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.  See a complete list of CISA instruments.

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. 

Resources and Techniques

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 volumes.

Instruments

Two massively parallel next-generation sequencing platforms (SOLiD 5500 series) are currently incorporated in users' research for transcriptomics...
Custodian(s): Galya Orr, Lye Meng Markillie
This instrument is housed in EMSL.  More details about this instrument will be available soon.
Custodian(s): Galya Orr, Dehong Hu
This microscopy system integrates seamlessly nonlinear two-photon excitation, laser-scanning confocal microscopy, and fluorescence lifetime imaging...
Custodian(s): Galya Orr, 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
The FEI Helios Nanolab dual-beam focused ion beam/scanning electron microscopy (FIB/SEM) microscope combines two important high-resolution...
Custodian(s): Bruce Arey

Publications

The interactions between proteins and surfaces are critical to a number of important processes including biomineralization, the biocompatibility of...
The interaction of nanomaterials with biomolecules, cells, and organisms is an enormously vital area of current research, with applications in...
Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of...
For applications of tristructural isotropic (TRISO) fuel particles in high temperature reactors, release of radioactive Ag isotope (110mAg) through...
Experimental verification of the microscopic origin of resistance switching in metal/oxide/metal heterostructures is needed for applications in non-...

Science Highlights

Posted: April 08, 2016
A research team from Pacific Northwest National Laboratory, or PNNL, EMSL and The Ohio State University studied microbial activity near rivers in an...
Posted: August 17, 2015
The Science With increasing emphasis on sustainable energy sources, lipid-derived biofuels have been proposed as a promising substitute for fossil...
Posted: April 17, 2015
The Science Archaea, a domain of single-celled microorganisms, represent a significant fraction of the earth’s biodiversity, yet they remain much...
Posted: February 06, 2015
The Science Cyanobacteria are of considerable interest as production organisms in biotechnology. They can grow by harvesting energy from sunlight,...
Posted: January 28, 2015
Scientists at Pacific Northwest National Laboratory, University of Puget Sound and EMSL used EMSL resources and capabilities to study the...

Instruments

The United States is one of the top nations for prematurely born infants and has a correspondingly high dayone infant mortality rate. Expanding our...
The United States is one of the top nations for prematurely born infants and has a correspondingly high dayone infant mortality rate. Expanding our...
Ciliate protists are ubiquitous in terrestrial environments, but their role in biomass deconstruction in these environments remains undefined. In...
Background: In the proposed work we will use a newly developed experimental system comprised of the alga Chrysochromulina tobin (Haptophyceae) and a...
We are requesting access to EMSL microscopy, including ETEM and STEM electron microscopes and the helium ion microscope, as well as the super...

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.  See a complete list of CISA instruments.

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. 

Resources and Techniques

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.

Ag Out-surface Diffusion In Crystalline SiC With An Effective SiO2 Diffusion Barrier.

Abstract: 

For applications of tristructural isotropic (TRISO) fuel particles in high temperature reactors, release of radioactive Ag isotope (110mAg) through the SiC coating layer is a safety concern. To understand the diffusion mechanism, Ag ion implantations near the surface and in the bulk were performed by utilizing different ion energies and energy-degrader foils. High temperature annealing was carried out on the as-irradiated samples to study the possible out-surface diffusion. Before and after annealing, Rutherford backscattering spectrometry (RBS) and secondary ion mass spectrometry (SIMS) measurements were employed to obtain the elemental profiles of the implanted samples. The results suggest little migration of buried Ag in the bulk, and an out-diffusion of the implanted Ag in the near-surface region of single crystal SiC. It is also found that a SiO2 layer, which was formed during annealing, may serve as an effective barrier to reduce or prevent Ag out diffusion through the SiC coating layer.

Citation: 
Xue H, HY Xiao, Z Zhu, V Shutthanandan, LL Snead, LA Boatner, WJ Weber, and Y Zhang.2015."Ag Out-surface Diffusion In Crystalline SiC With An Effective SiO2 Diffusion Barrier."Journal of Nuclear Materials 464:294-298. doi:10.1016/j.jnucmat.2015.05.001
Authors: 
Zhu Zihua
V Shutthanandan
Xue H
HY Xiao
Z Zhu
V Shutthanan
LL Snead
LA Boatner
WJ Weber
Y Zhang
Facility: 
Volume: 
Issue: 
Pages: 
Publication year: 
2015

Soft Landing of Bare PtRu Nanoparticles for Electrochemical Reduction of Oxygen .

Abstract: 

Magnetron sputtering of two independent Pt and Ru targets coupled with inert gas aggregation in a modified commercial source has been combined with soft landing of mass-selected ions to prepare bare 4.5 nm diameter PtRu alloy nanoparticles on glassy carbon electrodes with controlled size and morphology for electrochemical reduction of oxygen in solution. Employing atomic force microscopy (AFM) it is shown that the nanoparticles bind randomly to the glassy carbon electrode at a relatively low coverage of 7 x 104 ions µm-2 and that their average height is centered at 4 nm. Scanning transmission electron microscopy images obtained in the high-angle annular dark field mode (STEM-HAADF) further confirm that the soft-landed PtRu alloy nanoparticles are uniform in size and have a Ru core decorated with small regions of Pt on the surface. Wide-area scans of the electrodes using X-ray photoelectron spectroscopy (XPS) reveal the presence of both Pt and Ru in relative atomic concentrations of ~9% and ~33%, respectively. Deconvolution of the high energy resolution XPS spectra in the Pt4f and Ru3d regions indicates the presence of both oxidized Pt and Ru. The substantially higher loading of Ru compared to Pt and enrichment of Pt at the surface of the alloy nanoparticles is confirmed by wide-area analysis of the electrodes using time-of-flight medium energy ion scattering (TOF-MEIS) employing both 80 keV He+ and O+ ions. The activity of electrodes containing 7 x 104 ions µm-2 of bare 4.5 nm PtRu nanoparticles toward the electrochemical reduction of oxygen was evaluated employing cyclic voltammetry (CV) in 0.1 M HClO4 and 0.5 M H2SO4 solutions. In both electrolytes a pronounced reduction peak was observed during O2 purging of the solution that was not evident during purging with Ar. Repeated electrochemical cycling of the electrodes revealed little evolution in the shape or position of the voltammograms indicating high stability of the alloy nanoparticles supported on glassy carbon. The reproducibility of the nanoparticle synthesis and deposition was evaluated by employing the same experimental parameters to prepare nanoparticles on glassy carbon electrodes on three occasions separated by several days. Surfaces with almost identical electrochemical behavior were observed with CV, demonstrating the highly reproducible preparation of bare alloy nanoparticles using physical synthesis in the gas-phase combined with soft landing of mass-selected ions

Citation: 
Johnson GE, RJ Colby, MH Engelhard, DW Moon, and J Laskin.2015."Soft Landing of Bare PtRu Nanoparticles for Electrochemical Reduction of Oxygen ."Nanoscale 7(29):12379-12391. doi:10.1039/C5NR03154K
Authors: 
H Mark
Julia Laskin
Johnson GE
RJ Colby
MH Engelhard
DW Moon
J Laskin
Instruments: 
Volume: 
7
Issue: 
29
Pages: 
12379-12391
Publication year: 
2015

Molecular Characterization of Brown Carbon (BrC) Chromophores in Secondary Organic Aerosol Generated From Photo-Oxidation of

Abstract: 

Atmospheric Brown carbon (BrC) is a significant contributor to light absorption and climate forcing. However, little is known about a fundamental relationship between the chemical composition of BrC and its optical properties. In this work, light-absorbing secondary organic aerosol (SOA) was generated in the PNNL chamber from toluene photo-oxidation in the presence of NOx (Tol-SOA). Molecular structures of BrC components were examined using nanospray desorption electrospray ionization (nano-DESI) and liquid chromatography (LC) combined with UV/Vis spectroscopy and electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). The chemical composition of BrC chromophores and the light absorption properties of toluene SOA (Tol-SOA) depend strongly on the initial NOx concentration. Specifically, Tol-SOA generated under high-NOx conditions (defined here as initial NOx/toluene of 5/1) appears yellow and mass absorption coefficient of the bulk sample (MACbulk@365nm = 0.78 m2 g-1) is nearly 80 fold higher than that measured for the Tol-SOA sample generated under low-NOx conditions (NOx/toluene < 1/300). Fifteen compounds, most of which are nitrophenols, are identified as major BrC chromophores responsible for the enhanced light absorption of Tol-SOA material produced in the presence of NOx. The integrated absorbance of these fifteen chromophores accounts for 40-60% of the total light absorbance by Tol-SOA at wavelengths between 300 nm and 500 nm. The combination of tandem LC-UV/Vis-ESI/HRMS measurements provides an analytical platform for predictive understanding of light absorption properties by BrC and their relationship to the structure of individual chromophores. General trends in the UV/vis absorption by plausible isomers of the BrC chromophores were evaluated using theoretical chemistry calculations. The molecular-level understanding of BrC chemistry is helpful for better understanding the evolution and behavior of light absorbing aerosols in the atmosphere.

Citation: 
Lin P, J Liu, JE Shilling, SM Kathmann, J Laskin, and A Laskin.2015."Molecular Characterization of Brown Carbon (BrC) Chromophores in Secondary Organic Aerosol Generated From Photo-Oxidation of Toluene."Physical Chemistry Chemical Physics. PCCP 17(36):23312-23325. doi:10.1039/c5cp02563j
Authors: 
Lin Peng
Julia Laskin
Alexander Laskin
Lin P
J Liu
JE Shilling
SM Kathmann
J Laskin
A Laskin
Facility: 
Instruments: 
Volume: 
17
Issue: 
36
Pages: 
23312-23325
Publication year: 
2015

Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations.

Abstract: 

We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. These results could serve as guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.

Citation: 
Aidhy DS, B Liu, Y Zhang, and WJ Weber.2015."Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations."Computational Materials Science 99:298-305. doi:doi:10.1016/j.commatsci.2014.12.030
Authors: 
DS Aidhy
B Liu
Y Zhang
WJ Weber
Volume: 
Issue: 
Pages: 
Publication year: 
2015

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Leads

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Dr. Orr has been the lead PI on a grant funded by NIH-National Institute of Environmental Health Sciences (NIEHS), on a STAR grant funded by the EPA, and on a grant from the Air Force Research Laboratory, where quantitative super resolution and...