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

The Influx, a one-of-a kind flow cytometer/cell sorter, provides 5 laser lines simultaneously, powerful detection capabilities and diverse sorting...
Custodian(s): Galya Orr
The JEOL JEM-3000SFF was designed for high-resolution cryogenic transmission electron microscopy (cryo-EM) of biological samples and expands EMSL/...
This time-lapse fluorescence microscope with single molecule detection sensitivity is used to follow individual molecules or organelles in their...
Custodian(s): Galya Orr
This Laser Capture Microdissection system is equipped with 100 x objective lens for enriching distinct organelles, or isolating single cells from...
Custodian(s): Dehong Hu
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

Publications

The altered layer (i.e., amorphous hydrated surface layer and crystalline reaction products)represents a complex region, both physically and...
We record sequences of Raman spectra at a plasmonic junction formed by a gold AFM tip in contact with a silver surface coated with 4,4’-...
Photosynthetic microbes are of emerging interest as production organisms in biotechnology because they can grow autotrophically using sunlight, an...
Extended cycling of the Li-O2 battery under full discharge/charge conditions is achievable upon selection of appropriate electrode materials and...
To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist...

Science Highlights

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...
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....

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.

Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2.

Abstract: 

Photosynthetic microbes are of emerging interest as production organisms in biotechnology because they can grow autotrophically using sunlight, an abundant energy source, and CO2, a greenhouse gas. Important traits for such microbes are fast growth and amenability to genetic manipulation. Here we describe Synechococcus elongatus UTEX 2973, a unicellular cyanobacterium capable of rapid autotrophic growth, comparable to heterotrophic industrial hosts such as yeast. Synechococcus 2973 can be readily transformed for facile generation of desired knockout and knock-in mutations. Genome sequencing coupled with global proteomics studies revealed that Synechococcus 2973 is a close relative of the widely studied cyanobacterium Synechococcus elongatus PCC 7942, an organism that grows more than two times slower. A small number of nucleotide changes are the only significant differences between the genomes of these two cyanobacterial strains. Thus, our study has unraveled genetic determinants necessary for rapid growth of cyanobacterial strains of significant industrial potential.

Citation: 
Yu J, ML Liberton, P Cliften, R Head, JM Jacobs, RD Smith, DW Koppenaal, JJ Brand, and HB Pakrasi.2015."Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2."Scientific Reports 5:8132. doi:10.1038/srep08132
Authors: 
Yu J
ML Liberton
P Cliften
R Head
JM Jacobs
RD Smith
DW Koppenaal
JJ Br
HB Pakrasi
Facility: 
Volume: 
0
Issue: 
0
Pages: 
0
Publication year: 
2015

Vibronic Raman Scattering at the Quantum Limit of Plasmons.

Abstract: 

We record sequences of Raman spectra at a plasmonic junction formed by a gold AFM tip in contact with a silver surface coated with 4,4’-dimercaptostilbene (DMS). A 2D correlation analysis of the recorded trajectories reveals that the observable vibrational states can be divided into sub-sets. The first set comprises the totally symmetric vibrations of DMS (ag) that are neither correlated with each other nor to the fluctuating background, which is assigned to the signature of charge transfer plasmons tunneling through DMS. The second set consists of bu vibrations, which are correlated both with each other and with the continuum. Our findings are rationalized on the basis of the charge-transfer theory of Raman scattering, and illustrate how the tunneling plasmons modulate the vibronic coupling term from which the intensities of the bu states are derived.

Citation: 
El-Khoury PZ, and WP Hess.2014."Vibronic Raman Scattering at the Quantum Limit of Plasmons."Nano Letters 14(7):4114-4118. doi:10.1021/nl501690u
Authors: 
PZ El-Khoury
WP Hess
Facility: 
Volume: 
14
Issue: 
7
Pages: 
4114-4118
Publication year: 
2014

Modeling Interfacial Glass-Water Reactions: Recent Advances and Current Limitations.

Abstract: 

The altered layer (i.e., amorphous hydrated surface layer and crystalline reaction products)represents a complex region, both physically and chemically, sandwiched between two distinct boundaries - pristine glass surface at the inner most interface and aqueous solution at the outer most. The physico-chemical processes that control the development of this region have a significant impact on the long-term glass-water reaction. Computational models, spanning different length and time-scales, are currently being developed to improve our understanding of this complex and dynamic process with the goal of accurately describing the pore-scale changes that occur as the system evolves. These modeling approaches include Geochemical Reaction Path simulations, Glass Reactivity in Allowance for Alteration Layer simulations, Monte Carlo simulations, and Molecular Dynamics methods. Discussed in this manuscript are the advances and limitations of each modeling approach placed in the context of the glass water reaction and how collectively these approaches provide insights into the mechanisms that control the formation and evolution of altered layers; thus providing the fundamental data needed to develop pore-scale equations that enable more accurate predictions of nuclear waste glass corrosion in a geologic repository.

Citation: 
Pierce EM, P Frugier, LJ Criscenti, KD Kwon, and SN Kerisit.2014."Modeling Interfacial Glass-Water Reactions: Recent Advances and Current Limitations."International Journal of Applied Glass Science 5(4):421-435. doi:10.1111/ijag.12077
Authors: 
EM Pierce
P Frugier
LJ Criscenti
KD Kwon
SN Kerisit
Volume: 
5
Issue: 
4
Pages: 
421-435
Publication year: 
2014

Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model.

Abstract: 

To assess molecular responses to low doses of radiation that may be encountered during medical diagnostic procedures, nuclear accidents, or terrorist acts, a quantitative global proteomic approach was used to identify protein alterations in a reconstituted human skin tissue treated with 10 cGy of ionizing radiation. Subcellular fractionation was employed to remove highly abundant structural proteins and provide insight on radiation induced alterations in protein abundance and localization. In addition, peptides were post-fractionated using high resolution 2-dimensional liquid chromatography to increase the dynamic range of detection of protein abundance and translocation changes. Quantitative data was obtained by labeling peptides with 8-plex isobaric iTRAQ tags. A total of 207 proteins were detected with statistically significant alterations in abundance and/or subcellular localization compared to sham irradiated tissues. Bioinformatics analysis of the data indicated that the top canonical pathways affected by low dose radiation are related to cellular metabolism. Among the proteins showing alterations in abundance, localization and proteolytic processing was the skin barrier protein filaggrin which is consistent with our previous observation that ionizing radiation alters profilaggrin processing with potential effects on skin barrier functions. In addition, a large number of proteases and protease regulators were affected by low dose radiation exposure indicating that altered proteolytic activity may be a hallmark of low dose radiation exposure. While several studies have demonstrated altered transcriptional regulation occurs following low dose radiation exposures, the data presented here indicates post-transcriptional regulation of protein abundance, localization, and proteolytic processing play an important role in regulating radiation responses in complex human tissues.

Citation: 
Hengel S, JT Aldrich, KM Waters, L Pasa-Tolic, and DL Stenoien.2014."Quantitative Proteomic Profiling of Low Dose Ionizing Radiation Effects in a Human Skin Model."Proteomes 2(3):382-398. doi:10.3390/proteomes2030382
Authors: 
S Hengel
JT Aldrich
KM Waters
L Pasa-Tolic
DL Stenoien
Volume: 
2
Issue: 
3
Pages: 
382-398
Publication year: 
2014

Formation of Interfacial Layer and Long-Term Cylability of Li-O-2 Batteries.

Abstract: 

Extended cycling of the Li-O2 battery under full discharge/charge conditions is achievable upon selection of appropriate electrode materials and cycling protocol. However, the decomposition of the side products also contribute to the observed good cycling behavior of high capacity Li-O2 batteries. Quantitative analyses of the discharge and charge products reveals a quick switch from the predominant formation of Li2O2 to the predominant formation of side products during the first a few cycles of the Li-O2 batteries. After the switch, cycling stabilizes with a repeatable formation of Li2O2/side products at ~1:2 ratio. CNTs/Ru composite electrodes exhibits lower charge voltage and deliver 50 full discharge-charge cycles without sharp capacity drop. Ru coated glass carbon electrode can lead to more than 500 cycles without change in its cycling profiles. The better understanding on Li-O2 reaction processes developed in this work may lead to the further improvement on the long term cycling behavior of high capacity Li-O2 batteries.

Citation: 
Nasybulin EN, W Xu, BL Mehdi, EC Thomsen, MH Engelhard, RC Masse, P Bhattacharya, M Gu, WD Bennett, Z Nie, CM Wang, ND Browning, and J Zhang.2014."Formation of Interfacial Layer and Long-Term Cylability of Li-O-2 Batteries."ACS Applied Materials & Interfaces 6(16):14141-14151. doi:10.1021/am503390q
Authors: 
EN Nasybulin
W Xu
BL Mehdi
EC Thomsen
MH Engelhard
RC Masse
P Bhattacharya
M Gu
WD Bennett
Z Nie
CM Wang
ND Browning
J Zhang
Facility: 
Instruments: 
Volume: 
6
Issue: 
16
Pages: 
14141-14151
Publication year: 
2014

Reflection High-Energy Electron Diffraction Beam-Induced Structural and Property Changes on WO3 Thin Films.

Abstract: 

Reduction of transition metal oxides can greatly change their physical and chemical properties. Using deposition of WO3 as a case study, we demonstrate that reflection high-energy electron diffraction (RHEED), a surface-sensitive tool widely used to monitor thin-film deposition processes, can significantly affect the cation valence and physical properties of the films through electron-beam induced sample reduction. The RHEED beam is found to increase film smoothness during epitaxial growth of WO3, as well as change the electronic properties of the film through preferential removal of surface oxygen.

Citation: 
Du Y, H Zhang, T Varga, and SA Chambers.2014."Reflection High-Energy Electron Diffraction Beam-Induced Structural and Property Changes on WO3 Thin Films."Applied Physics Letters 105(5):051606. doi:10.1063/1.4892810
Authors: 
Du Y
H Zhang
T Varga
SA Chambers
Volume: 
Issue: 
Pages: 
Publication year: 
2014

Strong Room-temperature Negative Transconductance In An Axial Si/Ge Hetero-nanowire Tunneling Field-effect Transistor.

Abstract: 

We report on room-temperature negative transconductance (NTC) in axial Si/Ge hetero-nanowire tunneling field-effect transistors (TFETs). The NTC produces a current peak-to-valley ratio > 45, a high value for a Si-based device. We characterize the NTC characteristics over a range of gate VG and drain VD voltages, finding that NTC persists down to VD = –50 mV. The physical mechanism responsible for the NTC is the VG-induced depletion in the p-Ge section that eventually reduces the maximum electric field that triggers the tunneling ID, as confirmed via three-dimensional TCAD simulations.

Citation: 
Zhang P, ST Le, X Hou, A Zaslavsky, DE Perea, SA Dayeh, and ST Picraux.2014."Strong Room-temperature Negative Transconductance In An Axial Si/Ge Hetero-nanowire Tunneling Field-effect Transistor."Applied Physics Letters 105(6):Article No. 062106. doi:10.1063/1.4892950
Authors: 
P Zhang
ST Le
X Hou
A Zaslavsky
DE Perea
SA Dayeh
ST Picraux
Instruments: 
Volume: 
Issue: 
Pages: 
Publication year: 
2014

Bending-induced Symmetry Breaking of Lithiation in Germanium Nanowires .

Abstract: 

From signal transduction of living cells to oxidation and corrosion of metals, mechanical stress intimately couples with chemical reactions, regulating these biological and physiochemical processes. The coupled effect is particularly evident in electrochemical lithiation/delithiation cycling of high-capacity electrodes, such as silicon (Si), where on one hand lithiation-generated stress mediates lithiation kinetics, and on the other electrochemical reaction rate regulates stress generation and mechanical failure of the electrodes. Here we report for the first time the evidence on the controlled lithiation in germanium nanowires (GeNWs) through external bending. Contrary to the symmetric core-shell lithiation in free-standing GeNWs, we show bending GeNWs breaks the lithiation symmetry, speeding up lithaition at the tensile side while slowing down at the compressive side of the GeNWs. The bending-induced symmetry breaking of lithiation in GeNWs is further corroborated by chemomechanical modeling. In the light of the coupled effect between lithiation kinetics and mechanical stress in the electrochemical cycling, our findings shed light on strain/stress engineering of durable high-rate electrodes and energy harvesting through mechanical motion.

Citation: 
Gu M, H Yang, DE Perea, J Zhang, S Zhang, and CM Wang.2014."Bending-induced Symmetry Breaking of Lithiation in Germanium Nanowires ."Nano Letters 14(8):4622-4627. doi:10.1021/nl501680w
Authors: 
Gu M
H Yang
DE Perea
J Zhang
S Zhang
CM Wang
Facility: 
Instruments: 
Volume: 
14
Issue: 
8
Pages: 
4622-4627
Publication year: 
2014

Yeast cell surface display for lipase whole cell catalyst and its applications.

Abstract: 

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

Citation: 
Liu Y, R Zhang, Z Lian, S Wang, and AT Wright.2014."Yeast cell surface display for lipase whole cell catalyst and its applications."Journal of Molecular Catalysis B: Enzymatic 106:17-25. doi:10.1016/j.molcatb.2014.04.011
Authors: 
Y Liu
R Zhang
Z Lian
S Wang
AT Wright
Volume: 
Issue: 
Pages: 
Publication year: 
2014

In-situ Study of Nanostructure and Electrical Resistance of Nanocluster Films Irradiated with Ion Beams.

Abstract: 

An in-situ study is reported on the structural evolution in nanocluster films under He+ ion irradiation using an advanced helium ion microscope. The films consist of loosely interconnected nanoclusters of magnetite or iron-magnetite (Fe-Fe3O4) core-shells. The nanostructure is observed to undergo dramatic changes under ion-beam irradiation, featuring grain growth, phase transition, particle aggregation, and formation of nanowire-like network and nano-pores. Studies based on ion irradiation, thermal annealing and election irradiation have indicated that the major structural evolution is activated by elastic nuclear collisions, while both electronic and thermal processes can play a significant role once the evolution starts. The electrical resistance of the Fe-Fe3O4 films measured in situ exhibits a super-exponential decay with dose. The behavior suggests that the nanocluster films possess an intrinsic merit for development of an advanced online monitor for neutron radiation with both high detection sensitivity and long-term applicability, which can enhance safety measures in many nuclear operations.

Citation: 
Jiang W, JA Sundararajan, T Varga, ME Bowden, Y Qiang, JS McCloy, CH Henager, Jr, and RO Montgomery.2014."In-situ Study of Nanostructure and Electrical Resistance of Nanocluster Films Irradiated with Ion Beams."Advanced Functional Materials 24(39):6210-6218. doi:10.1002/adfm.201400553
Authors: 
W Jiang
JA Sundararajan
T Varga
ME Bowden
Y Qiang
JS McCloy
CH Henager
Jr
RO Montgomery
Instruments: 
Volume: 
24
Issue: 
39
Pages: 
6210-6218
Publication year: 
2014

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