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.

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.

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,...
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
The single-molecule optical microscope is designed to study complex reaction dynamics such as enzymatic reactions, protein-protein interactions, and...
Porous graphitic carbon (PGC) particles were functionalized/passivated in situ in packed beds at elevated temperature with neat di-tert-amylperoxide...
A series of cathodes using Pt supported onto graphene sheets with different contents of carbon black in the catalyst layer were prepared and...
Reaction barriers were calculated by using ab initio electronic structure methods for the reductive dechlorination of the polychlorinated ethylenes:...
P53 phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We report...
Phosphorylated p53 at serin 15 (phospho-p53-15) is a potential biomarker of Gamma-radiation exposure. In this paper, we described a new magnetic...
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.

7 Å Resolution in Protein 2-Dimentional-Crystal X-Ray Diffraction at Linac Coherent Light Source.

Abstract: 

Membrane proteins arranged as two-dimensional (2D) crystals in the lipid en- vironment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. X-ray diffraction from individual 2D crystals did not represent a suitable investigation tool because of radiation damage. The recent availability of ultrashort pulses from X-ray Free Electron Lasers (X-FELs) has now provided a mean to outrun the damage. Here we report on measurements performed at the LCLS X-FEL on bacteriorhodopsin 2D crystals mounted on a solid support and kept at room temperature. By merg- ing data from about a dozen of single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 °A, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase of resolution. The presented results pave the way to further X-FEL studies on 2D crystals, which may include pump-probe experiments at subpicosecond time resolution.

Citation: 
Pedrini B, CJ Tsai, G Capitani, C Padeste, M Hunter, NA Zatsepin, A Barty, H Benner, S Boutet, GK Feld, S Hau-Riege, R Kirian, C Kupitz, M Messerschmidt, JI Ogren, T Pardini, B Segelke, GJ Williams, JC Spence , R Abela, MA Coleman, JE Evans, G Schertler, M Frank, and XD Li.2014."7 Å Resolution in Protein 2-Dimentional-Crystal X-Ray Diffraction at Linac Coherent Light Source."Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 369(1647):Article No. 20130500. doi:10.1098/rstb.2013.0500
Authors: 
B Pedrini
CJ Tsai
G Capitani
C Padeste
M Hunter
NA Zatsepin
A Barty
H Benner
S Boutet
GK Feld
S Hau-Riege
R Kirian
C Kupitz
M Messerschmidt
JI Ogren
T Pardini
B Segelke
GJ Williams
JC Spence
R Abela
MA Coleman
JE Evans
G Schertler
M Frank
XD Li
Instruments: 
Publication year: 
2014

In situ molecular imaging of hydrated biofilm in a microfluidic reactor by ToF-SIMS.

Abstract: 

The first results of using a novel single channel microfluidic reactor to enable Shewanella biofilm growth and in situ characterization using time-of-flight secondary ion mass spectrometry (ToF-SIMS) in the hydrated environment are presented. The new microfluidic interface allows direct probing of the liquid surface using ToF-SIMS, a vacuum surface technique. The detection window is an aperture of 2 m in diameter on a thin silicon nitride (SiN) membrane and it allows direct detection of the liquid surface. Surface tension of the liquid flowing inside the microchannel holds the liquid within the aperture. ToF-SIMS depth profiling was used to drill through the SiN membrane and the biofilm grown on the substrate. In situ 2D imaging of the biofilm in hydrated state was acquired, providing spatial distribution of the chemical compounds in the biofilm system. This data was compared with a medium filled microfluidic reactor devoid of biofilm and dried biofilm samples deposited on clean silicon wafers. Principle Component Analysis (PCA) was used to investigate these observations. Our results show that imaging biofilms in the hydrated environment using ToF-SIMS is possible using the unique microfluidic reactor. Moreover, characteristic biofilm fatty acids fragments were observed in the hydrated biofilm grown in the microfluidic channel, illustrating the advantage of imaging biofilm in its native environment.

Citation: 
Hua X, XY Yu, Z Wang, L Yang, B Liu, Z Zhu, AE Tucker, WB Chrisler, EA Hill, S Thevuthasan, Y Lin, S Liu, and MJ Marshall.2014."In situ molecular imaging of hydrated biofilm in a microfluidic reactor by ToF-SIMS."Analyst 139:1609-1613. doi:10.1039/C3AN02262E
Authors: 
X Hua
XY Yu
Z Wang
L Yang
B Liu
Z Zhu
AE Tucker
WB Chrisler
EA Hill
S Thevuthasan
Y Lin
S Liu
MJ Marshall
Instruments: 
Publication year: 
2014

Absorption Mode FT-ICR Mass Spectrometry Imaging.

Abstract: 

Fourier transform ion cyclotron resonance mass spectrometry offers the highest mass resolving power for molecular imaging experiments. This high mass resolving power ensures that closely spaced peaks at the same nominal mass are resolved for proper image generation. Typically higher magnetic fields are used to increase mass resolving power. However, a gain in mass resolving power can also be realized by phase correction of the data for absorption mode display. In addition to mass resolving power, absorption mode offers higher mass accuracy and signal-to-noise ratio over the conventional magnitude mode. Here we present the first use of absorption mode for Fourier transform ion cyclotron resonance mass spectrometry imaging. The Autophaser algorithm is used to phase correct each spectrum (pixel) in the image and then these parameters are used by the Chameleon work-flow based data processing software to generate absorption mode ?Datacubes? for image and spectral viewing. Absorption mode reveals new mass and spatial features that are not resolved in magnitude mode and results in improved selected ion image contrast.

Citation: 
Smith DF, DP Kilgour, M Konijnenburg, PB O'Connor, and RM Heeren.2013."Absorption Mode FT-ICR Mass Spectrometry Imaging."Analytical Chemistry 85(23):11180-11184. doi:10.1021/ac403039t
Authors: 
DF Smith
DP Kilgour
M Konijnenburg
PB O'Connor
RM Heeren
Instruments: 
Volume: 
85
Issue: 
23
Pages: 
11180-11184
Publication year: 
2013

Current Understanding and Remaining Challenges in Modeling Long-Term Degradation of Borosilicate Nuclear Waste Glasses.

Abstract: 

Chemical durability is not a single material property that can be uniquely measured. Instead it is the response to a host of coupled material and environmental processes whose rates are estimated by a combination of theory, experiment, and modeling. High-level nuclear waste (HLW) glass is perhaps the most studied of any material yet there remain significant technical gaps regarding their chemical durability. The phenomena affecting the long-term performance of HLW glasses in their disposal environment include surface reactions, transport properties to and from the reacting glass surface, and ion exchange between the solid glass and the surrounding solution and alteration products. The rates of these processes are strongly influenced and are coupled through the solution chemistry, which is in turn influenced by the reacting glass and also by reaction with the near-field materials and precipitation of alteration products. Therefore, those processes must be understood sufficiently well to estimate or bound the performance of HLW glass in its disposal environment over geologic time-scales. This article summarizes the current state of understanding of surface reactions, transport properties, and ion exchange along with the near-field materials and alteration products influences on solution chemistry and glass reaction rates. Also summarized are the remaining technical gaps along with recommended approaches to fill those technical gaps.

Citation: 
Vienna JD, JV Ryan, S Gin, and Y Inagaki.2013."Current Understanding and Remaining Challenges in Modeling Long-Term Degradation of Borosilicate Nuclear Waste Glasses."International Journal of Applied Glass Science 4(4):283-294. doi:10.1111/ijag.12050
Authors: 
JD Vienna
JV Ryan
S Gin
Y Inagaki
Instruments: 
Volume: 
4
Issue: 
4
Pages: 
283-294
Publication year: 
2013

In situ chemical probing of the electrode-electrolyte interface by ToF-SIMS.

Abstract: 

A portable vacuum interface allowing direct probing of the electrode-electrolyte interface was developed. A classical electrochemical system consisting of gold working electrode, platinum counter electrode, platinum reference electrode, and potassium iodide electrolyte was used to demonstrate real-time observation of the gold iodide adlayer on the electrode and chemical species as a result of redox reactions using cyclic voltammetry (CV) and the time-of-flight secondary ion mass spectrometry (ToF-SIMS, a vacuum-based surface analytical technique) simultaneously. This microfluidic electrochemical probe provides a new way to investigate the surface region with adsorbed molecules and region of diffused layer with chemical speciation in liquids in situ by surface sensitive techniques.

Citation: 
Liu B, XY Yu, Z Zhu, X Hua, L Yang, and Z Wang.2014."In situ chemical probing of the electrode-electrolyte interface by ToF-SIMS."Lab on a Chip 14(5):855-859. doi:10.1039/C3LC50971K
Authors: 
B Liu
XY Yu
Z Zhu
X Hua
L Yang
Z Wang
Instruments: 
Volume: 
14
Issue: 
5
Pages: 
855-859
Publication year: 
2014

An international initiative on long-term behavior of high-level nuclear waste glass.

Abstract: 

Nations producing borosilicate glass as an immobilization material for radioactive wastes resulting from spent nuclear fuel reprocessing have reinforced scientific collaboration to obtain consensus on mechanisms controlling the long-term dissolution rate of glass. This goal is deemed to be crucial for the development of reliable performance assessment models for geological disposal. The collaborating laboratories all conduct fundamental and/or applied research with modern materials science techniques. The paper briefly reviews the radioactive waste vitrification programmes of the six participant nations and summarizes the state-of-the-art of glass corrosion science, emphasizing common scientific needs and justifications for on-going initiatives.

Citation: 
Gin S, A Abdelouas, LJ Criscenti, WL Ebert, K Ferrand, T Geisler, MT Harrison, Y Inagaki, S Mitsui, KT Mueller, JC Marra, CG Pantano, EM Pierce, JV Ryan, JM Schofield, CI Steefel, and JD Vienna.2013."An international initiative on long-term behavior of high-level nuclear waste glass."Materials Today 16(6):243-248. doi:10.1016/j.mattod.2013.06.008
Authors: 
S Gin
A Abdelouas
LJ Criscenti
WL Ebert
K Ferr
T Geisler
MT Harrison
Y Inagaki
S Mitsui
KT Mueller
JC Marra
CG Pantano
EM Pierce
JV Ryan
JM Schofield
CI Steefel
JD Vienna
Instruments: 
Volume: 
16
Issue: 
6
Pages: 
243-248
Publication year: 
2013

Monte Carlo Simulations of the Corrosion of Aluminoborosilicate Glasses.

Abstract: 

Aluminum is one of the most common components included in nuclear waste glasses. Therefore, Monte Carlo (MC) simulations were carried out to investigate the influence of aluminum on the rate and mechanism of dissolution of sodium borosilicate glasses in static conditions. The glasses studied were in the compositional range (70-2x)% SiO2 x% Al2O3 15% B2O3 (15+x)% Na2O, where 0 ≤ x ≤ 15%. The simulation results show that increasing amounts of aluminum in the pristine glasses slow down the initial rate of dissolution as determined from the rate of boron release. However, the extent of corrosion - as measured by the total amount of boron release - initially increases with addition of Al2O3, up to 5 Al2O3 mol%, but subsequently decreases with further Al2O3 addition. The MC simulations reveal that this behavior is due to the interplay between two opposing mechanisms: (1) aluminum slows down the kinetics of hydrolysis/condensation reactions that drive the reorganization of the glass surface and eventual formation of a blocking layer; and (2) aluminum strengthens the glass thereby increasing the lifetime of the upper part of its surface and allowing for more rapid formation of a blocking layer. Additional MC simulations were performed whereby a process representing the formation of a secondary aluminosilicate phase was included. Secondary phase formation draws dissolved glass components out of the aqueous solution, thereby diminishing the rate of condensation and delaying the formation of a blocking layer. As a result, the extent of corrosion is found to increase continuously with increasing Al2O3 content, as observed experimentally. For Al2O3 < 10 mol%, the MC simulations also indicate that, because the secondary phase solubility eventually controls the aluminum content in the part of the altered layer in contact with the bulk aqueous solution, the dissolved aluminum and silicon concentrations at steady state are not dependent on the Al2O3 content of the pristine aluminoborosilicate glass.

Citation: 
Kerisit SN, JV Ryan, and EM Pierce.2013."Monte Carlo Simulations of the Corrosion of Aluminoborosilicate Glasses."Journal of Non-crystalline Solids 378:273-281. doi:10.1016/j.jnoncrysol.2013.07.014
Authors: 
SN Kerisit
JV Ryan
EM Pierce
Instruments: 
Publication year: 
2013

Cold Crucible Induction Melter Studies for Making Glass Ceramic Waste Forms: A Feasibility Assessment.

Abstract: 

Glass ceramics are being developed to immobilize fission products, separated from used nuclear fuel by aqueous reprocessing, into a stable waste form suitable for disposal in a geological repository. This work documents the glass ceramic formulation at bench scale and for a scaled melter test performed in a pilot-scale (~1/4 scale) cold crucible induction meter (CCIM). Melt viscosity, electrical conductivity, and crystallization behavior upon cooling were measured on a small set of compositions to select a formulation for melter testing. Property measurements also identified a temperature range for melter operation and cooling profiles necessary to crystallize the targeted phases in the waste form. Bench scale and melter run results successfully demonstrate the processability of the glass ceramic using the CCIM melter technology.

Citation: 
Crum JV, V Maio, JS McCloy, C Scott, BJ Riley, B Benefiel, JD Vienna, K Archibald, CP Rodriguez, V Rutledge, Z Zhu, JV Ryan, and MJ Olszta.2014."Cold Crucible Induction Melter Studies for Making Glass Ceramic Waste Forms: A Feasibility Assessment."Journal of Nuclear Materials 444(1-3):481-492. doi:10.1016/j.jnucmat.2013.10.029
Authors: 
JV Crum
V Maio
JS McCloy
C Scott
BJ Riley
B Benefiel
JD Vienna
K Archibald
CP Rodriguez
V Rutledge
Z Zhu
JV Ryan
MJ Olszta
Instruments: 
Volume: 
444
Pages: 
481-492
Publication year: 
2014

High Mass Accuracy and High Mass Resolving Power FT-ICR Secondary Ion Mass Spectrometry for Biological Tissue Imaging.

Abstract: 

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for exact mass elemental formula assignment. We have recently reported a secondary ion mass spectrometer with the combination of a C60 primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm50% > 3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Citation: 
Smith DF, A Kiss, FE Leach, III, EW Robinson, L Pasa-Tolic, and RM Heeren.2013."High Mass Accuracy and High Mass Resolving Power FT-ICR Secondary Ion Mass Spectrometry for Biological Tissue Imaging."Analytical and Bioanalytical Chemistry 405(18):6069-6076. doi:10.1007/s00216-013-7048-1
Authors: 
DF Smith
A Kiss
FE Leach
III
EW Robinson
L Pasa-Tolic
RM Heeren
Instruments: 
Volume: 
405
Issue: 
18
Pages: 
6069-6076
Publication year: 
2013

Advanced Mass Calibration and Visualization for FT-ICR Mass Spectrometry Imaging.

Abstract: 

Mass spectrometry imaging by Fourier transform ion cyclotron resonance (FT-ICR) yields hundreds of unique peaks, many of which cannot be resolved by lower performance mass spectrometers. The high mass accuracy and high mass resolving power allow confident identification of small molecules and lipids directly from biological tissue sections. Here, calibration strategies for FT-ICR MS imaging were investigated. Sub-parts-per-million mass accuracy is demonstrated over an entire tissue section. Ion abundance fluctuations are corrected by addition of total and relative ion abundances for a root-mean-square error of 0.158 ppm on 16,764 peaks. A new approach for visualization of FT-ICR MS imaging data at high resolution is presented. The "Mosaic Datacube" provides a flexible means to visualize the entire mass range at a mass spectral bin width of 0.001 Da. The high resolution Mosaic Datacube resolves spectral features not visible at lower bin widths, while retaining the high mass accuracy from the calibration methods discussed.

Citation: 
Smith DF, A Kharchenko, M Konijnenburg, I Klinkert, L Pasa-Tolic, and RM Heeren.2012."Advanced Mass Calibration and Visualization for FT-ICR Mass Spectrometry Imaging."Journal of the American Society for Mass Spectrometry 23(11):1865-1872. doi:10.1007/s13361-012-0464-1
Authors: 
DF Smith
A Kharchenko
M Konijnenburg
I Klinkert
L Pasa-Tolic
RM Heeren
Instruments: 
Volume: 
23
Issue: 
11
Pages: 
1865-1872
Publication year: 
2012

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