Environmental Molecular Sciences Laboratory

A DOE Office of Science User Facility

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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 instruments available through CISA.

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 and 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 NextSeq550 for high capacity, parallel sequencing, and the Ion Proton and Ion S5 systems for long reads sequencing as well as diversity profiling of complex microbial communities. 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. In EMSL we also conduct ChIP-Seq and AmpliSeq analyses among other sequencing-based analyses.

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.

Plant growth resources to support the controlled growth and monitoring of plants or cell lines under defined conditions for further analyses. 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.

Other research resources found in EMSL and managed by Pacific Northwest National Laboratory include a super resolution fluorescence microscope:

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.

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, 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
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 cell culture facility is adjacent to the microscopy suite, where bio-hoods, CO2 incubators, benchtop centrifuges, 37°C water baths and a shakers...
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

Publications

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification (PTM) on cysteines that plays...
Wood-decomposing fungi efficiently decompose plant lignocellulose, and there is increasing interest in characterizing and perhaps harnessing the...
Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network ofbranching and fusing hyphae that are often considered to be...
Many studies have highlighted the role of dysregulated glucagon secretion in the etiology of hyperglycemia and diabetes. Accordingly, understanding...
Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems but factors governing its production are poorly understood. Recent...

Science Highlights

Posted: May 11, 2020
A team of researchers from Pacific Northwest National Laboratory (PNNL), EMSL, and Lawrence Berkeley National Laboratory (LBNL) used EMSL expertise...
Posted: October 30, 2019
The Science Bacteria in groundwater move in surprising ways. They can passively ride flowing groundwater, or they can actively move on their own in...
Posted: September 23, 2019
The Science Scientists hope to harness fungi that decompose the most abundant type of biomass in wood, lignocellulose. Lignocellulose could be used...
Posted: March 29, 2018
The Science For years, scientists have experimented with phages—the viruses that infect bacteria—to learn how they change their host. Because such...
Posted: December 19, 2017
The Science The production of biofuels from plant biomass is a highly promising source of energy, but researchers are trying to find microbes that...

Instruments

Methylmercury (MeHg), an organic Hg compound, is a potent toxin that bioaccumulates in food sources and is primarily produced by microorganisms in...
In nature, most microbes exist as members of complex multispecies communities that play vital roles promoting plant and soil health. Many of these...
Sugar conversion through central carbon catabolism in filamentous fungi is a complex progress that involves many pathways. During growth on plant...
Sorghum bicolor is an important bioenergy crop because it is nutrient and water use efficient, and produces a large quantity of biomass that can be...
Exosomes are powerful vehicles for local, distal and even transpecies cell-to-cell communication in diverse eukaryotes, but whether plant...

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 instruments available through CISA.

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 and 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 NextSeq550 for high capacity, parallel sequencing, and the Ion Proton and Ion S5 systems for long reads sequencing as well as diversity profiling of complex microbial communities. 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. In EMSL we also conduct ChIP-Seq and AmpliSeq analyses among other sequencing-based analyses.

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.

Plant growth resources to support the controlled growth and monitoring of plants or cell lines under defined conditions for further analyses. 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.

Other research resources found in EMSL and managed by Pacific Northwest National Laboratory include a super resolution fluorescence microscope:

Pages

Leads

Galya Orr
Dr. Orr is the capability lead for the Cell Isolation & Systems Analysis (CISA), where she leads the team in the development and applications of capabilities for biological research at the cellular and molecular level. Orr and her team have been...