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At least 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

Early detection of pathogens using nucleic acids in clinical samples often requires sensitivity at the single-copy level, which currently necessitates time-consuming and expensive nucleic acid amplification. Here, we describe 1) a redesigned flow cell in the shape of a trapezoid-subtracted geometric stadium, and 2) modified experimental procedures that allow for the measurement of sub-attomolar analytes in microliter quantities on a fluorescence-based waveguide biosensor. We verified our instrumental sensitivity with a 200-μL sample of a fluorescent streptavidin conjugate at 100 zM (100 zeptomolar, or 100·10 −21 mol L −1 ) and theoretically explored the applicability of this modified sensing platform in a sandwich immunoassay format using a Langmuir adsorption model. We present assays that demonstrate specific detection of synthetic influenza A DNA (in buffer) and RNA (in saliva) oligonucleotides at the single-copy level (200 μL at 10 zM) using a fluorescent molecular beacon. Lastly, we demonstrate detection of isolated genomic influenza A RNA at a clinically relevant concentration. This work constitutes a sensitivity improvement of over twelve orders of magnitude compared to our previous nucleic acid detection work, illustrating the significant enhancements that can be gained with optimized experimental design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

X-ray Fluorescence Microscopy to Develop Elemental Classifiers and Investigate Elemental Signatures in BALB/c Mouse Intestine a Week after Exposure to 8 Gy of Gamma Rays

Iron redistribution in the intestine after total body irradiation is an established phenomenon. However, in the literature, there are no reports about the use of X-ray fluorescence microscopy or equivalent techniques to generate semi-quantitative 2D maps of iron in sectioned intestine samples from irradiated mice. In this work, we used X-ray fluorescence microscopy (XFM) to map the elemental content of iron as well as phosphorus, sulfur, calcium, copper and zinc in tissue sections of the small intestine from eight-week-old BALB/c male mice that developed gastrointestinal acute radiation syndrome (GI-ARS) in response to exposure to 8 Gray of gamma rays. Seven days after irradiation, we found that the majority of the iron is localized as hot spots in the intercellular regions of the area surrounding crypts and stretching between the outer perimeter of the intestine and the surface cell layer of villi. In addition, this study represents our current efforts to develop elemental cell classifiers that could be used for the automated generation of regions of interest for analyses of X-ray fluorescence maps. Once developed, such a tool will be instrumental for studies of effects of radiation and other toxicants on the elemental content in cells and tissues. While XFM studies cannot be conducted on living organisms, it is possible to envision future scenarios where XFM imaging of single cells sloughed from the human (or rodent) intestine could be used to follow up on the progression of GI-ARS.

X-ray fluorescence microscopy↗
NASA NTRS1993

Optical spectroscopy of a highly fluorescent aggregate of bacteriochlorophyll c

Bacteriochlorophyll (BChl) c and a similar model compound, Mg-methyl bacteriopheophorbide d, form several types of aggregates in nonpolar solvents. One of these aggregates is highly fluorescent, with a quantum yield higher than that of the monomer. This aggregate is also unusual in that it shows a rise time in its fluorescence emission decay at certain wavelengths, which is ascribed to a change in conformation of the aggregate. An analysis of fluorescence depolarization data is consistent with either a linear aggregate of four or five monomers or preferably a cyclic arrangement of three dimers.

NASA Discipline Exobiology↗
NASA NTRS1993

Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures

Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.

NASA Discipline Number 52-30↗
NASA NTRS2000

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

Evaluation Studies↗
NASA NTRS2019

Remote Sensing of Solar-Induced Chlorophyll Fluorescence (SIF) in Vegetation: 50 Years of Progress

Remote sensing of solar-induced chlorophyll fluorescence (SIF) is a rapidly advancing front in terrestrial vegetation science, with emerging capability in space-based methodologies and diverse application prospects. Although remote sensing of SIF – especially from space – is seen as a contemporary new specialty for terrestrial plants, it is founded upon a multi-decadal history of research, applications, and sensor developments in active and passive sensing of chlorophyll fluorescence. Current technical capabilities allow SIF to be measured across a range of biological, spatial, and temporal scales. As an optical signal, SIF may be assessed remotely using highly-resolved spectral sensors and state-of-the-art algorithms to distinguish the emission from reflected and/or scattered ambient light. Because the red to far-red SIF emission is detectable non-invasively, it may be sampled repeatedly to acquire spatio-temporally explicit information about photosynthetic light responses and steady-state behaviour in vegetation. Progress in this field is accelerating with innovative sensor developments, retrieval methods, and modelling advances. This review distills the historical and current developments spanning the last several decades. It highlights SIF heritage and complementarity within the broader field of fluorescence science, the maturation of physiological and radiative transfer modelling, SIF signal retrieval strategies, techniques for field and airborne sensing, advances in satellite-based systems, and applications of these capabilities in evaluation of photosynthesis and stress effects. Progress, challenges, and future directions are considered for this unique avenue of remote sensing.

Review↗
DOE OSTI2019

Estimation of Sorption Behavior of Europium onto Ca-Al-Si Hydrate by Fluorescence Analysis - 19222

As a nuclide migration evaluation in the radioactive waste disposal system, the sorption behaviors of Eu, which is a multivalent metal ion, onto Ca-Al-Si hydrates (including Ca-Si hydrates) were examined in this study. In the sorption experiments, Ca-Al-Si hydrates were handled without drying process in consideration of saturated groundwater environment. The sorption behaviors of Eu onto Ca-Al-Si hydrate with various combinations of Ca/Si molar ratio and Al/Si molar ratio were monitored by using the Ca, Al, Si and Eu concentrations, the fluorescence emission spectrum and the fluorescence lifetime. In addition, the structural changes of Ca-Al-Si hydrates by Eu were evaluated by Raman spectrum measurement. In the results, the fluorescence lifetimes of most samples remarkably exceeded that of Eu(OH){sub 3}. This suggests that Eu ions are immobilized by not only Ca-Si hydrates but also Ca-Al-Si hydrates. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2011

The MicroAnalysis Toolkit: X-ray Fluorescence Image Processing Software

The MicroAnalysis Toolkit is an analysis suite designed for the processing of x-ray fluorescence microprobe data. The program contains a wide variety of analysis tools, including image maps, correlation plots, simple image math, image filtering, multiple energy image fitting, semi-quantitative elemental analysis, x-ray fluorescence spectrum analysis, principle component analysis, and tomographic reconstructions. To be as widely useful as possible, data formats from many synchrotron sources can be read by the program with more formats available by request. An overview of the most common features will be presented.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Characterization of a Pixelated Cadmium Telluride Detector System Using a Polychromatic X-Ray Source and Gold Nanoparticle-Loaded Phantoms for Benchtop X-Ray Fluorescence Imaging

In this paper, the imaging dose and scan time have been considered as the two major constraints for routine benchtop x-ray fluorescence computed tomography (XFCT) imaging. One way to address this issue is to acquire x-ray fluorescence (XRF) signals in parallel through a 2D array of single-crystal detectors or a pixelated detector along with the cone-beam x-ray source. To identify a detector system suitable for this purpose, a commercially available, fully spectroscopic cadmium telluride (CdTe) pixelated detector, HEXITEC (High-Energy X-ray Imaging Technology), was tested under the experimental conditions optimized for benchtop XFCT imaging of gold nanoparticles (GNPs). Specifically, two different parallel-hole stainless steel collimators were fabricated and coupled with the detector for seamless integration into our existing benchtop cone-beam XFCT system. After the detector deployment, this benchtop XFCT system was used to detect XRF photons from GNP-loaded phantoms. A pixel-merging algorithm was introduced to enhance the sensitivity of XRF photon detection thereby minimizing the scan time. The effect of pixel-level charge sharing correction algorithms was investigated within the context of benchtop XFCT imaging. The detector energy resolution, in terms of the full width at half maximum (FWHM) values at different gold K-shell XRF energies, was also determined. Of the two charge sharing correction algorithms examined, the charge sharing addition gave better sensitivity than the charge sharing discrimination (csd). On the other hand, under the current experimental conditions, the energy resolution of the HEXITEC detector was the best with the csd and estimated to be 1.56 keV FWHM at 66-69 keV photon energy. Overall, despite some degradation of the detector energy resolution (compared with typical single crystal CdTe detectors), the HEXITEC detector enabled parallel data acquisition under the experimental conditions typical of benchtop XFCT imaging and operated well within our benchtop XFCT setup.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2023

Overcoming Barriers Associated with Oral Delivery of Differently Sized Fluorescent Core‐Shell Silica Nanoparticles

Oral delivery, while a highly desirable form of nanoparticle-drug administration, is limited by challenges associated with overcoming several biological barriers. Here, in this study, the authors study how fluorescent and poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi-total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco-2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, [C’ dots]) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C’ dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C’ dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.

36 MATERIALS SCIENCE↗
DOE OSTI2016

Boron Difluoride Curcuminoid Fluorophores with Enhanced Two-Photon Excited Fluorescence Emission and Versatile Living-Cell Imaging Properties

The synthesis of boron difluoride complexes of a series of curcuminoid derivatives containing various donor end groups is described in this paper. Time-dependent (TD)-DFT calculations confirm the charge-transfer character of the second lowest-energy transition band and ascribe the lowest energy band to a “cyanine-like” transition. Photophysical studies reveal that tuning the donor strength of the end groups allows covering a broad spectral range, from the visible to the NIR region, of the UV–visible absorption and fluorescence spectra. Two-photon-excited fluorescence and Z-scan techniques prove that an increase in the donor strength or in the rigidity of the backbone results in a considerable increase in the two-photon cross section, reaching 5000 GM, with predominant two-photon absorption from the S 0 –S 2 charge-transfer transition. Direct comparisons with the hemicurcuminoid derivatives show that the two-photon active band for the curcuminoid derivatives has the same intramolecular charge-transfer character and therefore arises from a dipolar structure. Overall, this structure–relationship study allows the optimization of the two-photon brightness (i.e., 400–900 GM) with one dye that emits in the NIR region of the spectrum. Finally, in addition, these dyes demonstrate high intracellular uptake efficiency in Cos7 cells with emission in the visible region, which is further improved by using porous silica nanoparticles as dye vehicles for the imaging of two mammalian carcinoma cells type based on NIR fluorescence emission.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering

In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing

Abstract Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca 2+ ) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca 2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca 2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH 2 O) and a pneumatic high-speed pressure clamp (− 25 to 0 mmHg). To visualize Ca 2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca 2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Wang, Haoqing↗
DOE OSTI2017

Characterization of the external and internal flow structure of an aerated-liquid injector using X-ray radiography and fluorescence

In the present study, the internal flowfield of aerated-liquid fuel injectors is examined through x-ray radiography and x-ray fluorescence. An inside-out injector, consisting of a perforated aerating tube within an annular liquid stream, sprays into a quiescent environment at a fixed mass flow rate of water and nitrogen gas. The liquid is doped with bromine (in the form of NaBr) to create an x-ray fluorescence signal. This allows for reasonable absorption and fluorescence signals, and one or both diagnostics can be used to track the liquid distribution. The injector housing is fabricated from beryllium (Be), which allows the internal flowfield to be examined (as Be has relatively low x-ray attenuation coefficient). Two injector geometries are compared, illustrating the effects of aerating orifice size and location on the flow evolution. Time-averaged equivalent pathlength (EPL) and line-of-sight averaged density ρ(y) reveal the formation of the two-phase mixture, showing that the liquid film thickness along the injector walls is a function of the aerating tube geometry, though only upstream of the nozzle. These differences in gas and liquid distribution (between injectors with different aerating tube designs) are suppressed as the mixture traverses the nozzle contraction. The averaged liquid velocity (computed from the density and liquid mass flow rate) reveal a similar trend. This suggests that at least for the current configurations, the plume width, liquid mass distribution, and averaged liquid velocity for the time-averaged external spray are insensitive to the aerating tube geometry.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2022

Observation of Long-Lived UV-Induced Fluorescence from Environmental Materials Using the HVeV Detector as Developed for SuperCDMS

Here, we describe recent experiments using a SuperCDMS HVeV single-charge sensitive detector illuminated with an ultraviolet LED (275 nm) and a monochromatic laser(650 nm) using a dual fiber optic system installed in a small dilution refrigerator at Stanford University. We observed a population of fluorescence background events after UV exposure but not after exposure to the laser source. The fluorescence was likely due to scattered UV photons absorbed outside the detector. We discuss the possibility of fluorescence being a contributor to the low energy excess background observed in above-ground Dark Matter experiments.

36 MATERIALS SCIENCE↗
DOE OSTI2018

Assessment of the flagellar redox potential in Chlamydomonas reinhardtii using a redox-sensitive fluorescent protein, Oba-Qc

Highlights: • In vivo flagellar redox potential of Chlamydomonas reinhardtii was assessed. • Non-ratiometric redox biosensor was induced into flagella at the constant amount. • The standard curve could be drawn from the in vitro experiments. • Fluorescence of live cells was compared with the standard curve for quantification. • The in vivo redox potential changes depending on the light conditions for culture. Cellular reducing-oxidizing (redox) potential is mainly determined by the concentration ratio between reduced and oxidized glutathiones. It is normally kept at a moderately reduced state but affected to some extent by metabolic activities such as respiration and/or photosynthesis. Changes in redox potential induce many cellular activities collectively called redox responses. For an understanding of the dynamics of the cellular redox responses, redox potential must be accurately assessed in vivo. In this study, we developed a method to measure the in vivo redox potential in the green alga Chlamydomonas reinhardtii, using Oba-Qc, a recently developed redox-monitoring protein. Taking advantage of the periodic flagellar assembly, we introduced Oba-Qc molecules into the flagella at a constant density. Fluorescence signals from flagella in live cells, calibrated against the fluorescence from the samples in buffers of known redox potentials, determined the redox potential to be ∼-250 mV in the light and ∼-280 mV in the dark. Introduction of a sensor protein fused with a structural protein that assembles at a constant density will be also applicable for measurements of various kinds cellular signals in flagella.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

2D-imaging of sampling-probe perturbations in laminar premixed flames using Kr X-ray fluorescence

The perturbation of the temperature field caused by a quartz sampling probe has been investigated in a fuel-rich low-pressure premixed ethylene/oxygen/argon/krypton flame using X-ray fluorescence. The experiments were performed at the 7-BM beamline at the Advanced Photon Source (APS) at the Argonne National Laboratory where a continuous beam of X-rays at 15 keV was used to excite krypton atoms that were added to the unburnt flame gases in a concentration of 5% (by volume). The resulting krypton X-ray fluorescence at 12.65 keV was collected and the spatially resolved signal was subsequently converted into the local temperature of the imaged spot. One and two dimensional scans of the temperature field were obtained by translating the entire flame chamber through a pre-programmed sequence of positions on high precision translation stages and measuring the X-ray fluorescence at each location. Multiple measurements were performed at various separations between the burner surface and probe tip, representing sampling positions from the preheat, reaction, and postflame zones of the low-pressure flame. Distortions of up to 1000 K of the burner-probe centerline flame temperature were found with the tip of the probe in the preheat zone and distortions of up to 500 K were observed with it in the reaction and postflame zones. Furthermore, perturbations of the temperature field have been revealed that radially reach as far as 20 mm from the burner-probe centerline and about 3 mm in front of the probe tip. Finally, these results clearly reveal the limitations of one-dimensional models for predicting flame-sampling experiments and comments are made with regard to model developments and validations based on quantitative speciation data from low-pressure flames obtained via intrusive sampling techniques.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy

Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50 mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

62 RADIOLOGY AND NUCLEAR MEDICINE↗