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The author has identified the following significant results. Detection of short-lived events has continued. Forest fires, oil spills, vegetation damage, volcanoes, storm ridges, earthquakes, and floods have been detected and analyzed.
There are no author-identified significant results in this report.
Disclosed herein are systems, methods, systems and devices for measurement and visualization of chemical dynamics in living cells or tissues for diagnostic pathology. Devices can be open- or closed-channel microfluidic membrane devices for long-term IR spectroscopy of live adherent cells and ultimately for rapidly identifying time-dependent spectral features indicative of chemical abnormality in individual cells.
The search for life in our solar system relies on our ability to measure key biomarkers in situ and identify them as likely of biotic origin. One class of biomarkers that would be indicative of life is cell-like features such as the colocalization of key biochemicals, spatial organization of native UV fluorescent compounds, and polarization across membrane compartments. The Europa Lander Science Definition Team has emphasized the need for a light microscope to search for these features on Europa, and the Europa Light Microscope (ELM) has been developed to address this need. This work presents the development towards high launch readiness of ELM by demonstrating its optical resolution under brightfield illumination, deep UV fluorescence imaging of unstained living samples, and multicolor imaging of samples stained with exogenous fluorophores targeting proteins, lipids, and nucleic acids. This work also seeks to extend the capabilities of ELM by incorporating fluorescence-based assays for membrane potential as a key biomarker for living cellular material.
The methods utilized to perform an infrared thermography inspection of the bondline of structural reinforcing caps on X-33 liquid hydrogen (LH2) tank during the manufacture are described in this presentation. A total of thirty-six caps were bonded to the tank using a wet cobond process with vacuum bagging and low temperature curing. The thermal map of each bondline was acquired through the vacuum bag by applying heat to the cap and then viewing it during cool down. Poorly bonded regions, or voids, were characterized by localized hot spots in the thermal image. These regions were marked on the tank by referring to a live thermal image, then hand massaged in an attempt to remove the entrapped air. The cap was then reinspected and, if it was determined that the entrapped air could not be removed, the cap was removed and the adhesive cleaned away before it cured. Measures were then taken to better match the surface of the cap to the tank and the process repeated. After the bondline had cured sufficiently to allow vacuum bag removal, a "flash heating" thermographic Investigation was performed to verify that the cap had bonded properly to the tank. Any regions that had opened up after the preliminary inspection were marked and filled by drilling small holes in the cap and injecting resin. A final inspection was then performed to ensure the quality of the bondline.
Voyager encounter data are presented in computer animation (CA) and real (R) animation. The highlights include a view of 2 full rotations of Neptune. It shows spacecraft trajectory 'diving' over Neptune and intercepting Triton's orbit, depicting radiation and occulation zones. Also shown are a renegade orbit of Triton and Voyager's encounter with Neptune's Magnetopause. A model of the spacecraft's complex maneuvers during close encounters of Neptune and Triton is presented. A view from Earth of Neptune's occulation experiment is is shown as well as a recreation of Voyager's final pass. There is detail of Voyager's Image Compensation technique which produces Voyager images. Eighteen images were produced on June 22 - 23, 1989, from 57 million miles away. A 68 day sequence which provides a stroboscopic view - colorization approximates what is seen by the human eye. Real time images recorded live from Voyager on 8/24/89 are presented. Photoclinometry produced the topography of Triton. Three images are used to create a sequence of Neptune's rings. The globe of Neptune and 2 views of the south pole are shown as well as Neptune rotating. The rotation of a scooter is frozen in images showing differential motion. There is a view of rotation of the Great Dark Spot about its own axis. Photoclinometry provides a 3-dimensional perspective using a color mosaic of Triton images. The globe is used to indicate the orientation of Neptune's crescent. The east and west plumes on Triton are shown.
Abstract Photoprotection mechanisms are ubiquitous among photosynthetic organisms. The photoprotection capacity of the green alga Chlamydomonas reinhardtii is correlated with protein levels of stress-related light-harvesting complex (LHCSR) proteins, which are strongly induced by high light (HL). However, the dynamic response of overall thylakoid structure during acclimation to growth in HL has not been fully understood. Here, we combined live-cell super-resolution microscopy and analytical membrane subfractionation to investigate macroscale structural changes of thylakoid membranes during HL acclimation in Chlamydomonas . Subdiffraction-resolution live-cell imaging revealed that the overall thylakoid structures became thinned and shrunken during HL acclimation. The stromal space around the pyrenoid also became enlarged. Analytical density-dependent membrane fractionation indicated that the structural changes were partly a consequence of membrane unstacking. The analysis of both an LHCSR loss-of-function mutant , npq4 lhcsr1 , and a regulatory mutant that over-expresses LHCSR, spa1-1 , showed that structural changes occurred independently of LHCSR protein levels, demonstrating that LHCSR was neither necessary nor sufficient to induce the thylakoid structural changes associated with HL acclimation. In contrast, stt7-9 , a mutant lacking a kinase of major light-harvesting antenna proteins, had a slower thylakoid structural response to HL relative to all other lines tested but still showed membrane unstacking. These results indicate that neither LHCSR- nor antenna-phosphorylation-dependent HL acclimation are required for the observed macroscale structural changes of thylakoid membranes in HL conditions.
High resolution B-mode ultrasound images of the common carotid artery are obtained with an ultrasound transducer using a standardized methodology. Subjects are supine with the head counter-rotated 45 degrees using a head pillow. The jugular vein and carotid artery are located and positioned in a vertical stacked orientation. The transducer is rotated 90 degrees around the centerline of the transverse image of the stacked structure to obtain a longitudinal image while maintaining the vessels in a stacked position. A computerized methodology assists operators to accurately replicate images obtained over several spaced-apart examinations. The methodology utilizes a split-screen display in which the arterial ultrasound image from an earlier examination is displayed on one side of the screen while a real-time live ultrasound image from a current examination is displayed next to the earlier image on the opposite side of the screen. By viewing both images, whether simultaneously or alternately, while manually adjusting the ultrasound transducer, an operator is able to bring into view the real-time image that best matches a selected image from the earlier ultrasound examination. Utilizing this methodology, measurement of vascular dimensions such as carotid arterial IMT and diameter, the coefficient of variation is substantially reduced to values approximating from about 1.0% to about 1.25%. All images contain anatomical landmarks for reproducing probe angulation, including visualization of the carotid bulb, stacking of the jugular vein above the carotid artery, and initial instrumentation settings, used at a baseline measurement are maintained during all follow-up examinations.
A standardized acquisition methodology assists operators to accurately replicate high resolution B-mode ultrasound images obtained over several spaced-apart examinations utilizing a split-screen display in which the arterial ultrasound image from an earlier examination is displayed on one side of the screen while a real-time "live" ultrasound image from a current examination is displayed next to the earlier image on the opposite side of the screen. By viewing both images, whether simultaneously or alternately, while manually adjusting the ultrasound transducer, an operator is able to bring into view the real-time image that best matches a selected image from the earlier ultrasound examination. Utilizing this methodology, dynamic material properties of arterial structures, such as IMT and diameter, are measured in a standard region over successive image frames. Each frame of the sequence has its echo edge boundaries automatically determined by using the immediately prior frame's true echo edge coordinates as initial boundary conditions. Computerized echo edge recognition and tracking over multiple successive image frames enhances measurement of arterial diameter and IMT and allows for improved vascular dimension measurements, including vascular stiffness and IMT determinations.
Legionella is a genus of ubiquitous environmental pathogens found in freshwater systems, moist soil, and composted materials. More than four decades of Legionella research has provided important insights into Legionella pathogenesis. Although standard commercial microscopes have led to significant advances in understanding Legionella pathogenesis, great potential exists in the deployment of more advanced imaging techniques to provide additional insights. The lattice light sheet microscope (LLSM) is a recently developed microscope for 4D live cell imaging with high resolution and minimum photo-damage. We built a LLSM with an improved version for the optical layout with two path-stretching mirror sets and a novel reconfigurable galvanometer scanner ( RGS ) module to improve the reproducibility and reliability of the alignment and maintenance of the LLSM. We commissioned this LLSM to study Legionella pneumophila infection with a tailored workflow designed over instrumentation, experiments, and data processing methods. Our results indicate that Legionella pneumophila infection is correlated with a series of morphological signatures such as smoothness, migration pattern and polarity both statistically and dynamically. Our work demonstrates the benefits of using LLSM for studying long-term questions in bacterial infection. Our free-for-use modifications and workflow designs on the use of LLSM system contributes to the adoption and promotion of the state-of-the-art LLSM technology for both academic and commercial applications.
The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
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.
The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials, bioimaging, nanomedicine, and other protein-based technologies. Here, in this study, we describe lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate side chains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at preassociated amine-acid pairs. Indiscriminate cross-linking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain and that introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Abs, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with the potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.
Sucrose is the main saccharide used for longdistance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small ( 50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we report that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ~20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.
A multispectral scanner has been adapted to capture spectral images of living plants under various types of illumination for purposes of monitoring the health of, or monitoring the transfer of genes into, the plants. In a health-monitoring application, the plants are illuminated with full-spectrum visible and near infrared light and the scanner is used to acquire a reflected-light spectral signature known to be indicative of the health of the plants. In a gene-transfer- monitoring application, the plants are illuminated with blue or ultraviolet light and the scanner is used to capture fluorescence images from a green fluorescent protein (GFP) that is expressed as result of the gene transfer. The choice of wavelength of the illumination and the wavelength of the fluorescence to be monitored depends on the specific GFP.
Root biology is pivotal in addressing global challenges including sustainable agriculture and climate change. However, roots have been relatively understudied among plant organs, partly due to the difficulties in imaging root structures in their natural environment. Here we used microfabricated ecosystems (EcoFABs) to establish growing environments with optical access and employed nonlinear multimodal microscopy of third-harmonic generation (THG) and three-photon fluorescence (3PF) to achieve label-free, in situ imaging of live roots and microbes at high spatiotemporal resolution. THG enabled us to observe key plant root structures including the vasculature, Casparian strips, dividing meristematic cells, and root cap cells, as well as subcellular features including nuclear envelopes, nucleoli, starch granules, and putative stress granules. THG from the cell walls of bacteria and fungi also provides label-free contrast for visualizing these microbes in the root rhizosphere. With simultaneously recorded 3PF signal, we demonstrated our ability to investigate root-microbe interactions by achieving single-bacterium tracking and subcellular imaging of fungal spores and hyphae in the rhizosphere.
Autonomous experimentation is an emerging area of research, primarily related to autonomous vehicles, scientific combinatorial discovery approaches in materials science and drug discovery, and iterative research loops of planning, experimentation, and analysis. However, autonomous approaches developed in these contexts are difficult to apply to high-dimensional mapping technologies, such as scanning hyperspectral imaging of biological systems, due to sample complexity and heterogeneity. We briefly cover the history of adaptive sampling algorithms and surrogate modeling in order to define autonomous adaptive data acquisition as an objective-based, flexible building block for future biological imaging experimentation driven by intelligent infrastructure. We subsequently summarize the recent implementations of autonomous adaptive data acquisition (AADA) for scanning hyperspectral imaging, assess how these address the difficulties of autonomous approaches in hyperspectral imaging, and highlight the AADA design variation from a goal-oriented perspective. Finally, we present a modular AADA architecture that embeds AADA-driven flexible building blocks to address the challenge of time resolution for high-dimensional scanning hyperspectral imaging of nonequilibrium dynamical systems. In our example research-driven experimental design case, we propose an AADA infrastructure for time-resolved, noninvasive, and label-free scanning hyperspectral imaging of living biological systems. This AADA infrastructure can accurately target the correct state of the system for experimental workflows that utilize subsequent expensive, high-information-content analytical techniques.