spectroscopy in this work. We identified the superoxide radical anion (O2•-) as an intermediate in the ORR process using 5,5-dimethyl-pyrroline N-oxide as a spin trap, while no O2•- in OER was detected during the charge process. These findings provide insightful understanding on the fundamental oxygen reaction mechanisms in rechargeable nonaqueous Li-O2 batteries.

Citation: 
Cao R, ED Walter, W Xu, EN Nasybulin, P Bhattacharya, ME Bowden, MH Engelhard, and J Zhang.2014."The Mechanisms of Oxygen Reduction and Evolution Reactions in Nonaqueous Lithium-Oxygen Batteries."ChemSusChem 7(9):2436-2440. doi:10.1002/cssc.201402315
Authors: 
R Cao
ED Walter
W Xu
EN Nasybulin
P Bhattacharya
ME Bowden
MH Engelhard
J Zhang
Instruments: 
Volume: 
7
Issue: 
9
Pages: 
2436-2440
Publication year: 
2014

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

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