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DOE OSTI2023

Previously uncharacterized rectangular bacterial structures in the dolphin mouth

Much remains to be explored regarding the diversity of uncultured, host-associated microbes. Here, we describe rectangular bacterial structures (RBSs) in the mouths of bottlenose dolphins. DNA staining revealed multiple paired bands within RBSs, suggesting the presence of cells dividing along the longitudinal axis. Cryogenic transmission electron microscopy and tomography showed parallel membrane-bound segments that are likely cells, encapsulated by an S-layer-like periodic surface covering. RBSs displayed unusual pilus-like appendages with bundles of threads splayed at the tips. We present multiple lines of evidence, including genomic DNA sequencing of micromanipulated RBSs, 16S rRNA gene sequencing, and fluorescence in situ hybridization, suggesting that RBSs are bacterial and distinct from the genera Simonsiella and Conchiformibius (family Neisseriaceae), with which they share similar morphology and division patterning. Our findings highlight the diversity of novel microbial forms and lifestyles that await characterization using tools complementary to genomics such as microscopy.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1979

Splicing single-mode optical fibers

Approach used to weld multimode fibers has been adapted for more exacting splicing of single-mode fibers. Precision cleaver with tungsten carbide knife edge cuts single-mode optical fibers for welding. Welding apparatus includes micromanipulator, microscopes, and vacuum chucks.

Goldstein, R.↗

NASA NTRS1989

Subminiature Hot-Wire Probes

Class of improved subminiature hot-wire flow-measuring probes developed. Smaller sizes yield improved resolution in measurements of practical aerodynamic flows. Probe made in one-wire, two-perpendicular-wire, and three-perpendicular-wire version for measurement of one, two, or all three components of flow. Oriented and positioned on micromanipulator stage and viewed under microscope during fabrication. Tested by taking measurements in constant-pressure turbulent boundary layer. New probes give improved measurements of turbulence quantities near surfaces and anisotropies of flows strongly influence relative errors caused by phenomena related to spatial resolution.

Westphal, R. V.↗

NASA NTRS1993

Micropipet manipulation of lipid membranes: Direct measurement of the material properties of a cohesive structure that is only two molecules thick

The objectives are to demonstrate how we can make direct measurements of the mechanical properties of a special structure in biology, namely the lipid bilayer membrane, using a micromanipulation technique, and how these properties compare and contrast with 'more traditional' technological/engineering materials. Given that the investment in equipment and expertise to carry out these experiments is probably beyond the scope of most teaching labs, the described experiment is not intended as one that can actually be demonstrated in a student laboratory class. The intention behind presenting this work is to begin to raise awareness in the Material Science community about the material properties of biological material that form a new (to us) category of soft engineering materials that have dimensions on the nanoscale.

Needham, David↗

NASA NTRS1994

A highly redundant robot system for inspection

The work on the serpentine inspection system at JPL is described. The configuration of the inspection system consists of 20 degrees of freedom in total. In particular, the design and development of the serpentine micromanipulator end-effector tool which has 12 degrees of freedom is described. The inspection system is used for application in JPL's Remote Surface Inspection project and as a research tool in redundant manipulator control.

Lee, Thomas S.↗

NASA NTRS1996

Minimum surface-effect microgripper design for force-reflective telemanipulation of a microscopic environment

This paper describes the fundamental physical motivations for a minimum surface effect design, and presents a microgripper that incorporates a piezoelectric ceramic actuator and a flexure-based structure and transmission. The microgripper serves effectively as a one degree-of-freedom prototype of a minimum surface effect micromanipulator design. Data is presented that characterizes the microgripper performance under both pure position and pure force control, followed by a discussion of the attributes and limitations of flexure-based design. The microgripper is interfaced with a force reflective macrogripper, and the pair is controlled with a hybrid position/force scheme. Data is presented that illustrates the effective operation of the telerobotic pair.

Goldfarb, Michael↗

NASA NTRS1996

Minimum Surface-Effect Microgripper Design for Force-Reflective Telemanipulation of a Microscopic Environment

This paper describes the fundamental physical motivations for minimum surface effect design, and presents a microgripper that incorporates a piezoelectric ceramic actuator and a flexure-based structure and transmission. The microgripper serves effectively as a one degree-of-freedom prototype of minimum surface effect micromanipulator design. Data is presented that characterizes the microgripper performance under both pure position and pure force control, followed by a discussion of the attributes and limitations of flexure-based design. The microgripper is interfaced with a force-reflective macrogripper, and the pair controlled with a hybrid position/force scheme. Data is presented that illustrates the effective operation of the telerobotic pair.

Goldfarb, Michael↗

NASA NTRS2002

Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions

We have described a powerful tool for the study of mechanical interactions between cells and their physical environment. Although the approach has already been used in a variety of ways to measure traction forces and to characterize active and passive responses of cultured cells to mechanical stimulation, it can be extended easily and combined with other microscopic approaches, including fluorescent analog imaging (Beningo et al., 2001), photobleaching, calcium imaging, micromanipulation, and electrophysiology. This method will be particularly useful for studying the functions of various components at focal adhesions, and the effects of mechanical forces on focal adhesion-mediated signal transduction. In addition, the method can be extended to a 3D setting, e.g., by sandwiching cultured cells between two layers of polyacrylamide to create an environment mimicking that in the tissue of a multicellular organism. Whereas chemical interactions between cells and the environment have been investigated extensively, many important questions remain as to the role of physical forces in cellular functions and the interplay between chemical and physical mechanisms of communication. The present approach, as well as other approaches capable of probing physical interactions, should fill in this important gap in the near future.

NASA Discipline Cell Biology↗

NASA NTRS1999

Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development

Growth of Arabidopsis thaliana (L.) Heynh. in decreasing oxygen partial pressures revealed a linear decrease in seed production below 15 kPa, with a complete absence of seed production at 2.5 kPa oxygen. This control of plant reproduction by oxygen had previously been attributed to an oxygen effect on the partitioning between vegetative and reproductive growth. However, plants grown in a series of decreasing oxygen concentrations produced progressively smaller embryos that had stopped developing at progressively younger stages, suggesting instead that their growth is limited by oxygen. Internal oxygen concentrations of buds, pistils, and developing siliques of Brassica rapa L. and siliques of Arabidopsis were measured using a small-diameter glass electrode that was moved into the structures using a micromanipulator. Oxygen partial pressures were found to be lowest in the developing perianth (11.1 kPa) and pistils (15.2 kPa) of the unopened buds. Pollination reduced oxygen concentration inside the pistils by 3 kPa after just 24 h. Inside Brassica silique locules, partial pressures of oxygen averaged 12.2 kPa in darkness, and increased linearly with increasing light levels to 16.2 kPa. Measurements inside Arabidopsis siliques averaged 6.1 kPa in the dark and rose to 12.2 kPa with light. Hypoxia in these microenvironments is postulated to be the point of control of plant reproduction by oxygen.

Non-NASA Center↗

NASA NTRS1997

Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

NASA Discipline Cell Biology↗

NASA NTRS2005

Ultrasonic Micro-Blades for the Rapid Extraction of Impact Tracks from Aerogel

The science return of NASA's Stardust Mission with its valuable cargo of cometary debris hinges on the ability to efficiently extract particles from silica aerogel collectors. The current method for extracting cosmic dust impact tracks is a mature procedure involving sequential perforation of the aerogel with glass needles on computer controlled micromanipulators. This method is highly successful at removing well-defined aerogel fragments of reasonable optical clarity while causing minimal damage to the surrounding aerogel collector tile. Such a system will be adopted by the JSC Astromaterials Curation Facility in anticipation of Stardust s arrival in early 2006. In addition to Stardust, aerogel is a possible collector for future sample return missions and is used for capture of hypervelocity ejecta in high power laser experiments of interest to LLNL. Researchers will be eager to obtain Stardust samples for study as quickly as possible, and rapid extraction tools requiring little construction, training, or investment would be an attractive asset. To this end, we have experimented with micro-blades for the Stardust impact track extraction process. Our ultimate goal is a rapid extraction system in a clean electron beam environment, such as an SEM or dual-beam FIB, for in situ sample preparation, mounting and analysis.

Ishii, H. A.↗

NASA NTRS2001

Light Microscopy Module: An On-Orbit Microscope Planned for the Fluids and Combustion Facility on the International Space Station

The Light Microscopy Module (LMM) is planned as a fully remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of fluids and biology experiments within NASA Glenn Research Center's Fluids and Combustion Facility on the International Space Station. Within the Fluids and Combustion Facility, four fluids physics experiments will utilize an instrument built around a light microscope. These experiments are the Constrained Vapor Bubble experiment (Peter C. Wayner of Rensselaer Polytechnic Institute), the Physics of Hard Spheres Experiment-2 (Paul M. Chaikin of Princeton University), the Physics of Colloids in Space-2 experiment (David A. Weitz of Harvard University), and the Low Volume Fraction Colloidal Assembly experiment (Arjun G. Yodh of the University of Pennsylvania). The first experiment investigates heat conductance in microgravity as a function of liquid volume and heat flow rate to determine, in detail, the transport process characteristics in a curved liquid film. The other three experiments investigate various complementary aspects of the nucleation, growth, structure, and properties of colloidal crystals in microgravity and the effects of micromanipulation upon their properties. Key diagnostic capabilities for meeting the science requirements of the four experiments include video microscopy to observe sample features including basic structures and dynamics, interferometry to measure vapor bubble thin film thickness, laser tweezers for colloidal particle manipulation and patterning, confocal microscopy to provide enhanced three-dimensional visualization of colloidal structures, and spectrophotometry to measure colloidal crystal photonic properties.

Doherty, Michael P.↗

NASA NTRS2002

Light Microscopy Module: On-Orbit Microscope Planned for the Fluids Integrated Rack on the International Space Station

The Light Microscopy Module (LMM) is planned as a remotely controllable, automated, on-orbit facility, allowing flexible scheduling and control of physical science and biological science experiments within the Fluids Integrated Rack (FIR) on the International Space Station. Initially four fluid physics experiments in the FIR will use the LMM the Constrained Vapor Bubble, the Physics of Hard Spheres Experiment-2, Physics of Colloids in Space-2, and Low Volume Fraction Entropically Driven Colloidal Assembly. The first experiment will investigate heat conductance in microgravity as a function of liquid volume and heat flow rate to determine, in detail, the transport process characteristics in a curved liquid film. The other three experiments will investigate various complementary aspects of the nucleation, growth, structure, and properties of colloidal crystals in microgravity and the effects of micromanipulation upon their properties.

Motil, Susan M.↗

NASA NTRS2009

Measurement of Trap Length for an Optical Trap

The trap length along the beam axis for an optical trap formed with an upright, oil-immersion microscope was measured. The goals for this effort were twofold. It was deemed useful to understand the depth to which an optical trap can reach for purposes of developing a tool to assist in the fabrication of miniature devices. Additionally, it was desired to know whether the measured trap length favored one or the other of two competing theories to model an optical trap. The approach was to trap a microsphere of known size and mass and raise it from its initial trap position. The microsphere was then dropped by blocking the laser beam for a pre-determined amount of time. Dropping the microsphere in a free-fall mode from various heights relative to the coverslip provides an estimate of how the trapping length changes with depth in water in a sample chamber on a microscope slide. While it was not possible to measure the trap length with sufficient precision to support any particular theory of optical trap formation, it was possible to find regions where the presence of physical boundaries influenced optical traps, and determine that the trap length, for the apparatus studied, is between 6 and 7 m. These results allow more precise control using optical micromanipulation to assemble miniature devices by providing information about the distance over which an optical trap is effective.

Wrbanek, Susan Y.↗

NASA NTRS2010

Measurement of the Length of an Optical Trap

NASA Glenn has been involved in developing optical trapping and optical micromanipulation techniques in order to develop a tool that can be used to probe, characterize, and assemble nano and microscale materials to create microscale sensors for harsh flight environments. In order to be able to assemble a sensor or probe candidate sensor material, it is useful to know how far an optical trap can reach; that is, the distance beyond/below the stable trapping point through which an object will be drawn into the optical trap. Typically, to measure the distance over which an optical trap would influence matter in a horizontal (perpendicular to beam propagation) direction, it was common to hold an object in one optical trap, place a second optical trap a known distance away, turn off the first optical trap, and note if the object was moved into the second trap when it was turned on. The disadvantage of this technique is that it only gives information of trap influence distance in horizontal (x y) directions. No information about the distance of the influence of the trap is gained in the direction of propagation of the beam (the z direction). A method was developed to use a time-of-flight technique to determine the length along the propagation direction of an optical trap beam over which an object may be drawn into the optical trap. Test objects (polystyrene microspheres) were held in an optical trap in a water-filled sample chamber and raised to a pre-determined position near the top of the sample chamber. Next, the test objects were released by blocking the optical trap beam. The test objects were allowed to fall through the water for predetermined periods of time, at the end of which the trapping beam was unblocked. It was noted whether or not the test object returned to the optical trap or continued to fall. This determination of the length of an optical trap's influence by this manner assumes that the test object falls through the water in the sample chamber at terminal velocity for the duration of its fall, so that the distance of trap influence can be computed simply by: d = VTt, where d is the trap length (or distance of trap reach), VT is the terminal velocity of the test object, and t is the time interval over which the object is allowed to fall.

Wrbanek, Susan Y.↗

NASA NTRS2003

Preparation of Regular Specimens for Atom Probes

A method of preparation of specimens of non-electropolishable materials for analysis by atom probes is being developed as a superior alternative to a prior method. In comparison with the prior method, the present method involves less processing time. Also, whereas the prior method yields irregularly shaped and sized specimens, the present developmental method offers the potential to prepare specimens of regular shape and size. The prior method is called the method of sharp shards because it involves crushing the material of interest and selecting microscopic sharp shards of the material for use as specimens. Each selected shard is oriented with its sharp tip facing away from the tip of a stainless-steel pin and is glued to the tip of the pin by use of silver epoxy. Then the shard is milled by use of a focused ion beam (FIB) to make the shard very thin (relative to its length) and to make its tip sharp enough for atom-probe analysis. The method of sharp shards is extremely time-consuming because the selection of shards must be performed with the help of a microscope, the shards must be positioned on the pins by use of micromanipulators, and the irregularity of size and shape necessitates many hours of FIB milling to sharpen each shard. In the present method, a flat slab of the material of interest (e.g., a polished sample of rock or a coated semiconductor wafer) is mounted in the sample holder of a dicing saw of the type conventionally used to cut individual integrated circuits out of the wafers on which they are fabricated in batches. A saw blade appropriate to the material of interest is selected. The depth of cut and the distance between successive parallel cuts is made such that what is left after the cuts is a series of thin, parallel ridges on a solid base. Then the workpiece is rotated 90 and the pattern of cuts is repeated, leaving behind a square array of square posts on the solid base. The posts can be made regular, long, and thin, as required for samples for atom-probe analysis. Because of their small volume and regularity, the amount of FIB-milling time can be much less than that of the method of sharp shards. Individual posts can be broken off for mounting in a manner similar to that of the method of sharp shards. Alternatively, the posts can be left intact on the base and the base can be cut to a small square (e.g., 3 by 3 mm) suitable for mounting in an atom probe of a type capable of accepting multiple-tip specimens. The advantage of multiple-tip specimens is the possibility of analyzing many tips without the time-consuming interchange of specimens.

Kuhlman, Kim↗

NASA NTRS2012

Pristine Stratospheric Collections of Cosmic Dust

Since 1981, NASA has routinely collected interplanetary dust particles (IDPs) in the stratosphere by inertial impact onto silicone oil-coated flat plate collectors deployed on the wings of high-altitude aircraft [1]. The highly viscous oil traps and localizes the particles, which can fragment during collection. Particles are removed from the collectors with a micromanipulator and washed of the oil using organic solvents, typically hexane or xylene. While silicone oil is an efficient collection medium, its use is problematic. All IDPs are initially coated with this material (polydimethylsiloxane, n(CH3)2SiO) and traces of oil may remain after cleaning. The solvent rinse itself is also a concern as it likely removes indigenous organics from the particles. To avoid these issues, we used a polyurethane foam substrate for the oil-free stratospheric collection of IDPs.

Messenger, S.↗

NASA NTRS2018

Novel Applications of Focused Ion Beam Technique for Planetary Sample Analyses

We are using innovative FIB techniques to prepare samples of planetary materials for different types of coordinated analyses using ion microprobes, synchrotron beamlines, and specialized transmission electron microscopy (TEM) techniques. In these cases, the FIB sample preparation is the critical step in enabling these specialized analyses. We discuss several examples below utilizing the FEI Quanta3D instrument at the NASA Johnson Space Center. Trace element analyses utilizing synchrotron x-ray fluorescence. The trace element content of mineral grains in comet dust provides important clues on their formation and processing in the early solar system. We preformed coordinated analyses of a comet dust particle that had been prepared using ultramicrotomy for TEM analysis. Following the TEM analyses, we extracted a 70 nm thick section from a region of the carbon (C) film of the TEM grid, for additional analyses. A carbon ring ~2-3 μm thick was deposited on top of the C film using the FIB. The C film on the outer rim of the ring was milled away using various patterns to uniformly release the stresses on the film, preventing rupture and collapse, and was attached to the micromanipulator needle. We then isolated the ring completely and transferred the section to a silicon sample holder for analysis using the HXN (hard X-ray nanoprobe) beamline at NSLSII at Brookhaven National Lab. Coordinated Analyses of Presolar Grains. Rare sub-m presolar grains that originate in evolved stars and supernovae, occur in primitive astromaterials and are identified by their exotic isotopic compositions. Coordinated analyses of these grains using NanoSIMS, TEM, and other techniques on the same grain is enabled by innovative FIB sample preparation. In order to obtain subsequent isotopic analyses of Mg and Fe, contributions from surrounding grains were minimized. We precisely deposited a protective cap of Pt on top of the grain to preserve the grain of interest and then milled away about 5 μm diameter of the surrounding material. Following the isotopic analyses, the spindle was extracted and thinned to electron transparency for TEM microstructural analyses. In situ heating TEM experiments on lunar samples. We extracted a FIB thin section from Apollo 17 lunar rock 76015. To avoid ion-beam damage, e-beam deposition was used to deposit the first 500 nm of the C strap, followed by ion beam-assisted deposition of ~3 μm carbon. We performed an ex situ lift-out of the section and placed the section on one of the elements of a microelectromechanical systems (MEMS) - specialized heating substrate and attached the section to the substrate by depositing small C straps with the FIB. The heating chips utilize silicon nitride windows to support the samples and provide uniform heating while enabling TEM imaging. The heating chip was loaded into a Hitachi “Blaze” heating holder and analyzed using a Hitachi HF5000 at the University of Arizona.

Rahman, Z.↗