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

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...
The microstructure and chemistry of SmCo2Fe2B melt-spun alloy after multistage annealing was investigated using high resolution transmission electron...
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 °...

Science Highlights

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...
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...

Instruments

There are no related projects at this time.

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.

ToF-SIMS Depth Profiling Of Insulating Samples, Interlaced Mode Or Non-interlaced Mode?

Abstract: 

Dual beam depth profiling strategy has been widely adopted in ToF-SIMS depth profiling, in which two basic operation modes, interlaced mode and non-interlaced mode, are commonly used. Generally, interlaced mode is recommended for conductive or semi-conductive samples, whereas non-interlaced mode is recommended for insulating samples, where charge compensation can be an issue. Recent publications, however, show that the interlaced mode can be used effectively for glass depth profiling, despite the fact that glass is an insulator. In this study, we provide a simple guide for choosing between interlaced mode and non-interlaced mode for insulator depth profiling. Two representative cases are presented: (1) depth profiling of a leached glass sample, and (2) depth profiling of a single crystal MgO sample. In brief, the interlaced mode should be attempted first, because (1) it may provide reasonable-quality data, and (2) it is time-saving for most cases, and (3) it introduces low H/C/O background. If data quality is the top priority and measurement time is flexible, non-interlaced mode is recommended because interlaced mode may suffer from low signal intensity and poor mass resolution. A big challenge is tracking trace H/C/O in a highly insulating sample (e.g., MgO), because non-interlaced mode may introduce strong H/C/O background but interlaced mode may suffer from low signal intensity. Meanwhile, a C or Au coating is found to be very effective to improve the signal intensity. Surprisingly, the best analyzing location is not on the C or Au coating, but at the edge (outside) of the coating.

Citation: 
Wang Z, K Jin, Y Zhang, F Wang, and Z Zhu.2014."ToF-SIMS Depth Profiling Of Insulating Samples, Interlaced Mode Or Non-interlaced Mode?"Surface and Interface Analysis 46(S1):257-260. doi:10.1002/sia.5419
Authors: 
Z Wang
K Jin
Y Zhang
F Wang
Z Zhu
Volume: 
46
Issue: 
0
Pages: 
257-260
Publication year: 
2014

Stability Of Nanoclusters In 14YWT Oxide Dispersion Strengthened Steel Under Heavy Ion-irradiation By Atom Probe Tomography.

Abstract: 

14YWT oxide dispersion strengthened (ODS) ferritic steel was irradiated with of 5 MeV Ni2+ ions, at 300 °C, 450 °C, and 600 °C to a damage level of 100 dpa. The stability of Ti–Y–O nanoclusters was investigated by applying atom probe tomography (APT) in voltage mode, of the samples before and after irradiations. The average size and number density of the nanoclusters was determined using the maximum separation method. These techniques allowed for the imaging of nanoclusters to sizes well below the resolution limit of conventional transmission electron microscopy techniques. The most significant changes were observed for samples irradiated at 300 °C where the size (average Guinier radius) and number density of nanoclusters were observed to decrease from 1.1 nm to 0.8 nm and 12 × 1023 to 3.6 × 1023, respectively. In this study, the nanoclusters are more stable at higher temperature.

Citation: 
He J, F Wan, K Sridharan, TR Allen, AG Certain, V Shutthanandan, and Y Wu.2014."Stability Of Nanoclusters In 14YWT Oxide Dispersion Strengthened Steel Under Heavy Ion-irradiation By Atom Probe Tomography."Journal of Nuclear Materials 455(1-3):41-45. doi:10.1016/j.jnucmat.2014.03.024
Authors: 
He J
F Wan
K Sridharan
TR Allen
AG Certain
V Shutthanan
Y Wu
Facility: 
Instruments: 
Volume: 
455
Issue: 
0
Pages: 
41-45
Publication year: 
2014

The Effect of Eectronic Energy Loss on Irradiation-Induced Grain Growth in Nanocrystalline Oxides.

Abstract: 

Grain growth of nanocrystalline materials is generally thermally activated, but can also be driven by irradiation at much lower temperature. In nanocrystalline ceria and zirconia, contributions from both displacement damage and ionization to the grain growth are identified. Our atomistic simulations have revealed fast grain boundary (GB) movements due to the high density of disorder near GBs. Our experimental results have shown that irradiation-induced grain growth is a function of total energy deposited, where the excitation of target electrons and displacement of lattice atoms both contribute to the overall disorder and both play important roles in grain growth. The coupling of energy deposition to the electronic and lattice structures should both be taken into consideration when engineering nanostructural materials.

Citation: 
Zhang Y, DS Aidhy, T Varga, S Moll, PD Edmondson, F Namavar, K Jin, CN Ostrouchov, and WJ Weber.2014."The Effect of Eectronic Energy Loss on Irradiation-Induced Grain Growth in Nanocrystalline Oxides."Physical Chemistry Chemical Physics. PCCP 16(17):8051-8059. doi:10.1039/c4cp00392f
Authors: 
Y Zhang
DS Aidhy
T Varga
S Moll
PD Edmondson
F Namavar
K Jin
CN Ostrouchov
WJ Weber
Facility: 
Volume: 
16
Issue: 
17
Pages: 
8051-8059
Publication year: 
2014

Microstructure of Multistage Annealed Nanocrystalline SmCo2Fe2B Alloy with Enhanced Magnetic Properties.

Abstract: 

The microstructure and chemistry of SmCo2Fe2B melt-spun alloy after multistage annealing was investigated using high resolution transmission electron microscopy (HRTEM) and 3D atom probe tomography. The multistage annealing resulted in an increase in both the coercivity and magnetization. The presence of Sm(Co,Fe)4B (1:4:1) and Sm2(Co,Fe)17Bx (2:17:x) magnetic phases were confirmed using both techniques. Fe2B at a scale of ∼5 nm was found by HRTEM precipitating within the 1:4:1 phase after the second-stage annealing. Ordering within the 2:17:x phase was directly identified both by the presence of antiphase boundaries observed by TEM and the interconnected isocomposition surface network found in 3D atom probe results in addition to radial distribution function analysis. The variations in the local chemistry after the secondary annealing were considered pivotal in improving the magnetic properties.

Citation: 
Jiang X, A Devaraj, B Balamurugan, J Cui, and JE Shield.2014."Microstructure of Multistage Annealed Nanocrystalline SmCo2Fe2B Alloy with Enhanced Magnetic Properties."Journal of Applied Physics 115(6):Article No. 063902. doi:10.1063/1.4865298
Authors: 
X Jiang
A Devaraj
B Balamurugan
J Cui
JE Shield
Volume: 
0
Issue: 
0
Pages: 
0
Publication year: 
2014

1H NMR Metabolomics Study of Metastatic Melanoma in C57BL/6J Mouse Spleen.

Abstract: 

Melanoma is a malignant tumor of melanocytes. Although extensive investigations have been done to study metabolic changes in primary melanoma in vivo and in vitro, little effort has been devoted to metabolic profiling of metastatic tumors in organs other than lymph nodes. In this work, NMR-based metabolomics combined with multivariate data analysis is used to study metastatic B16-F10 melanoma in C57BL/6J mouse spleen. Principal Component Analysis (PCA), an unsupervised multivariate data analysis method, is used to detect possible outliers, while Orthogonal Projection to Latent Structure (OPLS), a supervised multivariate data analysis method, is employed to find important metabolites responsible for discriminating the control and the melanoma groups. Two different strategies, i.e., spectral binning and spectral deconvolution, are used to reduce the original spectral data before statistical analysis. Spectral deconvolution is found to be superior for identifying a set of discriminatory metabolites between the control and the melanoma groups, especially when the sample size is small. OPLS results show that the melanoma group can be well separated from its control group. It is found that taurine, glutamate, aspartate, O-Phosphoethanolamine, niacinamide ,ATP, lipids and glycerol derivatives are decreased statistically and significantly while alanine, malate, xanthine, histamine, dCTP, GTP, thymidine, 2'-Deoxyguanosine are statistically and significantly elevated. These significantly changed metabolites are associated with multiple biological pathways and may be potential biomarkers for metastatic melanoma in spleen.

Citation: 
Wang X, MY Hu, J Feng, M Liu, and JZ Hu.2014."1H NMR Metabolomics Study of Metastatic Melanoma in C57BL/6J Mouse Spleen."Metabolomics 10(6):1129-1144. doi:10.1007/s11306-014-0652-z
Authors: 
X Wang
MY Hu
J Feng
M Liu
JZ Hu
Volume: 
10
Issue: 
6
Pages: 
1129-1144
Publication year: 
2014

NanoSIMS Imaging Alternation Layers of a Leached SON68 Glass Via A FIB-made Wedged Crater.

Abstract: 

Currently, nuclear wastes are commonly immobilized into glasses because of their long-term durability. Exposure to water for long periods of time, however, will eventually corrode the waste form and is the leading potential avenue for radionuclide release into the environment. Because such slow processes cannot be experimentally tested, the prediction of release requires a thorough understanding the mechanisms governing glass corrosion. In addition, due to the exceptional durability of glass, much of the testing must be performed on high-surface-area powders. A technique that can provide accurate compositional profiles with very precise depth resolution for non-flat samples would be a major benefit to the field. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) depth profiling is an excellent tool that has long been used to examine corrosion layers of glass. The roughness of the buried corrosion layers, however, causes the corresponding SIMS depth profiles to exhibit erroneously wide interfaces. In this study, NanoSIMS was used to image the cross-section of the corrosion layers of a leached SON68 glass sample. A wedged crater was prepared by a focused ion beam (FIB) instrument to obtain a 5× improvement in depth resolution for NanoSIMS measurements. This increase in resolution allowed us to confirm that the breakdown of the silica glass network is further from the pristine glass than a second dissolution front for boron, another glass former. The existence of these two distinct interfaces, separated by only ~20 nm distance in depth, was not apparent by traditional ToF-SIMS depth profiling but has been confirmed also by atom probe tomography. This novel sample geometry will be a major benefit to efficient NanoSIMS sampling of irregular interfaces at the nanometer scale that would otherwise be obscured within ToF-SIMS depth profiles.

Citation: 
Wang YC, DK Schreiber, JJ Neeway, S Thevuthasan, JE Evans, JV Ryan, Z Zhu, and W Wei.2014."NanoSIMS Imaging Alternation Layers of a Leached SON68 Glass Via A FIB-made Wedged Crater."Surface and Interface Analysis 46(S1):233-237. doi:10.1002/sia.5585
Authors: 
YC Wang
DK Schreiber
JJ Neeway
S Thevuthasan
JE Evans
JV Ryan
Z Zhu
W Wei
Volume: 
46
Issue: 
Pages: 
233-237
Publication year: 
2014

Therapeutic potential of nanoceria in regenerative medicine.

Abstract: 

Tissue engineering and regenerative medicine aim to achieve functional restoration of tissue or cells damaged through disease, aging or trauma. Advancement of tissue engineering requires innovation in the field of 3D scaffolding, and functionalization with bioactive molecules. Nanotechnology offers advanced materials with patterned nano-morphologies for cell growth and different molecular substrates which can support cell survival and functions. Cerium oxide nanoparticles (nanoceria) can control intracellular as well as extracellular reactive oxygen and nitrogen species. Recent findings suggest that nanoceria can enhance long-term cell survival, enable cell migration and proliferation, and promote stem cell differentiation. Moreover, the self-regenerative property of nanoceria permits a small dose to remain catalytically active for extended time. This review summarizes the possibilities and applications of nanoceria in the field of tissue engineering and regenerative medicine.

Citation: 
Das S, S Chigurupati, J Dowding, P Munusamy, DR Baer, JF McGinnis, MP Mattson, W Self, and S Seal.2014."Therapeutic potential of nanoceria in regenerative medicine."Materials Research Bulletin 39(11):976-983. doi:10.1557/mrs.2014.221
Authors: 
S Das
S Chigurupati
J Dowding
P Munusamy
DR Baer
JF McGinnis
MP Mattson
W Self
S Seal
Volume: 
39
Issue: 
11
Pages: 
976-983
Publication year: 
2014

In Situ Study of CO2 and H2O Partitioning Between Na-Montmorillonite and Variably Wet Supercritical Carbon Dioxide.

Abstract: 

Shale formations play fundamental roles in large-scale geologic carbon sequestration (GCS) aimed primarily to mitigate climate change, and in smaller-scale GCS targeted mainly for CO2-enhanced gas recovery operations. In both technologies, CO2 is injected underground as a supercritical fluid (scCO2), where interactions with shale minerals could influence successful GCS implementation. Reactive components of shales include expandable clays, such as montmorillonites and mixed-layer illite/smectite clays. In this work, we used in situ X-ray diffraction (XRD) and in situ infrared (IR) spectroscopy to investigate the swelling/shrinkage and water/CO2 sorption of a pure montmorillonite, Na-SWy-2, when the clay is exposed to variably hydrated scCO2 at 50 °C and 90 bar. Measured interlayer spacings and sorbed water concentrations at varying levels of scCO2 hydration are similar to previously reported values measured in air at ambient pressure over a range of relative humidities. IR spectra show evidence of both water and CO2 intercalation, and variations in peak shapes and positions suggest multiple sorbed types with distinct chemical environments. Based on the intensity of the asymmetric CO stretching band of the CO2 associated with the Na-SWy-2, we observed a significant increase in sorbed CO2 as the clay expands from a 0W to a 1W state, suggesting that water props open the interlayer so that CO2 can enter. However, as the clay transitions from a 1W to a 2W state, CO2 desorbs sharply. These observations were placed in the context of two conceptual models concerning hydration mechanisms for expandable clays and were also discussed in light of recent theoretical studies on CO2-H2O-clay interactions. The swelling/shrinkage of expandable clays could affect solid volume, porosity, and permeability of shales. Consequently, the results from this work could aid predictions of shale caprock integrity in large-scale GCS, as well as methane transmissivity in enhanced gas recovery operations.

Citation: 
Loring JS, ES Ilton, J Chen, CJ Thompson, PF Martin, P Benezeth, KM Rosso, AR Felmy, and HT Schaef.2014."In Situ Study of CO2 and H2O Partitioning Between Na-Montmorillonite and Variably Wet Supercritical Carbon Dioxide."Langmuir 30(21):6120-6128. doi:10.1021/la500682t
Authors: 
JS Loring
ES Ilton
J Chen
CJ Thompson
PF Martin
P Benezeth
KM Rosso
AR Felmy
HT Schaef
Volume: 
30
Issue: 
21
Pages: 
6120-6128
Publication year: 
2014

Dendrimer-Encapsulated Ruthenium Nanoparticles as Catalysts for Lithium-O2 Batteries.

Abstract: 

Dendrimer-encapsulated ruthenium nanoparticles (DEN-Ru) have been used as catalysts in lithium-O2 batteries for the first time. Results obtained from UV-vis spectroscopy, electron microscopy and X-ray photoelectron spectroscopy show that the nanoparticles synthesized by the dendrimer template method are ruthenium oxide instead of metallic ruthenium reported earlier by other groups. The DEN-Ru significantly improve the cycling stability of lithium (Li)-O2 batteries with carbon black electrodes and decrease the charging potential even at low catalyst loading. The monodispersity, porosity and large number of surface functionalities of the dendrimer template prevent the aggregation of the ruthenium nanoparticles making their entire surface area available for catalysis. The potential of using DEN-Ru as stand-alone cathode materials for Li-O2 batteries is also explored.

Citation: 
Bhattacharya P, EN Nasybulin, MH Engelhard, L Kovarik, ME Bowden, S Li, DJ Gaspar, W Xu, and J Zhang.2014."Dendrimer-Encapsulated Ruthenium Nanoparticles as Catalysts for Lithium-O2 Batteries."Advanced Functional Materials 24(47):7510-7519. doi:10.1002/adfm.201402701
Authors: 
P Bhattacharya
EN Nasybulin
MH Engelhard
L Kovarik
ME Bowden
S Li
DJ Gaspar
W Xu
J Zhang
Facility: 
Volume: 
24
Issue: 
47
Pages: 
7510-7519
Publication year: 
2014

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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...