Cell Isolation and Systems Analysis

spatiotemporal bacterial colonization pattern in root
The spatiotemporal bacterial colonization pattern in root is studied using fluorescence in situ hybridization (FISH), targeting specific bacterial species by their ribosomal RNA (16S). The lower (left) and higher (right) magnification images show the root epidermal cells in blue, bacterial cells positive for the FISH probe in green, and the DNA in the plant and bacterial cells in red.

The Cell Isolation and Systems Analysis (CISA) capability provides technologies and expertise to study individual cells, microbial communities, fungi and plants at the cellular and molecular level.  CISA supports studies that help to translate genome to functions to better understand, predict and redesign biological systems critical for sustainable bioenergy and environmental processes. 

CISA’s expertise in quantitative high resolution fluorescence microscopy, isolation of distinct organelles, cells or subpopulations for further omics, and next generation sequencing for single cell and meta-transcriptome analyses, can shed new light on the cellular organization and molecular processes within and between microbes, communities, fungi and plants. See a complete list of CISA instruments.

Together with other EMSL omics and imaging modalities, CISA’s expertise 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 plant-microbe interactions in environmental processes such as carbon cycling, or identify molecular processes underlying plant response to environmental stressors, among other studies addressing national needs.

Resources and Techniques

High-resolution quantitative fluorescence microscopy techniques to study the spatial and temporal organization of cellular and molecular processes in intact or live cells, communities and plant tissue. These include laser scanning multi-spectral confocal, single molecule, atomic force and super resolution fluorescence microscopy, such as STORM/PALM, structured illumination (SIM) & confocal Airyscan.  CISA specializes in quantitative approaches, such as gene expression analysis using super resolution or single molecule fluorescence in situ hybridization (FISH), detecting molecular interaction dynamics using fluorescence resonance energy transfer (FRET), or sensing the intracellular environment using fluorescence lifetime imaging (FLIM), all in intact cells, communities or plant tissue.

Cell and organelle isolation techniques for isolating distinct cells or subpopulations from complex microbial communities or tissues, as well as distinct organelles, for further omic analysis, such as population or single cell RNA-Seq. These include the Influx flow cytometer cell sorter, which sorts dissociated cells or organelles from cell communities or tissues in high throughput, and a high-resolution laser capture microdissection microscope, which captures individual cells or cell clusters based on their spatial context in the community or tissue.

Next Generation Sequencing for transcriptomic analysis (RNA-Seq) to study regulation of gene expression, metabolic pathways and molecular processes underlying the functions of microbial cells, communities, fungi and plants and the interactions between them. These include the SOLiD 5500 systems for high capacity, massively parallel sequencing, and the Ion Proton systems for long reads sequencing. Together, these systems generate highly accurate sequencing with single base resolution, as well as support de novo assembly of transcriptomics data. EMSL specializes in meta-transcriptome analysis of complex environmental communities, as well as singe cell RNA-Seq for transcriptome analysis of individual eukaryotic cells.

Data analysis tools and expertise for interpreting, assimilating and visualizing experimental omics and microscopy data. For omics data, these include bioinformatics, modeling and statistical tools, such as samtools, Bioconductor RNA-Seq analysis packages, DESeq and edgeR, as well as htseq-qa for quality control. In addition, various R scripts developed at PNNL are used for functional enrichment statistics and visualization of pathway activity across samples. For fluorescence microscopy data, these include image analysis software such as Volocity, ImageJ or Zen, as well as Matlab routines developed for specific tasks, such as quantifying gene expression in intact cells using super resolution and single molecule FISH, detecting molecular interaction dynamics using FRET, or sensing the intracellular environment using FLIM.

Microbial and plant growth resources to support the controlled growth and monitoring of microbial cultures, plants or cell lines under defined conditions for further analyses. For microbial cultures, these include bioreactors and monitoring systems in volumes ranging from 100 µl to 10 liters. For plants, in addition to EMSL enclosed greenhouse, CISA provides a growth chamber with controlled temperature and light cycling, as well as a cell culture facility with biological hoods and incubators.

Description

Capability Details

  • Super resolution fluorescence structured illumination microscopy (SIM) & confocal Airyscan enable 3D imaging of live or intact hydrated cells and plant tissues with 120 -150 nm lateral resolution. These imaging systems resolve protein complexes and subcellular structures using any fluorescent protein or dye. The fast and efficient image acquisition of the Airyscan supports imaging dim signals and capturing dynamic process in live cells. These systems are used, for example, to study the spatial and temporal gene or protein expression patterns of specific enzymes responsible for wood degradation by fungal hyphae.
  • Stochastic optical reconstruction microscopy (STORM), also known as photo-activated localization microscopy (PALM), enables imaging protein complexes and subcellular structures in intact hydrated cells with 20-30 nm resolution. STORM is used, for example, to quantify gene expression in intact cells and plant tissue by fluorescence in situ hybridization (FISH) with high accuracy.
  • Combined atomic force microscopy (AFM) and STORM/PALM for 3D topographic mapping of the cell surface with 1-10 nm resolution coupled with the identification of distinct proteins and molecular complexes in the membrane of intact cells by fluorescence imaging using single molecule or super resolution microscopy. This system is used, for example, to detect the spatial organization of specific membrane receptors or transporters in mutated versus wildtype microorganisms specialized in high carbon assimilation or lipid production.
  • Confocal fluorescence microscopy system that seamlessly integrates laser scanning confocal microscopy, multi-spectral signal acquisition, fluorescence lifetime imaging (FLIM) and differential interference contrast (DIC) imaging. The system enables high-resolution 3D imaging and quantitative analysis of molecular interaction dynamics by FRET in live cells, cell communities or tissues. This system is used, for example, to identify distinct cell populations in complex microbial communities by multi-spectral imaging of endogenous pigments.
  • Live cell single molecule fluorescence imaging  uses total internal reflection fluorescence (TIRF) techniques and time-lapse acquisition system to track individual molecules or organelles in live cells. The system enables the study of subcellular processes over time, as well as molecular interaction dynamics using fluorescence resonance energy transfer (FRET) in live cells. This system is used, for example, to study the spatial and temporal expression patterns of specific enzymes in relations to lipid production and accumulation dynamics in distinct organelles.
  • SOLiD systems together with Ion Proton systems provide unbiased global transcriptome analyses (RNA-Seq) with high accuracy and throughput. The systems enable global gene expression analyses, novel gene or isoform identification, and regulation of gene expression studies, such as ChIP-Seq or non-coding RNA analyses in complex microbial communities (meta-transcriptomics), organisms, or single eukaryotic cells. These systems are used, for example, to understand the functional roles of distinct species comprising a complex microbial community in the context of carbon cycling.
  • Influx flow cytometer cell sorter uses multiple laser lines and a powerful detection capability for high throughput analysis and sorting of distinct cells or organelles. The system incorporates an advanced multi-parameter sorting technology based on the presence and content of distinct genes and proteins or intracellular structures. The Influx supports detection and sorting of nanoscale particles, making it highly suitable for sorting and analyzing organelles and single cells. The system is used, for example, to sort single cells from an environmental microbial community by their consumption of fluorescent cellulose nanocrystals for further single cell genomics.
  • High resolution laser capture microdissection microscope equipped with a 100x magnification objective lens and multiple fluorescence lines. The system enables the enrichment of distinct organelles or isolation of single cells and cell clusters from complex microbial communities or tissue sections based on their spatial context for further analyses. This system is used, for example, to capture individual pairs of stomatal guard cells for single cell RNA-Seq, or isolate s mall, spatially defined heterotroph clusters from the autotrophs for meta-transcriptomics.
  • CyTOF mass cytometer uses ICP and Time-of-Fight mass spectrometry in single cells to quantify the expression of multiple proteins or RNA species in high throughput. The CyTOF enables single cell analysis of the expression of multiple proteins using antibodies, or multiple genes using in situ hybridization probes, tagged with different stable metal isotopes.
  • Bioreactors for controlled growth and monitoring  of diverse microbial cells in volumes ranging from 100 µl wells to 10 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. A plant growth chamber is also available with controlled temperature and light cycling.

    Instruments

    The system combines the power of atomic force microscopy (AFM) and super resolution fluorescence microscopy to support studies of cell surface...
    Custodian(s): Galya Orr
    This microscopy system integrates seamlessly nonlinear two-photon excitation, laser-scanning confocal microscopy, and fluorescence lifetime imaging...
    Custodian(s): Galya Orr
    The CyTOF is a mass cytometer that uses time-of-flight mass spectrometry to quantify the presence of metal-tagged antibodies in single cells in high...
    Custodian(s): Galya Orr
    The Influx, a flow cytometer/cell sorter, provides 5 laser lines simultaneously, powerful detection capability and diverse sorting approaches for...
    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

    Publications

    Post-translational modifications (PTMs) play an important role in various biological processes through changing protein structure and function. Some...
    Experimental verification of the microscopic origin of resistance switching in metal/oxide/metal heterostructures is needed for applications in non-...
    Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism....
    Ultrathin films of barium oxide were grown on Ag(001) and Ag(111) using the evaporation of Ba metal in an O2 atmosphere by molecular beam epitaxy....
    Thermoproteales populations (phylum Crenarchaeota) are abundant in high-25 temperature (>70° C) environments of Yellowstone National...

    Science Highlights

    Posted: March 21, 2017
    Microbial communities have significant impact on Earth's biogeochemical and ecological processes. Scientists need to understand how microbes...
    Posted: March 20, 2017
    The Science Molds produce a wide range of both valuable and toxic molecules, which have important implications for energy production, agriculture...
    Posted: January 24, 2017
    The Science Chemical bonds in hydrogen gas can be harnessed to power fuel cells or internal combustion engines. Researchers have now reported the...
    Posted: August 01, 2016
    Cyanobacteria use the sun's energy to create food for themselves while consuming carbon dioxide and giving off oxygen, thus playing a role in Earth'...
    Posted: July 01, 2016
    Scientists at Pacific Northwest National Laboratory and Argonne National Laboratory used EMSL capabilities to better understand how microbes connect...

    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...
    Quantum information science is an up and coming area of research aimed to transform computing capabilities in the future. The use of quantum bits, or...
    Background: In the proposed work we will use a newly developed experimental system comprised of the alga Chrysochromulina tobin (Haptophyceae) and a...
    Peatbogs are unbalanced ecosystems that gain more carbon than is released making them a critical global sink for carbon. Biological nitrogen fixation...
    Remediation of metal and radionuclide contaminants in soil and groundwater systems is challenging because of their strong chemical interactions with...

    spatiotemporal bacterial colonization pattern in root
    The spatiotemporal bacterial colonization pattern in root is studied using fluorescence in situ hybridization (FISH), targeting specific bacterial species by their ribosomal RNA (16S). The lower (left) and higher (right) magnification images show the root epidermal cells in blue, bacterial cells positive for the FISH probe in green, and the DNA in the plant and bacterial cells in red.

    The Cell Isolation and Systems Analysis (CISA) capability provides technologies and expertise to study individual cells, microbial communities, fungi and plants at the cellular and molecular level.  CISA supports studies that help to translate genome to functions to better understand, predict and redesign biological systems critical for sustainable bioenergy and environmental processes. 

    CISA’s expertise in quantitative high resolution fluorescence microscopy, isolation of distinct organelles, cells or subpopulations for further omics, and next generation sequencing for single cell and meta-transcriptome analyses, can shed new light on the cellular organization and molecular processes within and between microbes, communities, fungi and plants. See a complete list of CISA instruments.

    Together with other EMSL omics and imaging modalities, CISA’s expertise 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 plant-microbe interactions in environmental processes such as carbon cycling, or identify molecular processes underlying plant response to environmental stressors, among other studies addressing national needs.

    Resources and Techniques

    High-resolution quantitative fluorescence microscopy techniques to study the spatial and temporal organization of cellular and molecular processes in intact or live cells, communities and plant tissue. These include laser scanning multi-spectral confocal, single molecule, atomic force and super resolution fluorescence microscopy, such as STORM/PALM, structured illumination (SIM) & confocal Airyscan.  CISA specializes in quantitative approaches, such as gene expression analysis using super resolution or single molecule fluorescence in situ hybridization (FISH), detecting molecular interaction dynamics using fluorescence resonance energy transfer (FRET), or sensing the intracellular environment using fluorescence lifetime imaging (FLIM), all in intact cells, communities or plant tissue.

    Cell and organelle isolation techniques for isolating distinct cells or subpopulations from complex microbial communities or tissues, as well as distinct organelles, for further omic analysis, such as population or single cell RNA-Seq. These include the Influx flow cytometer cell sorter, which sorts dissociated cells or organelles from cell communities or tissues in high throughput, and a high-resolution laser capture microdissection microscope, which captures individual cells or cell clusters based on their spatial context in the community or tissue.

    Next Generation Sequencing for transcriptomic analysis (RNA-Seq) to study regulation of gene expression, metabolic pathways and molecular processes underlying the functions of microbial cells, communities, fungi and plants and the interactions between them. These include the SOLiD 5500 systems for high capacity, massively parallel sequencing, and the Ion Proton systems for long reads sequencing. Together, these systems generate highly accurate sequencing with single base resolution, as well as support de novo assembly of transcriptomics data. EMSL specializes in meta-transcriptome analysis of complex environmental communities, as well as singe cell RNA-Seq for transcriptome analysis of individual eukaryotic cells.

    Data analysis tools and expertise for interpreting, assimilating and visualizing experimental omics and microscopy data. For omics data, these include bioinformatics, modeling and statistical tools, such as samtools, Bioconductor RNA-Seq analysis packages, DESeq and edgeR, as well as htseq-qa for quality control. In addition, various R scripts developed at PNNL are used for functional enrichment statistics and visualization of pathway activity across samples. For fluorescence microscopy data, these include image analysis software such as Volocity, ImageJ or Zen, as well as Matlab routines developed for specific tasks, such as quantifying gene expression in intact cells using super resolution and single molecule FISH, detecting molecular interaction dynamics using FRET, or sensing the intracellular environment using FLIM.

    Microbial and plant growth resources to support the controlled growth and monitoring of microbial cultures, plants or cell lines under defined conditions for further analyses. For microbial cultures, these include bioreactors and monitoring systems in volumes ranging from 100 µl to 10 liters. For plants, in addition to EMSL enclosed greenhouse, CISA provides a growth chamber with controlled temperature and light cycling, as well as a cell culture facility with biological hoods and incubators.

    Pages

    Leads

    (509) 371-6127

    Dr. Orr is the capability lead for the cell isolation and systems analysis (CISA), where she leads the team in the development and applications of capabilities for biological research at the molecular level. She has been initiating and leading...