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

Grain growth of nanocrystalline materials is generally thermally activated, but can also be driven by irradiation at much lower temperature. In...
14YWT oxide dispersion strengthened (ODS) ferritic steel was irradiated with of 5 MeV Ni2+ ions, at 300 °C, 450 °C, and 600 °...
The microstructure and chemistry of SmCo2Fe2B melt-spun alloy after multistage annealing was investigated using high resolution transmission electron...
Dual beam depth profiling strategy has been widely adopted in ToF-SIMS depth profiling, in which two basic operation modes, interlaced mode and non-...
Melanoma is a malignant tumor of melanocytes. Although extensive investigations have been done to study metabolic changes in primary melanoma in...

Science Highlights

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...
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...

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...
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...
Ocean microbes drive the biogeochemistry that fuels the planet, and their viruses impact microbes through mortality, horizontal gene transfer, and...
We propose to develop approaches that target metabolically active microorganisms and functions that drive carbon cycling in soils from bioenergy...

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.

Pages

Leads

(509) 371-6127

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...