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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Atomic Force Microscope Active Optical Probe for Single-Molecule Imaging and Time-Resolved Optical Spectroscopy (DOE SBIR Phase II/IIA Final Report)

Actoprobe LLC reports on the results of its DOE SBIR Phase II/IIA project on the development of Atomic Force Microscope Active Optical Probe for Single-Molecule Imaging and Time-Resolved Optical Spectroscopy. While chemistry science and technology greatly benefit from Atomic Force Microscopy in surface characterization, time-resolved chemical imaging on the single-molecule level lags far behind. Current scanning probe microscopy only obtains information about mechanical but not optical/chemical properties. To address this problem, the Actoprobe LLC research team has proposed a novel class of Atomic Force Microscopy probes, Ultra-Fast Pulsed Active Atomic Force Microscopy Optical Probes (UFP AAOPs), that will allow ultrafast time-resolved optical and chemical imaging at the nanoscale. As envisioned, these unique optical probes will perform the functions of conventional Atomic Force Microscopy probes and, in addition, will simultaneously provide chemical information about molecular scale interactions. This innovation is accomplished by integrating an ultrafast pulsed Quantum Dot laser source into an Atomic Force Microscopy probe. This report describes our progress with the fabrication of an ultrafast micrometer-size semiconductor laser, based on “artificial atoms” - Quantum Dots, integrated with an Atomic Force Microscopy probe. In this Phase II/IIA project, we have demonstrated the feasibility of the UFP AAOP concept by fabricating a first prototype of the UFP AAOP. The excellent performance of the probe has been proven in terms of AFM and optical spatial resolution through rigorous tests. The UFP AAOP provides pulses with less than 4 ps duration and higher than 11 GHz repetition rate, and spatial resolution better than 300 nm at 1240 nm wavelength. Technically, it is possible to reduce the pulse width to less than 1 ps and to improve lateral resolution to ~ 0.5 nm, which implies the potential capability for the probe to characterize chemical compounds with single-molecule resolution. The UFP AAOP fabrication procedure has been developed for wafer-scale production of multiple devices, with the yield of the process estimated to be lower than 1% with the limited fabrication capabilities and equipment available for use in the research project. However, using high-volume production tools and special GaAs processing equipment, the yield can be significantly improved, theoretically to ~ 50%. Finally, economic feasibility and scale-up manufacturing potential were analyzed for UFP AAOP and found to be very promising. In summary, the Actoprobe team has successfully demonstrated the feasibility of the UFP AAOP concept.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Single-molecule imaging of lignocellulose deconstruction by SCATTIRSTORM microscopy

The goal of this project is to build a multimodal optical microscope to measure the binding, processive degradation, and pausing behaviors of cellulases as they interact with and degrade both synthetic and naturally occurring lignocellulosic walls. To achieve this, we will use high spatio-temporal single-molecule imaging to track cellulases, while visualizing specific molecular components of cellulose, lignin and hemicellulose, that make up their lignocellulose substrate. The microscope will combine Interferometric Scattering (iSCAT), which provides unprecedented spatiotemporal resolution; Total Internal Reflection Fluorescence (TIRF), which provides single-molecule resolution of multiple fluorophore-labeled molecules; and Stochastic Reconstruction (STORM), which allows for three-dimensional super-resolution imaging of intact plant cell walls during degradation. Initial studies will investigate cellulase dynamics on in vitro-assembled cell wall analogs, and later work will progress to using native plant cell walls.

09 BIOMASS FUELS↗
DOE OSTI2024

LITE-SM: A Light Sheet Illuminator Compatible with Super Resolution and Single Molecule Imaging

The LITE-SM (hereinafter referred to as the Tilt-SM) is a light sheet illumination system purpose-built for single molecule (SM) imaging. SM imaging is a fluorescence imaging technique where individual molecules can be tracked in living cells/tissues in real time allowing for unprecedented insights into the intracellular dynamics of these molecules. The innovative all-mirror optical design greatly reduces/eliminates the aberrations that plague lens-based designs. This iteration of the Tilt-SM has significantly better optical performance then its predecessor with over 10x increase in optical power at the sample, allowing for the use of much smaller and more cost-effective lasers then the original Phase 1 system. Excellent STORM imaging as well as SM tracking has been achieved, satisfying the key objectives of this study.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms

Single-molecule superresolution microscopy is a powerful tool for the study of biological structures on size scales smaller than the optical diffraction limit. Imaging samples at cryogenic temperatures (77 K) reduces the quantum yield of photobleaching for many fluorescent labels, yielding localization precisions below 10 nm. Cryogenic imaging further enables correlation with cryogenic electron tomography. A key limitation in applying methods such as PALM and STORM to samples maintained at 77 K is the limited number of fluorophores known to undergo efficient turn-on and turn-off mechanisms necessary to control the sparsity of active emitters. Here we find that mApple, a red-emitting fluorescent protein, undergoes a novel turn-off mechanism in response to simultaneous illumination with two colors of light. This turn-off mechanism enables localization of many individual molecules in initially bright samples, but the final density of localizable emitters is limited by relatively inefficient turn-on (photoactivation). Bulk excitation and emission spectroscopy shows that mApple has access to two distinct emissive states as well as dark states accessible optically or through changes in pH. The bright and stable emission of mApple enables widefield collection of single-molecule emission spectra, which highlight the complex nature and environmental sensitivity of states observed in red fluorescent proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

In planta single-molecule imaging and holographic force spectroscopy to study real-time, multimodal turnover dynamics of polysaccharides and associated carbohydrate metabolites

Cellular production of polysaccharides from simple sugar molecules serves critical roles in a variety of metabolic processes essential to the survival of every living organism. In the context of photosynthetically fixed carbon, cell wall polysaccharides synthesis is particularly of interest for increased biomass accumulation for human food, animal feed, and bioenergy related applications. Despite a long history of research in plant cell biology, our understanding of in planta cell wall biosynthesis and its regulation is far from complete due to lack of a comprehensive microscopy toolkit that encompass the multiple spatial and temporal scales in which cell wall polysaccharides fibrils are synthesized and assembled into intricate cell wall networks. In this project, a multidisciplinary team of scientists from Rutgers University, Vanderbilt University, and Oak Ridge National Laboratory carried out innovative multimodal, multiscale microscopy studies of cell wall synthesis using single-molecule force-spectroscopy, super-resolution fluorescence microscopy, and in vivo live cell imaging. Our novel multimodal and holistic imaging approach revealed plant cell wall polysaccharides synthesis processes in unprecedented detail across multiple spatiotemporal scales, from a single-molecule to a single-cell. Besides, the team has made technical and scientific innovations across multiple research fields—microscopy, bioengineering, single-molecule biophysics, and plant biology, etc.—to accomplish the goals. The results from this project will greatly advance the mechanistic and holistic understanding of in planta cell wall synthesis, which will accelerate the development of better transgenic crops for bioenergy applications. Moreover, the new toolbox, combining powerful advanced microscopy assays with cell/protein engineering, will have broader impacts on molecular and cellular biology fields by paving the way for studying cellular processes occurring across multiple physical scales with multimodal microscopy methodologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Ultra-Fast Electron Microscopic Imaging of Single Molecules With a Direct Electron Detection Camera and Noise Reduction

Time-resolved imaging of molecules and materials made of light elements is an emerging field of transmission electron microscopy (TEM), and the recent development of direct electron detection cameras, capable of taking as many as 1,600 fps, has potentially broadened the scope of the time-resolved TEM imaging in chemistry and nanotechnology. However, such a high frame rate reduces electron dose per frame, lowers the signal-to-noise ratio (SNR), and renders the molecular images practically invisible. Here, we examined image noise reduction to take the best advantage of fast cameras and concluded that the Chambolle total variation denoising algorithm is the method of choice, as illustrated for imaging of a molecule in the 1D hollow space of a carbon nanotube with ~1 ms time resolution. Through the systematic comparison of the performance of multiple denoising algorithms, we found that the Chambolle algorithm improves the SNR by more than an order of magnitude when applied to TEM images taken at a low electron dose as required for imaging at around 1,000 fps. Open-source code and a standalone application to apply Chambolle denoising to TEM images and video frames are available for download.

47 OTHER INSTRUMENTATION↗
DOE OSTI2020

Exploring Plasmonic Photocatalysis via Single-Molecule Reaction Imaging

Plasmonic photocatalysis has emerged as a new frontier in heterogeneous catalysis due to its promise in harvesting light to drive reactions. Yet many mechanistic aspects remain to be unambiguously defined. Using single-molecule fluorescence imaging, Li et al. studied a fluorogenic and plasmon-enhanced reaction, amplex red oxidation, on single Au nanorods at subturnover resolution and under operando conditions. Both the rate-determining step and its activation energy were identified from the multiple elemental reactions. Here, the results provide insights into the mechanism of plasmonic photocatalysis that may help the rational design of heterogeneous catalysts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Early Career Award: Single Molecule Fluorescence Imaging for a Background-Free Neutrinoless Double Beta Decay Search

This project aimed to develop a suite of novel technologies targeted at detecting individual barium ions produced in neutrinoless double beta decays of 136 Xe, as part of the NEXT program of gaseous xenon detectors. We realized novel molecules which could sense Ba 2+ ions in dry conditions; new microscopy techniques operable in dry noble gases, including the required operating conditions of xenon at 10 bar pressure; and new radiofrequency ion transport methods (developed both theoretically and experimentally) that can transport ions in dense xenon gas. Together, these developments represent a major advance toward a background-free neutrinoless double beta decay experiment based on single molecule fluorescence imaging of Ba 2+ daughter ions.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2022

Super‐Resolution Lifetime Imaging of Single Molecules Near Gold Bowtie Nanoparticles

Interactions between light and matter serve as the basis of many technologies, but the quality of these devices is inherently limited by the optical properties of their constituents. Plasmonic nanoparticles are a highly versatile and tunable platform for the enhancement of such optical properties. However, the near-field nature of these effects has made thorough study and understanding of these mechanisms difficult. In this work, we introduce a fully confocal technique combining photoswitching super-resolution microscopy with fluorescence lifetime imaging microscopy to study single-molecule decay rate enhancement. We demonstrate that the technique combines a spatial resolution better than 20 nm, and a 16 ps temporal resolution. Simultaneously, an autocorrelation measurement is also performed to confirm that the data indeed originates from single molecules. This work provides insight into the various mechanisms of plasmon-enhanced emission, and allows the study of the correlation between emission intensity and lifetime enhancement. This complicated relationship is shown to be dependent upon the relative influence of various radiative and nonradiative decay pathways. Here, we provide a platform for further study of emission mislocalization, the position-dependent prominence of different decay pathways, and the direct super-resolved measurement of the local density of states.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Does Selectivity of Molecular Catalysts Change with Time? Polymerization Imaged by Single‐Molecule Spectroscopy

Abstract The chemoselectivity of molecular catalysts underpins much of modern synthetic organic chemistry. However, little is known about the selectivity of individual catalysts because this single‐catalyst‐level behavior is hidden by the bulk catalytic behavior. Here, for the first time, the selectivity of individual molecular catalysts for two different reactions is imaged in real time at the single‐catalyst level. This imaging is achieved through fluorescence microscopy paired with spectral probes that produce a snapshot of the instantaneous chemoselectivity of a single catalyst for either a single‐chain‐elongation or a single‐chain‐termination event during ruthenium‐catalyzed polymerization. Superresolution imaging of multiple selectivity events, each at a different single‐molecular ruthenium catalyst, indicates that catalyst selectivity may be unexpectedly spatially and time‐variable.

Garcia, IV, Antonio↗
DOE OSTI2022

X-ray multiphoton-induced Coulomb explosion images complex single molecules

Following structural dynamics in real time is a fundamental goal towards a better understanding of chemical reactions. Recording snapshots of individual molecules with ultrashort exposure times is a key ingredient towards this goal, as atoms move on femtosecond (10 -15 s) timescales. For condensed-phase samples, ultrafast, atomically resolved structure determination has been demonstrated using X-ray and electron diffraction. Pioneering experiments have also started addressing gaseous samples. However, they face the problem of low target densities, low scattering cross sections and random spatial orientation of the molecules. Therefore, obtaining images of entire, isolated molecules capturing all constituents, including hydrogen atoms, remains challenging. Here we demonstrate that intense femtosecond pulses from an X-ray free-electron laser trigger rapid and complete Coulomb explosions of 2-iodopyridine and 2-iodopyrazine molecules. We obtain intriguingly clear momentum images depicting ten or eleven atoms, including all the hydrogens, and thus overcome a so-far impregnable barrier for complete Coulomb explosion imaging—its limitation on molecules consisting of three to five atoms. In combination with state-of-the-art multi-coincidence techniques and elaborate theoretical modelling, this allows tracing ultrafast hydrogen emission and obtaining information on the result of intramolecular electron rearrangement. Our work represents an important step towards imaging femtosecond chemistry via Coulomb explosion.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2022

Single-molecule 3D imaging of HIV cellular entry by liquid-phase electron tomography

Enveloped viruses, including human immunodeficiency virus (HIV) and SARS-CoV-2, target cells through membrane fusion process. The detailed understanding of the process is sought after for vaccine development but remains elusive due to current technique limitations for direct three-dimensional (3D) imaging of an individual virus during its viral entry. Recently, we developed a simple specimen preparation method for real-time imaging of metal dynamic liquid-vaper interface at nanometer resolution by transmission electron microscopy (TEM). Here, we extended this method to study biology sample through snapshot 3D structure of a single HIV (pseudo-typed with the envelope glycoprotein of vesicular stomatitis virus, VSV-G) at its intermediate stage of viral entry to HeLa cells in a liquid-phase environment. By individual-particle electron tomography (IPET), we found the viral surface release excess lipids with unbound viral spike proteins forming ~50-nm nanoparticles instead of merging cell membrane. Moreover, the spherical-shape shell formed by matrix proteins underneath the viral envelope does not disassemble into a cone shape right after fusion. Further, the snapshot 3D imaging of a single virus provides us a direct structure-based understanding of the viral entry mechanism, which can be used to examine other viruses to support the development of vaccines combatting the current ongoing pandemic.

Kong, Lingli↗
DOE OSTI2024

Controlling the Fluctuating Tip-Enhanced Raman Spectra of Chloramben on Silver Nanocubes

Tip-enhanced Raman (TER) scattering from molecules residing at plasmonic junctions can be used to detect, identify, and image single molecules. This is most evident for flat molecules interrogated under extreme conditions of temperatures and pressure. It is also the case for (bio)molecular systems that feature preferred orientations/conformations under ambient laboratory conditions. More complex molecules that can adopt multiple conformations and/or that feature different protonation and/or charge states give rise to complex TER spectra. Here we illustrate how the latter can be controlled in the case of chloramben molecules coated onto plasmonic silver nanocubes. We show that characteristic molecular Raman spectra cannot be obtained when tunneling plasmons are operative, i.e., when the tip is in direct contact with the chemically functionalized plasmonic nanoparticles. We rationalize these observations and propose an approach to less invasive, and hence, more analytical TER spectral imaging.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Sub-10 nm upconversion nanocrystals for long-term single-particle tracking

Lanthanide-doped upconversion nanoparticles are attractive single-molecule imaging probes due to their high photostability and anti-Stokes luminescence. However, achieving both small particle size and strong brightness has remained a major challenge, as reducing size often leads to dimmer emission. Herein, we fabricate a sub-10 nm cascade actively protected upconversion nanoparticles, which shows a 33-fold enhanced upconversion efficiency at the single-particle level compared to larger ~19 nm conventional nanoparticles. Theoretical modeling and time-resolved measurements show that emission loss mainly comes from energy leakage of Er 3+ ions to surface defects. By introducing a NaYbF 4 layer as photon-harvesting and protective intermediate layer, we minimize this energy loss and significantly boost brightness. A monolayer of inert NaLuF 4 can effectively suppress the surface quenching to Yb 3+ . Using these ultra-small bright probes, we successfully tracked single epidermal growth factor receptor molecules on live cells for up to one hour, revealing dynamic switching between different diffusion modes.

Qiu, Xiaochen [Fudan Univ., Shanghai (China); Shen↗
DOE OSTI2022

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

59 BASIC BIOLOGICAL SCIENCES↗