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DOE OSTI2025

Ultra-high Information-content Chemical Imaging with Broadband Coherent Anti-Stokes Raman and Two-photon Fluorescence Lifetime Microscopy

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence(TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. Furthermore, this protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Xu, Haoyu [Georgia Institute of Technology, Atlant↗

DOE OSTI2020

Seawater pH reconstruction using boron isotopes in multiple planktonic foraminifera species with different depth habitats and their potential to constrain pH and <i>p</i>CO<sub>2</sub> gradients

Boron isotope systematics of planktonic foraminifera from core-top sediments and culture experiments have been studied to investigate the sensitivity ofδ11B of calcite tests to seawater pH. However, our knowledge of the relationship between δ11B and pH remains incomplete for many taxa. Thus, to expand the potential scope of application of this proxy, we report δ11B data for seven different species of planktonic foraminifera from sediment core tops. We utilize a method for the measurement of small samples of foraminifera and calculate the δ11B-calcite sensitivity to pH for Globigerinoides ruber, Trilobus sacculifer (sacc or without sacc), Orbulina universa, Pulleniatina obliquiloculata, Neogloboquadrina dutertrei, Globorotalia menardii, and Globorotalia tumida, including for unstudied core tops and species. These taxa have diverse ecological preferences and are from sites that span a range of oceanographic regimes, including some that are in regions of air–sea equilibrium and others that are out of equilibrium with the atmosphere. The sensitivity of δ11Bcarbonate to δ11Bborate (e.g., Δδ11Bcarbonate/Δδ11Bborate) in core tops is consistent with previous studies for T. sacculifer and G. ruber and close to unity for N. dutertrei, O. universa, and combined deep-dwelling species. Deep-dwelling species closely follow the core-top calibration fo rO. universa, which is attributed to respiration-driven microenvironments likely caused by light limitation and/or symbiont–host interactions. Our data support the premise that utilizing boron isotope measurements of multiple species within a sediment core can be utilized to constrain vertical profiles of pH andpCO2 at sites spanning different oceanic regimes, thereby constraining changes in vertical pH gradients and yielding insights into the past behavior of the oceanic carbon pumps.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1971

The identification of archaeological sites by false color infrared aerial photography

The study of color infrared photography of Tehuacan Valley, Mexico was made to determine the applicability of remotely sensed data to archeology. Photography was interpreted without prior knowledge of the area, followed by a field check to determine accuracy of the original interpretations and to evaluate causes of successes and failures. Results indicate that the visibility of sites depends primarily on its environmental situation, and also that the delineation of environments and microenvironments is especially easy with this type of film. Furthermore, the age and size of the sites are not necessarily the deciding factors in their discernment.

Gumerman, G. J.↗

NASA NTRS1971

The long winter model of Martian biology - A speculation.

A temporal microenvironment model is proposed for Martian biology that is based on an estimated mean thickness of nearly 1 km of frost in the Martian north polar cap summer remnant. If vaporized, this frost could yield not only 1 kg per sq cm of atmosphere, but also higher global temperatures through the greenhouse effect and a greatly increased likelihood of liquid water. Vaporization of such cap remnants may occur twice each equinoctial precession, and Martian organisms may now be in cryptobiotic repose awaiting the end of the long precessional winter. The Viking biology experiments might test this hypothesis.

Sagan, C.↗

NASA NTRS1981

Membrane lipids and the origin of life

The current state of knowledge regarding the development of biological systems is briefly reviewed. At a crucial stage concerning the evolution of such systems, the mechanisms leading to more complex structures must have evolved within the confines of a protected microenvironment, similar to those provided by the contemporary cell membranes. The major components found normally in biomembranes are phospholipids. The structure of the biomembrane is examined, and attention is given to questions concerning the availability of the structural components which are necessary in the formation of primitive lipid membranes. Two approaches regarding the study of protomembranes are discussed. The probability of obtaining ether lipids under prebiotic conditions is considered, taking into account the formation of cyclic and acyclic isoprenoids by the irradiation of isoprene with UV.

Oro, J.↗

NASA NTRS1984

Regulation of hematopoiesis in the suspended rat as a model for space flight

A series of studies was completed in which a variety of routine hematological and other parameters were obtained from sequential sampling of control and suspended rats. These data showed that, during suspension, the rats failed to gain weight at the same rate as the controls, ate and drank significantly less, demonstrated a transient increase in peripheral hematocrit and RBC count, a transient decrease in MCH, suppressed reticulocyte counts and a progressive decrease in MCV but no change in RBC shape. Leukocyte counts were variably decreased but no significant changes in platelet numbers were noted. Post-suspension, evidence of anemia was present from a reduced RBC count, hemoglobin, hematocrit, and MCV. A leukocytosis was also noted. Efforts directed to the collection of data aimed at understanding changes in blood volume during suspension are also discussed. As part of these studies the following parameters were investigated; RBC survival, in vitro leukocyte reactivity to PHA, bone marrow and spleen cellularity and morphology, ferrokinetics, and the hematopoietic inductive microenvironment.

Dunn, C. D. R.↗

NASA NTRS1984

The Antarctic cryptoendolithic ecosystem - Relevance to exobiology

Cryptoendolithic microorganisms in the Antarctic desert live inside porous sandstone rocks, protected by a thin rock crust. While the rock surface is abiotic, the microclimate inside the rock is comparatively mild. These organisms may have descended from early, pre-glaciation Antarctic life forms and thus may represent the last outpost of life in a gradually deteriorating environment. Assuming that life once arose on Mars, it is conceivable that, following the loss of water, the last of surviving organisms withdrew to similar insulated microenvironments. Because such microscopic pockets have little connection with the outside environment, their detection may be difficult. The chances that the Viking lander could sample cryptoendolithic microorganisms in the Antarctic desert would be infinitesimal.

Friedmann, E. I.↗

NASA NTRS1985

Evidence that Resorption of Bone by Rat Peritoneal Macrophages Occurs in an Acidic Environment

Skeletal loss in space, like any form of osteoporosis, reflects a relative imbalance of the activities of cells resorbing (degrading) or forming bone. Consequently, prevention of weightlessness induced bone loss may theoretically be accomplished by (1) stimulating bone formation or (2) inhibiting bone resorption. This approach, however, requires fundamental understanding of the mechanisms by which cells form or degrade bone, information not yet at hand. An issue central to bone resorption is the pH at which resorption takes place. The pH dependent spectral shift of a fluorescent dye (fluorescein isothiocyanate) conjugated to bone matrix was used to determine the pH at the resorptive cell bone matrix interface. Devitalized rat bone was used as the substrate, and rat peritoneal macrophages were used as the bone resorbing cells. The results suggest that bone resorption is the result of generation of an acidic microenvironment at the cell matrix junction.

Blair, H. C.↗

NASA NTRS1985

Aerobic sulfur-oxidizing bacteria: Environmental selection and diversification

Sulfur-oxidizing bacteria oxidize reduced inorganic compounds to sulfuric acid. Lithotrophic sulfur oxidizer use the energy obtained from oxidation for microbial growth. Heterotrophic sulfur oxidizers obtain energy from the oxidation of organic compounds. In sulfur-oxidizing mixotrophs energy are derived either from the oxidation of inorganic or organic compounds. Sulfur-oxidizing bacteria are usually located within the sulfide/oxygen interfaces of springs, sediments, soil microenvironments, and the hypolimnion. Colonization of the interface is necessary since sulfide auto-oxidizes and because both oxygen and sulfide are needed for growth. The environmental stresses associated with the colonization of these interfaces resulted in the evolution of morphologically diverse and unique aerobic sulfur oxidizers.

Caldwell, D.↗

NASA NTRS1985

Microbial colonization and growth on metal sulfides and other mineral surfaces

To determine whether a bacterial film forms on sulfur minerals in situ, various sulfur containing and other minerals were incubated in Penitencia Creek. The rate of cell growth and attachment within the surface microenvironment of mineral surfaces was also determined. To determine whether surfaces enriched with soluble sulfur substrates (cysteine, glutathione, thioglycolate, sulfite, and thiosulfate) increased the rate of growth or attachment of natural communities, membrane enrichments were incubated. These rates were determined as described by Caldwell et al. (1981, 1983). The growth of Pseudomonas fluorescens, a heterotrophic sulfur oxidizer, was studied in batch cell suspensions and in continuous culture. In batch culture the cells were oxygen limited (growth rate 0.33 per hour under oxygen limitations and 0.52 per hour when vigorously aerated). Growth within the film was glucose limited. Several behavioral phenomena were observed for cells growing within the hydrodynamic boundary layer. Despite a flow of 10 cm per second in the environment, the bacteria were able to move freely in both directions within the hydrodynamic boundary layer.

Caldwell, D.↗

NASA NTRS1987

Numerical simulation of the flow field and fuel sprays in an IC engine

A two-dimensional model for axisymmetric piston-cylinder configurations is developed to study the flow field in two-stroke direct-injection Diesel engines under motored conditions. The model accounts for turbulence by a two-equation model for the turbulence kinetic energy and its rate of dissipation. A discrete droplet model is used to simulate the fuel spray, and the effects of the gas phase turbulence on the droplets is considered. It is shown that a fluctuating velocity can be added to the mean droplet velocity every time step if the step is small enough. Good agreement with experimental data is found for a range of ambient pressures in Diesel engine-type microenvironments. The effects of the intake swirl angle in the spray penetration, vaporization, and mixing in a uniflow-scavenged two-stroke Diesel engine are analyzed. It is found that the swirl increases the gas phase turbulence levels and the rates of vaporization.

Nguyen, H. L.↗

NASA NTRS1988

Martian regolith geochemistry and sampling techniques

Laboratory study of samples of the intermediate and fine-grained regolith, including duricrust peds, is a fundamental prerequisite for understanding the types of physical and chemical weathering processes on Mars. The extraordinary importance of such samples is their relevance to understanding past changes in climate, availability (and possible physical state) of water, eolian forces, the thermal and chemical influences of volcanic and impact processes, and the inventory and fates of Martian volatiles. Fortunately, this regolith material appears to be ubiquitous over the Martian surface, and should be available at many different landing sites. Viking data has been interpreted to indicate a smectite-rich regolith material, implying extensive weathering involving aqueous activity and geochemical alteration. An all-igneous source of the Martian fines has also been proposed. The X-ray fluorescence measurement data set can now be fully explained in terms of a simple two-component model. The first component is silicate, having strong geochemical similarities with Shergottites, but not other SNC meteorites. The second component is salt. Variations in these components could produce silicate and salt-rich beds, the latter being of high potential importance for microenvironments in which liquid water (brines) could exist. It therefore would be desirable to scan the surface of the regolith for such prospects.

Clark, B. C.↗

NASA NTRS1990

Carbon-rich particles in Comet Halley

The majority of particles detected in the coma of Comet Halley contain carbon atoms; many of these grains appear to consist preponderately or only of light elements. These light-element particles may be composed of organic compounds. Of the possible combinations of the elements hydrogen, carbon, nitrogen, and oxygen, numerous examples are found of particles containing the combinations (H,C,O,N), (H,C,N), (H,C,O), and (H,C). These results may bear on the recent detection of polyoxymethylene fragments, the observation of cyanojets (CN patterns consistent with release from solid particles), the possible presence of cyanopolyacetylenes or HCN polymer and the make-up of the CHON particles. If cometary matter could reach the surface of the earth without complete disruption, these diverse organic and mixed particles could create unique microenvironments, possibly with significant or even pivotal prebiotic chemical activity. Here a speculative insight into possible relationships between carbon in comets and carbon in life is given, as well as a brief overview of on-going analysis of data from the highly successful Particle Impact Analyzer (PIA) experiment flown on the Giotto spacecraft for the flyby of Comet Halley (development and implementation of PIA was under the direction of J. Kissel of the Max Planck Institute for Kernphysik, Heidelberg). PIA is a time-of-flight analyzer which obtains mass spectra of ions from individual particles impacting on a Pt-Ag foil target within the instrument.

Clark, Benton C.↗

NASA NTRS1991

Exposure to stressful environments - Strategy of adaptive responses

Stresses such as hypoxia, water lack, and heat exposure can produce strains in more than a single organ system, in turn stimulating the body to adapt in multiple ways. Nevertheless, a general strategy of the various adaptive responses emerges when the challenges are divided into three groups: (1) conditions that affect the supply of essential molecules, (2) stresses that prevent the body from regulating properly the output of waste products such as CO2 and heat, and (3) environments that disrupt body transport systems. Problems may arise when there is a conflict between two stresses requiring conflicting adaptive changes. An alternative to adaptation, creation of microenvironment, is often favored by the animal.

Farhi, Leon E.↗

NASA NTRS1992

Silver Teflon blanket: LDEF tray C-08

A study of the Teflon blanket surface at the edge of tray C-08 illustrates the complexity of the microenvironments on the Long Duration Exposure Facility (LDEF). The distribution of particulate contaminants varied dramatically over a distance of half a centimeter (quarter of an inch) near the edge of the blanket. The geometry and optical effects of the atomic oxygen erosion varied significantly over the few centimeters where the blanket folded over the edge of the tray resulting in a variety of orientations to the atomic oxygen flux. A very complex region of combined mechanical and atomic oxygen damage occurred where the blanket contacted the edge of the tray. A brown film deposit apparently fixed by ultraviolet light traveling by reflection through the Teflon film was conspicuous beyond the tray contract zone. Chemical and structural analysis of the surface of the brown film and beyond toward the protected edge of the blanket indicated some penetration of energetic atomic oxygen at least five millimeters past the blanket-tray contact interface.

Crutcher, E. Russ↗

NASA NTRS1992

LDEF materials data analysis: Representative examples

Results of measurements on silverized teflon, heat shrink tubing and nylon tie downs on the wire harness clamps, silvered hex nuts, and contamination deposits are presented. We interpret the results in terms of our microenvironments exposure model and locations on the Long Duration Exposure Facility (LDEF). Distinct changes in the surface properties of FEP were observed as a function of UV exposure. Significant differences in outgassing characteristics were detected for hardware on the interior row 3 relative to identical hardware on the interior row 3 relative to identical hardware on nearby rows. The implications for in service performance are reviewed.

Pippin, H. Gary↗

NASA NTRS1993

Characterization of Minnesota lunar simulant for plant growth

Processing of lunar regolith into a plant growth medium is crucial in the development of a regenerative life support system for a lunar base. Plants, which are the core of such a system, produce food and oxygen for humans and, at the same time, consume carbon dioxide. Because of the scarcity of lunar regolith, simulants must be used to infer its properties and to develop procedures for weathering and chemical analyses. The Minnesota Lunar Simulant (MLS) has been identified to date as the best available simulant for lunar regolith. Results of the dissolution studies reveal that appropriately fertilized MLS can be a suitable medium for plant growth. The techniques used in conducting these studies can be extended to investigate the suitability of actual lunar regolith as a plant growth medium. Dissolution experiments were conducted using the MLS to determine its nutritional and toxicity characteristics for plant growth and to develop weathering and chemical analysis techniques. Two weathering regimes, one with water and one with dilute organic acids simulating the root rhizosphere microenvironment, were investigated. Elemental concentrations were measured using inductively-coupled-plasma (ICP) emission spectrometry and ion chromatography (IC). The geochemical speciation model, MINTEQA2, was used to determine the major solution species and the minerals controlling them. Acidification was found to be a useful method for increasing cation concentrations to meaningful levels. Initial results indicate that MLS weathers to give neutral to slightly basic solutions which contain acceptable amounts of the essential elements required for plant nutrition (i.e., potassium, calcium, magnesium, sulfur, zinc, sodium, silicon, manganese, copper, chlorine, boron, molybdenum, and cobalt). Elements that need to be supplemented include carbon, nitrogen, and perhaps phosphorus and iron. Trace metals in solution were present at nontoxic levels.

Oglesby, James P.↗