Search NASA⌕ Search

SEARCH · Search NASA

Results for “live cell imaging”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2018

Design and Development of Modules to Support Live Microscopic Imaging on Ground-Based Microgravity Simulators

In space, astronauts are exposed to environmental stressors that often result in physiological changes. One prominent stressor in spaceflight is microgravity, and research has shown that long term microgravity exposure causes muscle atrophy, bone loss, cardiovascular concerns, and vision impairment. It is critical to understand how altered gravity affects physiology on the cellular, molecular, and gene level in order to accurately assess health risks and to develop effective countermeasures. Ground-based microgravity simulators such as random positioning machines (RPMs) are used to produce some of the biological effects of altered gravity on different cell types and organisms. Real-time imaging during simulations are of particular interest as we can study how basic cell functions such as cell division, cell migration, and proliferation progress under microgravity conditions. However, design limitations of present microgravity simulators such as susceptibility to parasitic vibration and displacement of the sample from the center of rotation challenge the accuracy of experiment results and live images. We have developed a cell culture sample holder module suitable for live microscopic imaging on an RPM. CAD modeling and 3D printing technology were used to implement modifications to the sample holder and to install a digital microscope to perform live bright-field and fluorescent imaging. Vibration damping materials were also investigated to allow for stable imaging while the microgravity simulator was within a cell culture incubator. Novel methods and hardware modifications for improving live cell imaging on ground-based microgravity simulators were proposed and discussed.

3D printing↗
NASA NTRS1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗
NASA NTRS2002

Overexpression of neurofilament H disrupts normal cell structure and function

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS2007

Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

Cucinotta, Francis, A.↗
NASA NTRS2016

Chapter 7: Fluorescence Imaging of the Cytoskeleton in Plant Roots

During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

Actin↗
NASA NTRS2003

Gravity-regulated differential auxin transport from columella to lateral root cap cells

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.

Non-NASA Center↗
NASA NTRS1999

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

NASA Discipline Plant Biology↗
NASA NTRS1995

Compact, Automated, Frequency-Agile Microspectrofluorimeter

Compact, reliable, rugged, automated cell-culture and frequency-agile microspectrofluorimetric apparatus developed to perform experiments involving photometric imaging observations of single live cells. In original application, apparatus operates mostly unattended aboard spacecraft; potential terrestrial applications include automated or semiautomated diagnosis of pathological tissues in clinical laboratories, biomedical instrumentation, monitoring of biological process streams, and portable instrumentation for testing biological conditions in various environments. Offers obvious advantages over present laboratory instrumentation.

Fernandez, Salvador M.↗
NASA NTRS2010

Polarization Imaging Apparatus

A polarization imaging apparatus has shown promise as a prototype of instruments for medical imaging with contrast greater than that achievable by use of non-polarized light. The underlying principles of design and operation are derived from observations that light interacts with tissue ultrastructures that affect reflectance, scattering, absorption, and polarization of light. The apparatus utilizes high-speed electro-optical components for generating light properties and acquiring polarization images through aligned polarizers. These components include phase retarders made of OptoCeramic (registered TradeMark) material - a ceramic that has a high electro-optical coefficient. The apparatus includes a computer running a program that implements a novel algorithm for controlling the phase retarders, capturing image data, and computing the Stokes polarization images. Potential applications include imaging of superficial cancers and other skin lesions, early detection of diseased cells, and microscopic analysis of tissues. The high imaging speed of this apparatus could be beneficial for observing live cells or tissues, and could enable rapid identification of moving targets in astronomy and national defense. The apparatus could also be used as an analysis tool in material research and industrial processing.

Zou, Yingyin K.↗
NASA NTRS1997

Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

Non-NASA Center↗
NASA NTRS2002

Supergranule Rotation Rates and Lifetimes

Previous measurements of the rotation rate of the supergranule Doppler pattern have revealed three interesting characteristics. 1) The supergranule pattern rotates faster than the plasma at the surface and, at each latitude, it rotates faster than the plasma at any level below the surface. 2) Larger cells rotate more rapidly than smaller cells. 3) Faster rotation rates are found when using larger time lags between Doppler images. These last two characteristics are consistent with the idea that large cells live longer and extend deeper into the Sun where the rotation rate is faster. A re-examination of the rotation rates and lifetimes of the Doppler patterns seen with the Michelson Doppler Imager (MDI) instrument on Solar and Heliospheric Observatory (SOHO) confirms these characteristics. However, a simulation of the data using a spectrum for the cellular flows that matches the observed spectrum shows that these characteristics can be largely reproduced by cellular patterns that rotate at the same rate without any dependence upon cell size, The rotation rate , and its dependence on latitude, is nonetheless still faster than the surface or internal rotation rate. The difference in rotation rates as functions of cell size and time lag between observations is attributed to projection effects on the line-of-sight Doppler signal. This data simulation is also used to determine characteristic lifetimes for the cellular patterns as a function of cell size. These lifetime determinations are also affected by projection effects on the line-of-sight Doppler signal.

Hathaway, David H.↗
NASA NTRS1992

Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗
NASA NTRS2003

Laboratory and In-Flight In-Situ X-ray Imaging and Scattering Facility for Materials, Biotechnology and Life Sciences

We propose a multifunctional X-ray facility for the Materials, Biotechnology and Life Sciences Programs to visualize formation and behavior dynamics of materials, biomaterials, and living organisms, tissues and cells. The facility will combine X-ray topography, phase micro-imaging and scattering capabilities with sample units installed on the goniometer. This should allow, for the first time, to monitor under well defined conditions, in situ, in real time: creation of imperfections during growth of semiconductors, metal, dielectric and biomacromolecular crystals and films, high-precision diffraction from crystals within a wide range of temperatures and vapor, melt, solution conditions, internal morphology and changes in living organisms, tissues and cells, diffraction on biominerals, nanotubes and particles, radiation damage, also under controlled formation/life conditions. The system will include an ultrabright X-ray source, X-ray mirror, monochromator, image-recording unit, detectors, and multipurpose diffractometer that fully accommodate and integrate furnaces and samples with other experimental environments. The easily adjustable laboratory and flight versions will allow monitoring processes under terrestrial and microgravity conditions. The flight version can be made available using a microsource combined with multilayer or capillary optics.

Source record↗
NASA NTRS1996

Design, Fabrication and Testing of Multilayer Coated X-Ray Optics for the Water Window Imaging X-Ray Microscope

Hoover et. al. built and tested two imaging Schwarzschild multilayer microscopes. These instruments were constructed as prototypes for the "Water Window Imaging X-Ray Microscope," which is a doubly reflecting, multilayer x-ray microscope configured to operate within the "water window." The "water window" is the narrow region of the x-ray spectrum between the K absorption edges of oxygen (lamda = 23.3 Angstroms) and of carbon (lamda = 43.62 Angstroms), where water is relatively highly transmissive and carbon is highly absorptive. This property of these materials, thus permits the use of high resolution multilayer x-ray microscopes for producing high contrast images of carbon-based structures within the aqueous physiological environments of living cells. We report the design, fabrication and testing of multilayer optics that operate in this regime.

Spencer, Dwight C.↗
NASA NTRS2004

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

Fischer, David G.↗
NASA NTRS2005

Morphology and Elemental Composition of Recent and Fossil Cyanobacteria

Cyanobacteria (cyanophyta, cyanoprokaryota, and blue-green algae) are an ancient, diverse and abundant group of photosynthetic oxygenic microorganisms. Together with other bacteria and archaea, the cyanobacteria have been the dominant life forms on Earth for over 3.5 billion years. Cyanobacteria occur in some of our planets most extreme environments - hot springs and geysers, hypersaline and alkaline lakes, hot and cold deserts, and the polar ice caps. They occur in a wide variety of morphologies. Unlike archaea and other bacteria, which are typically classified in pure culture by their physiological, biochemical and phylogenetic properties, the cyanobacteria have historically been classified based upon their size and morphological characteristics. These include the presence or absence of heterocysts, sheath, uniseriate or multiseriate trichomes, true or false branching, arrangement of thylakoids, reproduction by akinetes, binary fission, hormogonia, fragmentation, presence/absence of motility etc. Their antiquity, distribution, structural and chemical differentiation, diversity, morphological complexity and large size compared to most other bacteria, makes the cyanobacteria ideal candidates for morphological biomarkers in returned Astromaterials. We have obtained optical (nomarski and phase contrast)/fluorescent (blue and green excitation) microscopy images using an Olympus BX60 compound microscope and Field Emission Scanning Electron Microscopy images and EDAX elemental compositions of living and fossil cyanobacteria. The S-4000 Hitachi Field Emission Scanning Electron Microscope (FESEM) has been used to investigate microfossils in freshly fractured interior surfaces of terrestrial rocks and the cells, hormogonia, sheaths and trichomes of recent filamentous cyanobacteria. We present Fluorescent and FESEM Secondary and Backscattered Electron images and associated EDAX elemental analyses of recent and fossil cyanobacteria, concentrating on representatives of the genera Calothnx, Leptolyngbya, Lyngbya, Planktolyngbya and Oscillatoria.

SaintAmand, Ann↗
NASA NTRS2009

Optoelectronic tweezers for microparticle and cell manipulation

An optical image-driven light induced dielectrophoresis (DEP) apparatus and method are described which provide for the manipulation of particles or cells with a diameter on the order of 100 .mu.m or less. The apparatus is referred to as optoelectric tweezers (OET) and provides a number of advantages over conventional optical tweezers, in particular the ability to perform operations in parallel and over a large area without damage to living cells. The OET device generally comprises a planar liquid-filled structure having one or more portions which are photoconductive to convert incoming light to a change in the electric field pattern. The light patterns are dynamically generated to provide a number of manipulation structures that can manipulate single particles and cells or groups of particles/cells. The OET preferably includes a microscopic imaging means to provide feedback for the optical manipulation, such as detecting position and characteristics wherein the light patterns are modulated accordingly.

Wu, Ming Chiang↗