Bioimaging Science Program 2021 Principal Investigator Meeting Proceedings - Biomolecular Characterization and Imaging Science

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Bioimaging Science Program 2021 Principal Investigator Meeting Proceedings - Biomolecular Characterization and Imaging Science
Biomolecular Characterization and Imaging Science
Bioimaging Science Program
2021 Principal Investigator
Meeting Proceedings

                        Office of Biological and Environmental Research

                                                          June 2021
Bioimaging Science Program 2021 Principal Investigator Meeting Proceedings - Biomolecular Characterization and Imaging Science
Biomolecular Characterization and Imaging Science

                                    Bioimaging Science Program
                                2021 Principal Investigator Meeting

                                                    February 22–23, 2021

                                                     Program Manager
                                                    Prem C. Srivastava
                                    Office of Biological and Environmental Research
                                                     Office of Science
                                                U.S. Department of Energy

                                                    Meeting Co-Chairs

                             Tuan Vo-Dinh                                 Marit Nilsen-Hamilton
                             Duke University                               Iowa State University

                          Jeffrey Cameron                                     James Evans
                    University of Colorado–Boulder               Pacific Northwest National Laboratory

About BER
The Biological and Environmental Research (BER) program advances fundamental research and scientific user facilities to support
Department of Energy missions in scientific discovery and innovation, energy security, and environmental responsibility. BER seeks to
understand biological, biogeochemical, and physical principles needed to predict a continuum of processes occurring across scales,
from molecular and genomics-controlled mechanisms to environmental and Earth system change. BER advances understanding of
how Earth’s dynamic, physical, and biogeochemical systems (atmosphere, land, oceans, sea ice, and subsurface) interact and affect
future Earth system and environmental change. This research improves Earth system model predictions and provides valuable infor-
mation for energy and resource planning.

Cover Images
Image 1: Medicago truncatula, see p. 31; Image 2: XRF confocal image of a leaf treated with AuNR@Ag, see p. 3; Image 3: The
combined nonlinear wide-field and in situ-LE-ME imaging system, see p. 38; Image 4: In situ imaging of lignin removal and enzyme
accessibility, see p. 5; Image 5: Plants expressing Pen3:GFP label intracellular and extracellular vesicles, see p. 20.

Digital Download
science.osti.gov/-/media/ber/bioimaging-technology/pdf/2021/Bioimaging_Science_PI_Meeting2021.pdf
Bioimaging Science Program 2021 Principal Investigator Meeting Proceedings - Biomolecular Characterization and Imaging Science
Biomolecular Characterization and Imaging Science

                                   Bioimaging Science Program
                     2021 Principal Investigator Meeting Proceedings

                                                  Published June 2021

                               Office of Biological and Environmental Research

Prepared for the                                            Prepared by
U.S. Department of Energy                                   Biological and Environmental Research Information System
Office of Science                                           Oak Ridge National Laboratory
Office of Biological and Environmental Research             Oak Ridge, TN 37830
Germantown, MD 20874-1290                                   Managed by UT-Battelle, LLC
                                                            For the U.S. Department of Energy
                                                            Under contract DE-AC05-00OR22725
Bioimaging Science Program 2021 Principal Investigator Meeting Proceedings - Biomolecular Characterization and Imaging Science
2021 PI Meeting Proceedings

     Contents
     Preface .......................................................................................................................................................................................................................................iii
     Bioimaging Science Program Projects..........................................................................................................................................................................iv
     Project Map...............................................................................................................................................................................................................................v
     Executive Summary..............................................................................................................................................................................................................vi
     Abstracts.....................................................................................................................................................................................................................................1
           Multimodal Single-Cell/Particle Imaging and Engineering for Energy Conversion in Bacteria..........................................................................1
           Plasmonics-Enhanced Optical Imaging Systems for Bioenergy Research..................................................................................................................3
            eal-Time Imaging and Quantification of Plant Cell Wall Constituents Using
           R
           Cavity-Dumped Stimulated Raman Scattering (cdSRS) Microscopy.............................................................................................................................5
           Single-Molecule Imaging of Lignocellulose Deconstruction by SCATTIRSTORM Microscopy............................................................................7
           Time-Resolved 3D Multi-Resolution Microscopy for Real-Time Cellulase Actions In Situ......................................................................................9
           In Planta Multimodal Single-Molecule Imaging to Study Real-Time Turnover Dynamics
            of Polysaccharides and Associated Carbohydrate Metabolites...................................................................................................................................11
           Development of Broadband Infrared Nano-Spectroscopy of Biological Materials in Fluid...............................................................................13
           Inorganic Voltage Nanosensors as Tools for Bioelectricity Studies in DOE-Relevant Bacteria and Their Communities..........................14
           Tracking Lignocellulosic Breakdown by Anaerobic Fungi and Fungal Cellulosomes..........................................................................................15
           Understanding Plant Signaling via Innovations in Probe Delivery and Imaging..................................................................................................17
           Spatiotemporal Dynamics of Photosynthetic Metabolism in Single Cells at Subcellular Resolution............................................................18
           Quantum Dot Toolkit for Multimodal Hyperspectral Bioimaging...............................................................................................................................19
           Live-Cell, Quantum Dot-Based Tracking of Plant and Microbial Extracellular Vesicles........................................................................................20
            orrelative Imaging of Enzyme and Metabolome Dynamics for Yield and Titer Co-Optimization
           C
           in Biofuel-Producing Microorganisms...................................................................................................................................................................................22
            evelopment and Implementation of an In Situ High-Resolution Isotopic Microscope
           D
           for Measuring Metabolic Interactions in Soil Mesocosms.............................................................................................................................................24
           E xpanding the Utility and Range of Quantum and Polymer Dots for Multiplexed
            Super-Resolution Fluorescence Imaging in Plants...........................................................................................................................................................25
           Hyperspectral Light-Sheet Raman Imaging of Leaf Metabolism................................................................................................................................26
           Metaoptics-Enabled Multifunctional Imaging...................................................................................................................................................................27
           Multiparametric Optical Label-Free Imaging to Analyze Plant Cell Wall Assembly and Metabolism............................................................28
           Detecting Chemical Signals in the Soil with 4DMAPS, an Integrated Aptasensor Assembly...........................................................................30
           A Quantum-Enhanced X-ray Microscope.............................................................................................................................................................................31
            evelopment of a Full-Field X-ray Fluorescence Imaging System for Near Real-Time
           D
           Trace Element Microanalysis of Complex Biological Systems......................................................................................................................................33
           T he 3DQ Microscope: A Novel System Using Entangled Photons to Generate Volumetric
            Fluorescence and Scattering Images for Bioenergy Applications..............................................................................................................................34
           I lluminating the Rhizosphere: Developing an Adaptive Optics, Multiphoton Microscope
            for 3D Label-Free Live Imaging of Microbes and Organic Matter in Soil and Roots.............................................................................................35
           Quantum Ghost Imaging of Water Content and Plant Health with Entangled Photon Pairs............................................................................36
           I ntrinsically Coregistered Chemical Imaging of Living Plant and Microbial Systems
            via 3D Nonlinear Optical Mapping and In Situ-Liquid Extraction-Mass Spectrometry........................................................................................38
           Probing Photoreception with New Quantum-Enabled Imaging.................................................................................................................................39
           Multimodal Chemical Imaging Across Scales to Visualize Metabolic Pathways in Live Plants and Microbial Systems...........................40
     References...............................................................................................................................................................................................................................41

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Preface
As part of the 2021 Biological Systems Science Division (BSSD) Principal Investigator (PI) Meeting, the Bioimaging
Science program (BSP), within BSSD's Biomolecular Characterization and Imaging Science portfolio, held its
annual PI meeting virtually February 22–23.

BSP's mission is to understand the translation of genomic information into the mechanisms that power living
cells, communities of cells, and whole organisms. The goal of BSP is to develop new imaging and measurement
technologies to visualize the spatial and temporal relationships of key metabolic processes governing pheno-
typic expression in plants and microbes.

BSP convenes annual PI meetings to bring together its contributing investigators to review progress and current
state-of-the-art bioimaging research. Holding the BSP meeting as part of the broader BSSD PI meeting allowed
researchers to interact with the extended Genomic Science program community. This convergence provided a
platform for networking and exchange of ideas, helping to forge new multidisciplinary collaborations among
investigators from the two sister programs.

An important highlight of the BSP meeting was the keynote presentation “Enhancing Fluorescence Microscopy
with Computation” by Dr. Hari Shroff of the NIH National Institute of Biomedical Imaging and Bioengineering. All
the BSP PIs made presentations describing their research focus and progress, and these were followed by round-
table discussions of each project. The meeting’s proceedings provide an outline of the program’s current state
and potential future directions and opportunities.

Prem C. Srivastava, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
301.903.4071; prem.srivastava@science.doe.gov

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2021 PI Meeting Proceedings

     Bioimaging Science Program Projects
                                                                   Development and Implementation of an In Situ High-­
     Universities                                                  Resolution Isotopic Microscope for Measuring Metabolic
     Multimodal Single-Cell/Particle Imaging and Engineering       Interactions in Soil Mesocosms
     for Energy Conversion in Bacteria                             Elizabeth A. Shank, University of Massachusetts Medical School
     Peng Chen, Cornell University
                                                                   Expanding the Utility and Range of Quantum and Polymer
     Plasmonics-Enhanced Optical Imaging Systems for               Dots for Multiplexed Super-Resolution Fluorescence Imaging
     Bioenergy Research                                            in Plants
     Tuan Vo-Dinh, Duke University                                 Gary Stacey, University of Missouri–Columbia

     Real-Time Imaging and Quantification of Plant Cell Wall       Hyperspectral Light-Sheet Raman Imaging of
     Constituents Using Cavity-Dumped Stimulated Raman             Leaf Metabolism
     Scattering (cdSRS) Microscopy                                 Keith Lidke, David Hanson, Jerilyn Ann Timlin, and Jamey Young
     Shi-You Ding, Michigan State University                       University of New Mexico

     Single-Molecule Imaging of Lignocellulose Deconstruction by   Metaoptics-Enabled Multifunctional Imaging
     SCATTIRSTORM Microscopy                                       Paul Bohn, Anthony Hoffman, and Joshua Shrout
     William Hancock, The Pennsylvania State University            University of Notre Dame

     Time-Resolved 3D Multi-Resolution Microscopy for Real-Time    Multiparametric Optical Label-Free Imaging to Analyze Plant
     Cellulase Actions In Situ                                     Cell Wall Assembly and Metabolism
     Haw Yang, Princeton University                                Marisa S. Otegui and Kevin W. Eliceiri, University of
                                                                   Wisconsin–Madison
     In Planta Multimodal Single-Molecule Imaging to Study
     Real-Time Turnover Dynamics of Polysaccharides and
     Associated Carbohydrate Metabolites                           National Laboratories
     Sang-Hyuk Lee, Rutgers University                             Detecting Chemical Signals in the Soil with 4DMAPS,
                                                                   an Integrated Aptasensor Assembly
     Development of Broadband Infrared Nanospectroscopy of         Marit Nilsen-Hamilton, Ames Laboratory
     Biological Materials in Fluid
     Tina Jeoh, University of California–Davis                     A Quantum-Enhanced X-ray Microscope
                                                                   Sean McSweeney, Brookhaven National Laboratory
     Inorganic Voltage Nanosensors as Tools for Bioelectricity
     Studies in DOE-Relevant Bacteria and Their Communities        Development of a Full-Field X-ray Fluorescence Imaging
     Shimon Weiss, University of California–Los Angeles            System for Near Real-Time Trace Element Microanalysis of
                                                                   Complex Biological Systems
     Tracking Lignocellulosic Breakdown by Anaerobic Fungi and     Ryan Tappero, Brookhaven National Laboratory
     Fungal Cellulosomes
     Michelle O’Malley, University of California–Santa Barbara     3DQ Microscope: A Novel System Using Entangled Photons to
                                                                   Generate Volumetric Fluorescence and Scattering Images for
     Understanding Plant Signaling via Innovations in Probe        Bioenergy Applications
     Delivery and Imaging                                          Ted A. Laurence, Lawrence Livermore National Laboratory
     Jean T. Greenberg, The University of Chicago
                                                                   Illuminating the Rhizosphere: Developing an Adaptive Optics,
     Spatiotemporal Dynamics of Photosynthetic Metabolism in       Multiphoton Microscope for 3D Label-Free Live Imaging of
     Single Cells at Subcellular Resolution                        Microbes and Organic Matter in Soil and Roots
     Jeffrey Cameron, University of Colorado–Boulder               Peter K. Weber, Lawrence Livermore National Laboratory

     Quantum Dot Toolkit for Multimodal                            Quantum Ghost Imaging of Water Content and Plant Health
     Hyperspectral Bioimaging                                      with Entangled Photo Pairs
     Prashant Nagpal, University of Colorado–Boulder               James Werner, Los Alamos National Laboratory

     Live-Cell, Quantum Dot–Based Tracking of Plant and            Intrinsically Coregistered Chemical Imaging of Living Plant
     Microbial Extracellular Vesicles                              and Microbial Systems via 3D Nonlinear Optical Mapping and
     Jeffrey L. Caplan, University of Delaware                     In Situ–Liquid Extraction–Mass Spectrometry
                                                                   John F. Cahill, Oak Ridge National Laboratory
     Correlative Imaging of Enzyme and Metabolome Dynamics
     for Yield and Titer Co-Optimization in Biofuel-Producing      Probing Photoreception with New Quantum-Enabled Imaging
     Microorganisms                                                James E. Evans, Pacific Northwest National Laboratory
     Andreas E. Vasdekis, University of Idaho
                                                                   Multimodal Chemical Imaging Across Scales to Visualize
                                                                   Metabolic Pathways in Live Plants and Microbial Systems
                                                                   Scott Lea, Pacific Northwest National Laboratory

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Project Map

      University of
      California—Davis
                                                                    The University         University of
                                         University of                 of Chicago          Notre Dame
             Pacific Northwest           Colorado–Boulder (2)
             National Laboratory (2)                                                                    Rutgers                 University of
                                                             University of                            University                Massachusetts
                                                       Wisconsin–Madison                                                        Medical School
                      University                                                              Michigan State
                      of Idaho                             Ames                               University
                                                       Laboratory
                                                                                                  Cornell
                                                                                                University

                                                                                                                                 Brookhaven National
                                                                                                                                 Laboratory (2)
                                                                                                                           Princeton
                                                                                                                           University
                                                                                                                          University
                                                                                                                          of Delaware
                                                                                                                          The Pennsylvania
                                                                                                                          State University

                                                                                                                   Duke
                                                                                                                   University

          University of
          California—Los Angeles

       University of California—
       Santa Barbara                                                                               Oak Ridge
                                             Los Alamos                                            National Laboratory
                                             National Laboratory               University of
                                                                               Missouri—Columbia
 Lawrence Livermore
 National Laboratory (2)               University of
                                       New Mexico

                                                                    University Project               National Laboratory Project

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     Executive Summary
     T   he U.S. Department of Energy’s Bioimaging
         Science program (BSP) supports fundamental
     research to develop and apply new and enhanced
                                                              and nonlinear optical techniques. These techniques
                                                              include surface-enhanced Raman scattering (SERS),
                                                              stimulated Raman scattering (SRS), hyperspectral
     bioimaging and measurement capabilities that             stimulated SRS (hsSRS), and tip-enhanced Raman
     enable scientists to study the biological functions of   scattering (TERS), as well as nano-Fourier transform
     plant and microbial systems relevant to bioenergy        infrared (FTIR) and X-ray microscopies.
     research. The program—within the Biomolecular
     Characterization and Imaging Science portfolio           BSP researchers are developing spectroscopic
     of DOE’s Office of Biological and Environmental          techniques to image dynamic events and molecular
     Research (BER)—currently sponsors multidisciplinary      processes in situ, enhancing various combinations
     research at 9 national laboratories and 19 universi-     of nondestructive and destructive approaches to
     ties (see List of Funded Projects and map, pp. vi–vii)   image laboratory-prepared or fixed samples, and
     with the goal of understanding the mechanisms that       creating inorganic voltage nanosensors to study
     power living cells, communities of cells, and whole      bacterial communities. Optical modalities are non-
     organisms. BSP researchers are developing instru-        invasive and include infrared/ultraviolet absorption
     ments and imaging systems from the ground up and         and adaptive optics multiphoton microscopy, fluo-
     are enhancing existing capabilities with new or trans-   rescence, and Raman techniques (e.g., conventional,
     formational improvements. These novel capabilities,      nonlinear, and plasmonics-enhanced). Recently,
     design-based technologies, and improved or inno-         BSP added quantum-­enabled bioimaging science
     vative uses of established methodologies will enable     research projects at national laboratories. These
     new fundamental discoveries and provide solutions        projects encompass state-of-the-art quantum-based
     to challenges in plant and microbial systems biology.    techniques such as quantum-enhanced X-ray micros-
     These challenges cross a range of scales—from single     copy, quantum ghost imaging, three-dimensional
     molecules to small unicellular organisms to complex      (3D) quantum microscopy, and quantum-enabled
     microbial and fungal community interactions with         imaging using entangled photons. Individual research
     plants. Together, BSP-supported researchers are          programs focused on multidisciplinary projects are
     creating an extensive and versatile toolbox enabling     complemented by research and development at
     real-time dynamic imaging of metabolic pathways,         DOE-­sponsored user facilities, which are building and
     material transport within and between cellular organ-    applying various technologies, such as ion micros-
     elles, plant-root and organism interactions, enzyme      copy and full-field X-ray fluorescence imaging.
     functions, and cellular structures.
                                                              BSP researchers are further enhancing co-application
                                                              of mass spectrometry and spectrochemical imaging
     Overview of Current BSP Research                         capabilities to yield highly selective, sensitive, and
     Expansion of New and Existing Technologies               quantitative chemical maps that identify intra- and
                                                              extracellular molecular gradients and the distribu-
     BSP has significantly expanded since its inception in    tions, abundances, and fates of stable isotopes, nat-
     2015. The program recently added an extensive range      ural elements, and metabolites. Using conventional
     of novel bioimaging technologies and cutting-edge        microscopies for correlated structural and chemical
     sensing approaches, including super-resolution           imaging, this work supports simultaneous observa-
     microscopy, hyperspectral light-sheet imaging, adap-     tion and interpretation of the biological function of
     tive optics, code-aperture methods, quantum entan-       living plant and microbial systems.
     glement and quantitative phase imaging, correlative
     imaging, and holographic force spectroscopy. These       Researchers also are significantly expanding the
     new technologies are complementary to and syner-         performance and impact of label-based and label-free
     gistic with ongoing developments in instrumentation      sensing and imaging technologies by developing
     involving molecular, optical, fluorescence, Raman,       unique probes, such as quantum and polymer dots,

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2021 PI Meeting Proceedings

as well as plasmonic nanoprobes equipped with              images. While these imaging approaches focus on
various bioreceptors (e.g., antibodies, aptamers, and      events inside cells, an alternative imaging approach
gene probes). These probes can specifically detect         uses aptamers as sensors to image specific molecular
important biomarkers, including metabolites, pro-          species present around cells. These imaging modali-
teins, and genomic markers, related to particular proc­    ties will be complemented by advanced technologies
esses and metabolic pathways in microbial and plant        such as high-speed atomic force microscopy (AFM),
systems relevant to bioenergy research. Development        interferometric scattering microscopy, infrared, and
of these unique probes and sensors is expanding the        vibrational sum frequency generation. Researchers
applicability of the new instrumentation by enabling       also are applying plasmonic infrared nanofocusing
researchers to dynamically track targeted cells, organ-    gratings combined with microfluidics to map cellu-
elles, enzymes, biomarkers, and small molecules and        lose surface fibrils with cellulose at the nanoscale.
to test and validate cellular processes and genome-
based models of cellular metabolism.                       Raman and Mass Spectrometry–Based Approaches
With the new instrumentation and optical probes            Other important portfolio components are various
developed under BSP sponsorship, these investiga-          Raman spectroscopy–based approaches, including
tions are expected to result in a better understanding     spontaneous, far-field sub-diffraction, TERS, coherent
of the spatial and temporal distributions of metab-        anti-Stokes (CARS), SRS, SERS, spatially offset Raman
olites associated with growing microbial and plant         spectroscopy (SORS), shifted-excitation Raman differ-
systems. Also anticipated are new insights into the        ence spectroscopy (SERDS), and cavity-­dumped SERS.
fundamental biology of many macro events, such as          BSP researchers also have developed a multimodal
nutrient utilization and community and ecosystem           microscope integrating CARS, SRS, and two-photon
interactions that include soil water retention caused      excitation systems and adaptive optics. The combi-
by the presence or absence of particular organisms or      nation of SERDS with hyperspectral Raman imaging
biomass. This comprehensive portfolio will improve         (HSRI) demonstrated the possibility of directly
understanding of the molecular underpinnings               imaging microRNA biotargets in intact living plants
of a diverse array of biological and environmental         under ambient light conditions.
processes.
                                                           Added to these imaging modalities will be a capa-
Multimodal Microscopy Techniques                           bility that enables researchers to capture samples
                                                           for profiling metabolites using several forms of mass
New BSP instruments span a wide range of modalities.
                                                           spectrometry, including laser ablation electrospray
Microscopy approaches include optical methods,
                                                           ionization mass spectrometry (LAESI-MS) and LAESI-
such as luminescence, confocal, adaptive optics
                                                           Fourier-transform ion cyclotron resonance mass
multiphoton, fluorescence scattering, reflected/
                                                           spectrometry (FTICR-MS) using a 21 Tesla magnet. To
transmitted light extinction spectroscopy, entangled
                                                           provide 3D spatiotemporal chemical information in
photon, and total internal reflection fluorescence
                                                           bulk and at the interfaces of biological systems, BSP
(TIRF). Also included are full-field X-ray fluorescence,
                                                           researchers developed a nonlinear optical mapping
imaging, polarimetry, entangled X-ray imaging, and
                                                           and in situ liquid extraction–mass spectrometry
novel single-molecule sensing methods, such as
stochastic optical reconstruction microscopy (STORM)       (LE-MS) capability utilizing a porous membrane
and photo-activated localization microscopy (PALM).        microfluidic surface in combination with a continuous
In addition, BSP researchers are increasing imaging        LE sampling probe. Also developed was a wide-field
throughput rates and resolution of single cells            CARS microscope for rapid and simultaneous acquisi-
through quantitative phase imaging (QPI) combined          tion of CARS images across an entire field of view.
with light-sheet fluorescence microscopy-based
optical quasi-lattice technology. Dark-field fluores-
                                                           Imaging Using Nucleic Acids
cence-based hyperspectral imaging is enabling the          In a different approach, BSP researchers are devel-
collection of high signal-to-noise images and will         oping electrochemical impedance spectroscopy with
allow multiplex collections of multi-fluorophore           nucleic acid aptamer sensors. This technology will

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       enable scientists to monitor nutrient transformations     cultivate and analyze biosystems from single cells to
       and microbial metabolic activities in the rhizosphere     complex communities.
       that contribute to plant growth and health and to
       investigate plant-microbe interactions that involve       Characterizing Diverse Molecules Across Scales
       chemical communications that travel through the rhi-      BSP research teams are developing capabilities to
       zosphere. Other applications of nucleic acids to image    study molecular signatures and processes that are
       plant and microbial activities include the detection      highly diverse and cover a broad range of length
       of microRNAs using silver-coated gold nanorods and        scales. These include atomic isotopes, metabolites,
       SERS sensing, as well as the detection of riboswitches    plant hormones, silica, trace elements, redox metabo-
       that act as metabolite reporters using quantitative       lism, microbial electron transfer, membrane potential,
       phase imaging that leverages a light-sheet fluores-       intercellular trafficking, cellulose and lignin synthesis
       cence technique and Raman imaging.                        and degradation, microRNAs that regulate lignifica-
                                                                 tion, enzymes and other proteins secreted by plants,
       Tracking Molecules In Situ and in Real Time               and quorum-sensing molecules.
       BSP researchers are focusing on understanding a
       variety of biological systems for better controlling      The functional dimensional scales in biological sys-
       plant health and growth to improve bioenergy              tems are vast, spanning molecules to multiorganismal
       resources. The subject organisms include plants,          systems. Because these systems are hierarchical in
       bacteria, fungi, and their combinations. Teams are        nature, activities on longer length and time scales are
       developing sophisticated instruments to image             built on activities and structures on shorter length
       metabolism in a single organism, gene expression,         and time scales. Therefore, processes must be fully
       and regulatory molecules (e.g., microRNAs, quorum-­       explained at the molecular level to be fully under-
       sensing molecules, and protein kinases) that operate      stood at the organismal or multiorganismal level.
       in intact organisms or are involved in communica-         Recognizing this need, BSP supports some innovative
       tion between organisms. Several approaches are            cross-scale imaging approaches that include plas-
       underway to capitalize on the advantageous spec-          monic nanoprobes to track single molecules. Also
       troscopic properties offered by semiconductors,           supported is 3D tracking with high-speed AFM and
       polymers, and quantum dots. Nuclear-based imaging         optical tweezers to control molecules or microbes,
       technologies such as Positron Emission Tomography         enable force measurements, or track molecules such
       enable scientists to visualize and quantify the move-     as cellulose synthase as it moves along the mem-
       ment of radiolabeled nutrients, plant hormones, and       brane or cellulase as it moves along cell walls. These
       other signal molecules within intact live plants. How-    studies will answer important questions regarding the
       ever, the widespread use of these technologies has        mechanisms of cellulose synthesis and degradation.
       been hampered by access to the limited number of          Understanding such mechanisms will, in turn, enable
       facilities that have the unique capabilities to produce   the development of biomass feedstocks that more
       these specialized agents. BSP-supported researchers       readily can be converted to biofuels and bioproducts.
       are developing instruments that will address the
       critical need of measuring these features directly,       Quantum-Enabled Techniques
       enabling a future in which molecular signatures can       BSP has recently added state-of-the-art quantum-­
       be tracked in real time and over time periods consis-     enabled bioimaging projects at national laboratories.
       tent with the biological processes under study. These     Quantum-enhanced X-ray microscopy uses entangled
       developments will include the capability of visual-       X-rays beams. With the ghost imaging technique,
       izing biosystems as they respond to external stressors    samples are illuminated using less-intense beams with
       and perturbations such as nutrient starvation and         energy more suitable for maintaining biological integ-
       chemical exchanges. BSP researchers also are using        rity. Furthermore, the quantum nature of the imaging
       synthetic rhizosphere microhabitats, transparent soil     process enables visualization of details impossible to
       microcosms, and versatile nanofluidic and microflu-       detect with classical methods. BSP researchers also are
       idic imaging and sampling devices simultaneously to       developing a new microscope using entangled photon

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2021 PI Meeting Proceedings

pairs to visualize water, lignocellulose, and lipids in    struction, or boosting feedstock sustainability and
plants. To probe samples, this system uses a wave-         plant drought tolerance. Organisms under study
length that can be in the near- or mid-infrared range      include:
where vibrational fingerprinting to identify key molec-
ular species is possible. Detection and imaging are        • Living plants — Arabidopsis thaliana, Medicago
then performed with visible light using high-­efficiency      truncatula, Brachypodium distachyon, Populus sp.,
and low-noise imaging detectors.                              Pinus taeda, and Zea mays.

Research teams are developing a high-quality 3D            • Microbial chemotrophs — Bacillus subtilis, Yarrowia
imaging modality that uses quantum-entangled                  lipolytica, and Pantoea sp.
photon pairs to obtain more information on fluores-        • Microbial phototrophs — Cyanothece sp.,
cence and scattering events than is available with            Rhodopseudomonas palustris, Ostreococcus tauri,
standard fluorescence or scattering measurements.             Chlamydomonas reinhardtii, and Synechococcus sp.
The system uses two separate 2D detectors to obtain
three- and four-dimensional information about the          • Systems for studying plant-microbe interactions
same photon, providing 3D optical imaging at high             — Arbuscular mycorrhizal symbioses, Glycine
frame rates to monitor dynamic host-bacterial interac-        max with Bradyrhizobium japonicum, and Suillus
tions in bioenergy algal pond and plant systems.              brevipes with P. taeda.

Also under development is a hybrid quantum-­enabled
imaging platform that combines advances in adap-
                                                           Research Challenges and
tive optics, quantum entanglement, coincidence             Future Opportunities
detection, ghost imaging, quantum phase-contrast           Multidisciplinary Research Teams
microscopy, and multidimensional nonlinear coherent
(nonentangled) photons and four-wave mixing. This          Biological imaging is inherently transdisciplinary,
system will enable researchers to visualize photore-       and successful teams need to continue to reflect this
ception in phytotropin and phytochrome proteins and        approach to advance BSP programmatic goals. Mul-
other quantum coherent processes that occur natu-          tidisciplinary teams are needed to integrate imaging
rally within biosystems, improving the ability to track    results with the corresponding genomic, proteomic,
ultrafast protein dynamics and the flow of metabolites     lipidomic, and metabolomic changes within cells to
between biological compartments in real time.              further understand biological complexity and hetero-
                                                           geneity. Achieving this understanding requires com-
Moving Toward More Complex Systems                         bining the expertise from researchers in conventional
                                                           and quantum-enabled imaging technology, nano-
Although many of the initial samples BSP researchers
                                                           science, computer science, structural biology, bio-
use to test new instruments and methods may be
                                                           chemistry, plant physiology, microbiology, genomic
from canonical model systems, the program should
                                                           science, ecology, soil science, and biogeochemistry.
continue to evaluate and adapt to real-world biosys-
                                                           This cross-disciplinary approach will be a critical step
tems as well. For example, label-free identification of
                                                           toward connecting phenotypes with genotypes and
microbes obtained from the environment remains
                                                           translating laboratory-developed technologies into
a grand challenge in biology, so extending BSP-­           the natural environment.
developed label-free approaches to such microbes in
the long term is a next frontier.                          Further Integration of Technologies into
In addition, while focusing on high-resolution             Multimodal Hybrid Instruments
imaging, some BSP-supported projects are applicable        BSP-supported development of individual imaging
to more complex biological systems and challenges          techniques is making significant strides. These
relevant to bioenergy and the environment, such            technologies range from complementary targeted
as understanding quorum sensing, improving lipid           and untargeted methods to destructive and nonde-
feedstock yields, enhancing lignocellulosic decon-         structive imaging modalities (e.g., optical, scanning

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2021 PI Meeting Proceedings

    probe, mass spectrometric, X-ray, and ion-based            important advances are integrated data processing
    approaches) that cover a wide range of spatial and         algorithms, visualizations, and modeling, which are
    temporal scales. The recent addition of cutting-edge,      key components for properly interpreting the diverse
    sophisticated and laboratory-based imaging methods         sets of imaging data, omics-based organismal models,
    (e.g., quantum entanglement and super-resolution           and other information emanating from BER genomics
    techniques such as STORM and PALM) strongly com-           research.
    plement the sensing and imaging approaches more
    suitable for general laboratory and field use.             Advances in Data Management and Analytics
                                                               To enable effective extraction of critical biological
    In addition to pursuing advances within each of these
                                                               and environmental information from experimental
    techniques, a major programmatic focus moving
                                                               data, major advances are needed in data storage,
    forward should be on making the developments
                                                               processing, and visualization. BSP’s long-term goal
    robust, easy to use, and accessible to the BER research
                                                               is to develop enabling capabilities that can generate
    community. One approach toward meeting this goal
    could be to further integrate these different and          spatially and time-resolved snapshots of relevant
    complementary approaches into hybrid all-in-one            cellular metabolism, including both primary and
    instruments. Multimodal spectral imaging in a single       secondary metabolites as well as genomic biomarkers
    and user-friendly setup across nano-, micro-, meso-,       and internal and secreted compounds. Achieving
    and macroscopic spatial domains will be a useful and       real-time data collection and interpretation of these
    versatile tool for future users. There is also a need to   integrated data will lead to major advances in bio-
    develop highly specialized, sophisticated instrumen-       imaging technology that will improve monitoring
    tation for fundamental research in the laboratory as       and phenotyping of plant and microbial systems and
    well as portable and easy-to-use instrumentation           expand the understanding of molecular and genomic
    for large-scale monitoring applications in the field.      pathways, in both the laboratory and in complex nat-
    Previously unachievable studies of microbes, plants,       ural environments. These advancements will require
    and other species in their environments will be pos-       new methods and algorithms to handle increasingly
    sible due to the new capabilities provided by these        challenging volumes of data, along with automated
    instruments. Results of these studies are expected to      and machine learning approaches to rapidly analyze
    reveal new insights on how to optimize development         this data and identify biologically and environmen-
    of sustainable bioenergy resources.                        tally meaningful signals.

                                                               Of interest is a central clearinghouse for archiving
    Cross-Platform Data Fusion and Integration
                                                               experimental and simulation data that incorporates
    With BSP’s expansion and the rapid increase in mon-        a standardized output and imaging framework for
    itoring modalities, data integration across multiple       different and potentially widely adoptable analytical
    technologies and approaches remains a high priority.       modalities. Such a data repository could be indepen-
    Data fusion (i.e., linking complementary data from dif-    dent or integrated with the DOE Systems Biology
    ferent techniques) will produce a more holistic picture    Knowledgebase (KBase) and take advantage of
    and better understanding of the biological systems         advances in artificial intelligence to extract patterns
    being imaged. Facilitating cross-platform bioimaging       from raw data for improved organization, interpreta-
    systems will require indexing and registering images       tion, and representation.
    (e.g., multifunctional tracers, probes, and sensors to
    serve as cross-platform fiducial markers) and mean-        Another opportunity for improving data interpret-
    ingfully co-referencing and co-registering disparate       ability is to leverage computer science (CS) graduate
    datasets for the same sample but of different for-         programs to help accelerate image processing or
    mats, magnifications, or resolutions. Also needed          data analysis pipelines for the large datasets collected
    are models capable of integrating multimodal data          within BSP. Many CS programs require students to
    spanning a wide range of spatial and temporal scales       gain access to and experience with real-world data by
    to effectively extract causality from observations and     building new software or other algorithms for more
    understand complex biological phenomena. Other             effective analytics. Using the plethora of BSP data,

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principal investigators (PIs) could sponsor CS grad-      Field-Deployable Capabilities for Whole Organisms
uate students to develop the next frontier of bioim-      and Complex Communities
aging analytics tools.
                                                          Another important challenge for the near term is
New Probes and Quantum-Enabled Techniques                 the extension of laboratory-based approaches into
to Expand Investigations                                  applications for whole organisms and plants in their
                                                          natural environments and under field settings. This
In parallel with BSP’s instrumentation development        expansion will require incorporating the dynamics
efforts, there is also a critical need for probe devel-   of microbially driven biogeochemistry (e.g., within
opment that enables identification, sensing, and          the rhizosphere, biofilms, and other key biological
functional imaging of various targets within complex      interfaces) into the imaging process. Although there
biological systems, ranging from key metabolites          has been progress in imaging genomic biotargets
to molecular and genomic biotargets (e.g., mRNA,          in living plants, advances are needed in imaging
microRNA, proteins, and regulatory small mole-            complex native microbial communities to decipher
cules). Relevant key advances would include the           their organization and the multiple metabolic pro-
simultaneous marking, spatially resolved tracking,        cesses occurring simultaneously in space and time.
and sensing of multiple players (e.g., elements,          Concerted efforts will also be needed to develop the
isotopes, enzymes, metabolites, and other molec-          ability to probe inherent signals within nontractable
ular biomarkers) in a given biological system. BSP’s      microbes in the environment and to create pathways
wide range of biosensing and imaging capabilities         that enable in situ microbial synthesis of probes for
are expected to provide the essential flexibility to      assaying function and activity. Furthermore, in addi-
broaden the scope of investigations, opening new          tion to sophisticated lab-based analytical methods,
possibilities to discover yet-unknown key biomarkers      portable instrumentation and practical techniques
or intermediates.                                         will allow the detection of weak optical signals from
Probing a sample inherently perturbs it, yet methods      whole-organismal data containing strongly inter-
based on selective probe-induced perturbations            fering background signals such as fluorescence,
of key biotargets or metabolic pathways of specific       ambient light, vibrations, and fixed-pattern noise
organisms could provide opportunities to investigate      encountered under field conditions.
and understand biological processes that otherwise
would be difficult to unveil. BSP researchers are also
                                                          Correlative Frozen or Fixed-Sample Imaging
pursuing an approach to minimize perturbation:            Finally, it is important to realize the benefits of com-
the incorporation of quantum-enabled science and          bining additional approaches that may be destructive
technologies. The potential of using ghost imaging        or applicable only to frozen or fixed samples, which
for bioimaging applications is intriguing because this    are typically outside the scope of the BSP portfolio.
approach can image a sample by detecting a photon         Many current BSP capabilities are based on optical
that never interacted with the sample. Furthermore,       approaches that empower real-time or in situ obser-
the ability of quantum-entangled two-photon               vations of living systems, but they do not provide
imaging to provide higher detection efficiency and        a complete picture of the sample or a whole-cell
decrease the total photon flux needed to observe a        context. Some science questions require more holistic
high-contrast image, and thereby permit very low          imaging and analysis to decipher complex associ-
dose imaging that could minimize photodamage              ations within or between living cells. Combining
effects, would facilitate longer-term, time-resolved      current BSP approaches with sequential downstream
imaging of biosystems. Deeper penetration by X-rays       frozen or fixed-sample correlative imaging (such as
combined with X-ray-entangled imaging will enable         cryo-electron microscopy or nano-secondary ion
imaging in thicker biological samples. The develop-       mass spectrometry) can provide additional spatial,
ment and integration of these and other quantum-­         ultrastructural, or chemical context needed for critical
enabled imaging technologies or sensors into the          scientific breakthroughs related to cellular sensing
BSP portfolio could significantly expand the range of     and metabolite response, flow, and fate. Such multi­
scientific questions the program addresses.               modal and correlative imaging approaches should

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      be encouraged within BSP to accelerate the under-         an approach would also facilitate continued tech-
      standing of biosystem complexity and organization         nological developments through the important
      and their impact on dynamics.                             user-developer feedback loop and the synergistic
                                                                interactions between imaging scientists and facil-
      Summary of Opportunities and Needed                       ities. These interactions would expand the scope
      Developments                                              of research being conducted using BSP-developed
                                                                capabilities.
      In summary, several advances are needed in key areas:
      • Integrating bioimaging techniques with advanced         Bioimaging Science Program Annual Meeting
         probes and delivery mechanisms that expand the         BSP’s annual PI meeting provides an important
         monitoring capability for important biotargets         avenue for the program to increase the cross-­platform,
         ranging from key metabolites to molecular and          cross-disciplinary, and multiscale synergies needed to
         genomic biomarkers.                                    achieve its goals. Scheduling this meeting proximal
      • Developing new or improved conventional or              to the DOE Genomic Science program (GSP) annual PI
         quantum-­enabled imaging technologies capable          meeting creates invaluable opportunities for syner-
         of monitoring biological systems in their natural      gistic interactions with that community. Furthermore,
         states or as they respond to environmental pertur-     inviting imaging experts external to BSP as keynote
         bations and stressors.                                 speakers injects novel perspectives and approaches
                                                                into discussions during the program’s annual meeting.
      • Developing new or improved biosensing and               Additional interactions across BSP’s research teams
         bioimaging approaches that enable real-time data       (e.g., through teleconferencing or web conferencing)
         collection across the full range of relevant spatial   could help maintain this interactive momentum and
         scales in the laboratory and under field conditions.   catalyze new directions of investigation.
      • Correlating multimodal dynamic and static “snap-
         shot” imaging methods, both destructive and
                                                                Additional Cross-Program Interactions and
         nondestructive, to provide a holistic understanding    Community Engagement
         of chemical-structural-functional linkages.            A new mechanism that allows supplemental funding
                                                                could foster even more direct cross-fertilization and
      • Establishing cross-platform protocols for sample
                                                                interaction between the GSP and BSP research com-
         preparation, calibration, indexing and spatial
                                                                munities. The envisioned new class of funding could
         registration, data verification, and correlation to
                                                                supplement the travel and supply costs of embed-
         increase the suite of complementary analyses
                                                                ding a graduate student or postdoctoral researcher
         that can be conducted on a given sample or suite
                                                                from a GSP-funded research group into a BSP-funded
         of samples.
                                                                research group for 1 to 6 months. This arrangement
      • Developing methods to increase throughput for           would stimulate more direct collaboration and cross-
         more mature imaging technologies that can be           talk between the two programs, yielding benefits for
         used for new applications.                             both. For GSP researchers, this collaboration would
                                                                give them access to cutting-edge technology that
      Expanding BSP’s Impact and Interactions                   otherwise may have been beyond reach, leading
                                                                to new scientific discoveries. For BSP researchers, it
      Community Access to BSP-Developed Technologies            would provide access to new science and samples
      Through User Facilities                                   they could use for adapting, benchmarking, and
      User accessibility to new BSP technologies and            evaluating the performance of their newly developed
      approaches will be a key factor for the program’s         instrumentation and methods. This funding mecha-
      success and longevity. Deploying some BSP imaging         nism would be very similar to DOE’s Office of Science
      capabilities to DOE scientific user facilities would      Graduate Student Research opportunity. However,
      expand the research community’s access to these           instead of enabling researchers to pursue part of their
      technologies, thereby increasing their impact. Such       graduate thesis research at a DOE national labora-

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tory or user facility, it would support GSP researchers   nity of scientists who could use the program’s
who want to visit and use the new technologies            bioimaging approaches. As part of outreach to BER
developed by BSP-funded groups at universities and        researchers, the portal would detail BSP’s diverse
national laboratories.                                    technological approaches, highlight the applications
                                                          for which they are best suited, and provide a forum
Finally, the creation of a bioimaging capability portal   for information dissemination, tutorials, and training
could enhance BSP’s impact on a wider commu-              opportunities.

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Abstracts
Multimodal Single-Cell/Particle Imaging and Engineering for Energy Conversion in Bacteria
Principal Investigators: Peng Chen (PI), Tobias Hanrath,
and Buz Barstow
Institution: Cornell University
Email: pc252@cornell.edu

Research Plans and Progress: This project’s research
aims to combine quantum materials synthesis, bacterial
synthetic biology, and multimodal single-entity imaging
to quantitatively study how hybrid quantum dot (QD)–
bacteria systems convert light to value chemicals at the
single- to subcell level, with the overall goal of gaining
insights to guide the engineering of QDs and bacterial         In this schematic of bacterial cells sitting on a layer of
genetics for more efficient bioenergy conversion.              quantum dots (QDs) on top of a transparent electrode,
                                                               focused laser beams excite local regions on the QD
On quantum materials, the project focused on developing        layer. The excited electrons can be donated to the
semiconductor cadmium sulfide (CdS) thin films on indium       bacteria for subsequent reduction of CO2 to biomass,
tin oxide (ITO) as photosensitizers; for that, CdS’s energy    in which a photoelectrochemical current is generated.
                                                               The QDs and the bacterial and photoelectrochemical
gap and redox potential can be tuned by its size and           processes can all be imaged. Courtesy Tobias Hanrath,
surface chemistry. The project examined partial surface        Cornell University.
oxidation and ligand-exchange processes and character-
ized them using spectroscopy and photoelectrochemical
measurements. The project also studied using PEDOT:PSS
between the ITO electrode and the QD thin film to ensure
                                                              and/or PhaP1 that decorates the surface of biomass PHB
that photoexcited electrons flow toward the microbe, thus
                                                              granules, with a (photoactivatable) fluorescent protein.
focusing on photoreduction (rather than photooxidation)
                                                              Under H2/CO2/air lithoautotrophic growth, researchers
processes in the QD/microbe hybrids.
                                                              determined: (1) the intracellular concentrations of MBH
On bacterial biology, the team has completed a systematic     and SH; (2) MBH and SH concentrations both have strong
survey of thermodynamic constraints on electromicrobial       positive correlations with PHB accumulation, with SH
conversion of CO2 and electricity to bioproducts, encom-      having slightly stronger correlation; and (3) biomass
passing microbes that uptake electricity by H2-oxidation      accumulation remains unchanged upon deleting MBH
(Ralstonia eutropha) and by extracellular electron uptake     but decreases by ~95% upon deleting SH, suggesting
(Shewanella oneidensis). Team members demonstrated            SH’s role in supplying reducing equivalents toward bio-
that both methods of electron uptake have comparable          mass synthesis.
high maximum conversion efficiencies. This theoretical
                                                              Team members further examined the photoelectrochem-
analysis allowed for building a 10-point roadmap for the
                                                              ical current across single semiconductor-cell interfaces
development of electromicrobial production technology.
                                                              for individual R. eutropha cells in contact with a semi-
The project has also identified genes encoding an electron
                                                              conductor film. With CdS (n-type), researchers measured
uptake pathway in S. oneidensis. Using a high-throughput
                                                              single­-interface photoelectrochemical currents at anodic
screening, researchers discovered 150 genes that affect
                                                              conditions to quantify the cells’ ability to accept pho-
electron uptake; four of them are indispensable. This set
                                                              togenerated holes (i.e., donate electrons). Many cells
of genes provides a portable electron uptake module,
                                                              show enhanced or suppressed photocurrent relative
transferrable to highly engineerable microbes to enable
                                                              to CdS films alone, suggesting pronounced cell-to-cell
electron uptake and power CO2 fixation.
                                                              heterogeneities and highlighting the need of single-­
On multimodal single-entity imaging in the R. eutropha        entity experiments. Researchers examined one-on-one
chromosome, researchers have tagged the membrane-­            correlations between cell-induced photocurrent changes
bound hydrogenase (MBH), the soluble hydrogenase (SH)         and the characteristics of the associated single cells (e.g.,

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    cell size/shape, the amounts of hydrogenase/PHB). Team            aper: Salimijazi, F., et al. 2020. “Constraints on
                                                                  3. P
    members observed a clear correlation between cell­               the Efficiency of Engineered Electromicrobial
    induced photocurrent changes and cell size. The team             Production,” Joule 4(10), 2101–30. DOI: 10.1016/j.
    also employed Cu2WS4 (p-type) and measured single-­              joule.2020.08.010.
    interface photocurrents at cathodic conditions. Many cells
                                                                  4. P
                                                                      reprint: Rowe, A. R., et al. 2021. “Identification of a
    are associated with cathodic photocurrent enhancement,
                                                                     Pathway for Electron Uptake in Shewanella oneiden-
    indicating that under this condition most cells exhibit
                                                                     sis,” bioRxiv. DOI: 10.1101/2021.01.12.426419.
    strong electron­-accepting capabilities.
                                                                 Potential Benefits and Applications: This research will
    Current and/or Anticipated Accomplishments and
                                                                 provide quantitative knowledge to understand the basic
    Deliverables:
                                                                 materials and biological factors as well as guiding princi-
     1. Image analysis software to find electron uptake         ples to engineer and improve such systems. If successful,
         genes in S. oneidensis: github.com/barstowlab/          this research will transform the study of hybrid inorganic­
         macroscope-imageanalyzer.                               bacterial systems for energy and chemical conversions.
     2. C
         ode for calculating electromicrobial production        The proposed experiments should break new scientific
        efficiency: github.com/barstowlab/rewiredcarbon.         grounds and open unforeseen opportunities.

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Plasmonics-Enhanced Optical Imaging Systems for Bioenergy Research
Principal Investigators: Tuan Vo-Dinh1 (PI), Tai-Ping        (iMSs) that can be monitored using surface-enhanced
Sun,1 and Kenneth Kemner2                                    Raman scattering (SERS). The team is currently develop-
Institutions: 1Duke University and 2Argonne National         ing innovative imaging technologies for visualization and
Laboratory                                                   quantitative characterization of biomarkers related to
Email: tuan.vodinh@duke.edu                                  molecular proc­esses and cellular function within living
                                                             plants, namely Multimodal Optical Sensing and Imaging
The goal of this project is aimed at addressing the DOE      Combinatory (MOSAIC) System. The advanced MOSAIC
Funding Opportunity Announcement need to develop             system will provide much-needed biofuel research tools
innovative and improved imaging instrumentation that         such as elucidating the regulation of the pathway to
can enable visualization and quantitative characterization   synthesize photosynthetic terpenes more efficiently for
of biomarkers and their dynamic role in cellular functions   biofuel production and tracking pathways of carbon
in living plants relevant to DOE bioenergy programs.         fixation in plants.

Research Plans and Progress, Including Objectives            Current and/or Anticipated Accomplishments/
and Goals for the Project Period: Monitoring gene            Deliverables for the Project Period: The project
expression in whole plants is a key requirement in           has developed a strategy for efficient delivery of iMS
many important fields, ranging from fundamental plant        nanoprobes into plant cells using silver-coated gold
biology to biofuel development. However, current             nanostars (AuNR@Ag) for SERS sensing. Figure panels
methods to monitor gene expression in plants cannot be       A and B show the transmission electron microscopy
performed directly in vivo. To overcome these limita-        (TEM) image of AuNR@Ag (A) and the SERS detec-
tions, the project has developed in vivo imaging and         tion of microRNAs (miRNAs) (B) using AuNR@Ag-iMS
biosensing of nucleic acid biotargets using plasmonic        nanoprobes. Also shown is the confocal imaging coregis-
nanoprobes referred to as inverse molecular sentinels        tration of iMS nanoprobes inside tobacco cells (C).

                                                                            (D)

  (A) TEM image of silver-coated gold nanostars (AuNR@Ag). (B) SERS spectra of AuNR@Ag-iMS in the presence
  (bottom spectrum) or absence (top spectrum) of target miRNAs. (C) Representative confocal microscopy images
  of Cy3-labeled AuNR@Ag-iMS infiltrated into tobacco plants expressing GFP fluorophore in the cytoplasm.
  (D) Overlay of gold (Au; green), zinc (Zn; blue), and manganese (Mn; red) distributions in XRF confocal image of
  200 μ × 200 μ area of leaf treated with AuNR@Ag. White lines drawn as an aid to identify four leaf cells within the
  field of view. Dashed white lines drawn to delineate where the confocal plane of the image transitions from inside
  to outside of the cell. Courtesy Tuan Vo Dinh, Duke University; and Ken Kemner, Argonne National Laboratory.

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