Sample Mounting Techniques Research Articles

Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

Long-wavelength Mesh&Collect native SAD phasing from microcrystals.

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 µm3 for native phasing requires careful experimental planning. The type of anomalous scatterers that are naturally present in the sample, such as sulfur, phosphorus and calcium, will dictate the beam energy required and determine the level of radiation sensitivity, while the crystal size will dictate the beam size and the sample-mounting technique, in turn indicating the specifications of a suitable beamline. On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of ∼20 µm or less using an accordingly sized microbeam at a wavelength of 1.892 Å (6.551 keV, close to the Mn edge at 6.549 keV) increases the expected Bijvoet ratio to 2.1% from an expected 0.7% at 12.6 keV (Se K edge), thus allowing experimental phase determination using the anomalous signal from naturally present Mn2+ and Ca2+ ions. Dozens of crystals were harvested and flash-cryocooled in micro-meshes, rapidly screened for diffraction (less than a minute per loop) and then used for serial Mesh&Collect collection of about 298 partial data sets (10° of crystal rotation per sample). The partial data sets were integrated and scaled. A genetic algorithm for combining partial data sets was used to select those to be merged into a single data set. This final data set showed high completeness, high multiplicity and sufficient anomalous signal to locate the anomalous scatterers, and provided phasing information which allowed complete auto-tracing of the polypeptide chain. To allow the complete experiment to run in less than 2 h, a practically acceptable time frame, the diffractometer and detector had to run together with limited manual intervention. The combination of several cutting-edge components allowed accurate anomalous signal to be measured from small crystals.

Conformal atomic layer deposition of alumina on millimeter tall, vertically-aligned carbon nanotube arrays.

Atomic layer deposition (ALD) can be used to coat high aspect ratio and high surface area substrates with conformal and precisely controlled thin films. Vertically aligned arrays of multiwalled carbon nanotubes (MWCNTs) with lengths up to 1.5 mm were conformally coated with alumina from base to tip. The nucleation and growth behaviors of Al2O3 ALD precursors on the MWCNTs were studied as a function of CNT surface chemistry. CNT surfaces were modified through a series of post-treatments including pyrolytic carbon deposition, high temperature thermal annealing, and oxygen plasma functionalization. Conformal coatings were achieved where post-treatments resulted in increased defect density as well as the extent of functionalization, as characterized by X-ray photoelectron spectroscopy and Raman spectroscopy. Using thermogravimetric analysis, it was determined that MWCNTs treated with pyrolytic carbon and plasma functionalization prior to ALD coating were more stable to thermal oxidation than pristine ALD coated samples. Functionalized and ALD coated arrays had a compressive modulus more than two times higher than a pristine array coated for the same number of cycles. Cross-sectional energy dispersive X-ray spectroscopy confirmed that Al2O3 could be uniformly deposited through the entire thickness of the vertically aligned MWCNT array by manipulating sample orientation and mounting techniques. Following the ALD coating, the MWCNT arrays demonstrated hydrophilic wetting behavior and also exhibited foam-like recovery following compressive strain.

MacCHESS: Structural Biology at Cornell's High-Energy Synchrotron Source

"MacCHESS (""Macromolecular diffraction at CHESS"") is an NIH funded facility at the Cornell High Energy Synchrotron Source; we provide a user facility with an exceptional level of support, as well as pursuing research to benefit the entire structural biology community. CRYSTALLOGRAPHY: High-flux monochromatic beamlines outfitted with state-of-the-art equipment are available. BSL-2 biohazards can be handled. Research activities include on-line confocal microscopy, working with multiple small crystals, use of graphene to reduce background, etc. BIOSAXS: A dedicated beamline features: a dual SAXS/WAXS setup using 2 Pilatus detectors; an integrated computer-controlled flow system including robotic sample loading from 96-well trays, custom-made disposable, transparent sample cells, and a convenient graphical interface; a well-equipped wet lab for sample monitoring and final preparation; an in-line SEC-MALS/DLS-SAXS option. Microfluidic "lab-on-a-chip" units are under development. Periodic workshops are held to educate users in the intricacies of BioSAXS. PRESSURE CRYOCOOLING: Cryocooling crystals under pressure reduces both cooling-induced degradation and the need for penetrating cryoprotectants, and can stabilize mobile ligands and possibly reaction intermediates. We offer pressure-cryocooling as a service to CHESS users, while continuing to develop the method. Several sample mounting techniques are now available, and the technique has promise for use with biological samples other than crystals. FMI: To request beamtime, fill out a simple on-line proposal form at http://www.chess.cornell.edu. Mail-in service is available, and remote data collection is supported for experienced crystallography users. We welcome a chance to collaborate on "non-standard" experiments. For more information, contact User Administrator Kathy Dedrick (kd73@cornell.edu)."

An improved instrumented indentation technique for single microfibers

Abstract

Macchess: Unique Opportunities for Structural Biology at a Synchrotron Source

MacCHESS (“Macromolecular diffraction at CHESS”) is an NIH funded facility at the Cornell High Energy Synchrotron Source; we provide a user facility with exceptional staff support. A special MacCHESS strength is developing new x-ray methods to benefit the entire structural biology community.Crystallography - High-flux monochromatic beamlines equipped with large CCD detectors, air bearing-based goniometers, selection of 100-µm or 20-µm beam size, cryocooling, excellent crystal centering systems, and crystal automounters are available. BSL-2 biohazards can be handled. Methods for collecting and processing multi-crystal data sets are under development.BioSAXS - A dedicated beamline features a dual SAXS/WAXS setup using 2 Pilatus detectors and an integrated computer-controlled flow system including robotic sample loading from 96-well trays, a capillary cell with a replaceable insert, and a convenient graphical interface. An HPLC system allows sample preparation immediately before taking data. Microfluidic “lab-on-a-chip” units are under development. Periodic workshops are held to educate users in the intricacies of BioSAXS.Pressure cryocooling - A new method for cryocooling crystals under pressure reduces both cooling-induced degradation and the need for penetrating cryoprotectants, and can stabilize mobile ligands and possibly reaction intermediates. We offer pressure-cooling as a service to CHESS users, while continuing to develop the method. Several sample mounting techniques are now available, and the technique has promise for use with biological samples other than crystals.More information - To request beamtime, fill out a simple on-line proposal form at http://www.chess.cornell.edu. Mail-in service is available for crystallography. We welcome a chance to collaborate on “non-standard” experiments. For more information, contact User Administrator Kathy Dedrick (kd73@cornell.edu).

Determination of water content in silicate glasses using Raman spectrometry: Implications for the study of explosive volcanism

Raman spectroscopy can measure water concentrations of hydrous silicate glasses with several advantages such as: (1) high-spatial resolution of 1–2 μm2; (2) non-destructive character; and (3) easy access, without any specific sample preparation or mounting techniques. The latter reasons render Raman highly suitable for studying natural products, such as volcanic pumice and scoriae fragments. Two spectral regions can be distinguished in Raman spectra of hydrated silicate glasses: a low-wavenumber region (15–1500 cm−1), which corresponds to vibrations of the silicate network, and a high-wavenumber region (3100–3750 cm−1), corresponding to the OH stretching vibrations of H2O molecules and OH groups. Behrens et al. (2006) have published empirical equations relating the area ratio between these two regions and the water content. However, the proposed internal calibrations depend on chemical composition of the glasses. In this paper, we reinvestigated the previous procedures to improve the background subtraction. Our results allow us to present a more general and linear calibration. Water concentrations up to 13 wt% can be measured for a broad range of natural silicate melts, from basalts to rhyolite (40 up to 80 wt% SiO2), using a single calibration curve with an absolute error of 0.2 wt%.

Photoluminescence of gold, copper and niobium as a function of temperature

A specially constructed instrument for measuring the low intensity photoluminescence emission spectra of metals is described. It uses low luminescence optical components and dedicated sample mounting techniques. Room temperature measurements agree closely with literature spectra for high-purity gold and are found to be sensitive to 100 ppm impurities. Detailed spectra are presented, which are weakly temperature dependent, for gold, copper and unpolished niobium between room temperature and 100 K. We conclude that this work provides accurate luminescence data for Au from 300 K down to 100 K. Although the (variable temperature) luminescence data for Cu are consistent both with the room temperature experimental data in the literature and theory, we conclude the role of surface adsorbates and/or oxides cannot be ruled out. Theory suggests that Nb has a factor ∼50 lower luminescence intensity than Au and Cu because the real part of the refractive index is a factor ∼5 higher and the density of states ∼2 eV below the Fermi energy is a factor of ∼4 lower than Au and Cu. Measurements are presented for unpolished Nb, but given the lack of signal detection for polished Nb and that theory predicts very weak signals, we conclude that the luminescence signals from pure Nb still remain below the sensitivity of our instrument.

Testing semiconductor devices at extremely high operating temperatures

We developed a dedicated measurement set-up for the electrical and electrothermal characterization of semiconductor devices and microsystems under very high-temperature conditions. The set-up consists of several modules comprising a vacuum system as basic unit and a number of alternative sample stages. Currently it enables measurements in the temperature range between room temperature and about 700 °C. We give a detailed description of the measurement system, sample mounting techniques, and exemplary measurements on SiC devices.

A new sample mounting technique for room-temperature macromolecular crystallography

A new method for mounting protein crystals and other environmentally sensitive samples for room-temperature diffraction measurements is described. A crystal is retrieved using a microfabricated sample mount as recently reported, and the mount is inserted into a modified goniometer-compatible base. A transparent thin-wall polyester tube sealed at one end and filled with stabilizing liquid is then drawn over the crystal and sealed to the goniometer base. Compared with mounting using glass capillaries, this method can provide lower-background X-ray scattering, especially at higher resolutions; dramatically improved ease of crystal mounting with minimal chance of damage; accurate and reproducible crystal positioning relative to the goniometer base; improved crystal visibility and ease of alignment, especially for very small crystals; and compatibility with high-throughput approaches. Crystals can be rapidly screened and eliminated earlier in the data collection pipeline, and the cause of poor low-temperature diffraction can be diagnosed.

Site-specific SIMS backside analysis

For maximum SIMS depth resolution for any layer in a sample, the depth profile should begin only as far from the layer as necessary to establish a constant implant concentration of the primary ion beam species. Depth resolution and detection limit can be severely degraded if it is necessary to sputter through an over layer having non uniform sputtering properties or containing a high level of the impurity of interest prior to reaching the layer of interest. A SIMS backside analysis method based on mechanical polishing is extended to allow site-specific SIMS backside depth profile analysis. Optical microscopy employing a red filter was used to allow direct viewing of the site to be depth profiled both during polishing and in situ during SIMS analysis. Depth profile analyses were performed on 100 μm×100 μm device test structures. Sample charging resulting from insulator layers present in these device test structures was alleviated using a modified sample mounting technique. Backside SIMS depth profile analysis using an O 2 + primary ion beam having an impact energy of 1.25 keV was used to determine if boron had penetrated a thin SiO 2 layer.

Extending RBS analysis to very fragile, nanometer-thick foils

We have applied Rutherford backscattering (RBS) to the analysis of very thin and very fragile, free-standing foils. These foils consisted of a thin metal or other layer encapsulated inside thin layers of a polycarbonate-like polymer. Thicknesses of the middle layers were ∼10 nm of Fe, ∼2 nm of Au and ∼7 nm of Ni with ∼12 nm of NaF. The encapsulating, polymer layers each had a thickness of about 75 nm. Application of appropriate sample mounting and RBS techniques has led to spectra with essentially no background. We determined the amounts and depth distributions of all elements with atomic numbers Z⩾6. Very low current densities (∼2 nA/mm 2) of 2.3 MeV He + ions were used to minimize heating by the beam and yet obtain useful statistics in a reasonable time. To monitor any changes in the foils during analysis, spectra were obtained in a sequence of low-fluence bombardments. After each bombardment the beam was interrupted, and the data was examined and stored. This procedure permitted us to monitor the data for any significant changes in the foil and allowed cooling of the target during the beam-off interval.

Silicon bonding for ultrahigh vaccuum surface science studies

A novel silicon bonding technique and its application for surface science studies in a wide temperature range of 30–1400 K is presented. The silicon single crystal is bonded on a polished tantalum plate via thin silver and tungsten interlayers deposited by evaporation in high vacuum. Upon annealing the silicon sample is bonded with a strong mechanical and good thermal contact. Several problems common to alternative sample mounting techniques are solved by this procedure. Excellent temperature control and small temperature gradients across the surface well below 1 K are demonstrated by thermal desorption data for two different adsorbates: ethane and hydrogen on Si(001)-(2×1).

Use of X-Ray Transmission Diffractometry for the Study of Clay-Particle Orientation at Different Water Contents

AbstractHomoionic Ca-saturated clay pastes were prepared and drying curves were obtained by applying suction pressures from 1 kPa to 100 MPa, A transmission device was used to study particle orientation by placing the clay in a cell specially designed to obtain diagrams corresponding to different sample orientations. The 00l and hk0 reflections were compared to determine the best reflections for studying clay-particle orientation. Depending on the clay, 00/ reflections or the 020 reflection and/or hkl bands can be used to analyze orientation. In many cases the 020 reflection is preferred because the intensity of the peak is high and appears to be independent of the H2O content and the degree of stacking order of layers along the [001] direction.For interstratified clays, the conditions required to obtain 00l reflections depended on several factors, the most important of which is the water content. Also, the intensity relating to particie orientation depends on (1) particle extension (size) in the (001) plane and (2) the crystal structure. Illite crystals of <1000 A gave a poorly oriented clay matrix. In contrast, large aggregates of illite, smectite, and kaolinite particles (>10,000 Å) showed a strongly oriented system. The particles of smectites may be curved and the dry material was poorly oriented owing to weak cohesion forces between the layers in comparison to illite.The study of the orientation of particles by X-ray diffraction on hydrated samples may be affected by sample mounting techniques. Any change in the content or the way the sample is mounted may modify the microstructure of a material.Clay containing a high water content affects the disorientation of particles, whereas, for the dry samples, pore size, pore volume, and solid continuity are associated with the geometry and crystal structure of the clay matrix.

A tensile stage to study thin polymer films in an environmental scanning electron microscope

An economical and easily constructed tensile stage to study thin polymer films in an environmental scanning electron microscope has been developed. Surface features of the film and the morphology in the vicinity of a crack tip while under stress may be studied using this stage. The construction, sample mounting technique, and some of the photomicrographs obtained using this tensile stage have been illustrated and described.

A Fast-Result Method of Planar Polishing for Optical Microscopy

Abstract The planarizing technique of materials lapping and polishing shows many benefits in providing samples for optical microscopy in failure analysis, quality control and related fields. A method is described below which provides both rapid and accurate micro-sections of pcb's, wafers, packaged components and other processed materials, with the use of a novel approach involving a 'calibrated' polishing base and a 'Micropositioner' head. Other benefits include the ability to halt material removal at a predetermined process endpoint and convenient sample mounting techniques.

Critical current measurements of Nb 3Sn superconductors: NBS contribution to the VAMAS interlaboratory comparison

Critical current measurements on several Nb 3Sn superconductors were made as part of an interlaboratory comparison (round robin). These measurements were made in conjunction with twenty-four laboratories from the European Economic Community, Japan and the USA as part of the Versailles Agreement on Advanced Materials and Standards (VAMAS). The results of the NBS measurements, including the effect of sample mounting techniques on the measured critical current, are given. A systematic study of the effect of measurement mandrel (tubular sample-holder made from G10 fibreglass-epoxy composite) geometry revealed that a seemingly small change in that geometry can result in a 40% change in the measured critical current at a magnetic field of 12 T. Specifically, the radial thermal contraction of the measurement mandrel depends on its wall thickness and, thus, so does the conductor prestrain (at 4 K) and, ultimately, the measured critical current. Techniques for reducing variation in the measured critical current are suggested.

Nb/sub 3/Sn critical-current measurements using tubular fiberglass-epoxy mandrels

A systematic study of the effect of sample mounting techniques on the superconducting critical-current measurement was made. A seemingly small change in mandrel geometry can result in a 40% change in the measured critical current of a Nb/sub 3/Sn sample at 12 T. This is a result of a change in the conductor prestrain at 4 K caused by variation in thermal contraction between thick-and thin-walled fiberglass-epoxy composite (G-10) tubes. An approximate measure of the vibrations in thermal contraction (from room to liquid-nitrogen temperature) indicate a 0.2% greater contraction for the thick-walled tube. This difference, combined with strain sensitivity measurements, is consistent with the observed decrease in critical current.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Reactive sputter sectioning: A tool for polymer film analysis

Reactive sputter etching has been used to create thinned ‘‘sections’’ of polymer films for use in microstructural analysis. This paper emphasizes the features of the sputter etching process which must be controlled to permit efficient sectioning without the introduction of artifacts, such as microstructural changes induced by heating and surface texture which might obscure subsequent analysis. Sample mounting techniques which allow oxygen sputter etching rates of 20 μm/h without heating of the film above 50 °C and simple electrode surface design rules which allow etching for long periods with very low levels of surface texturing are described. The minimum oxygen flow rates which must be supplied to prevent a depletion of the oxygen reactant and a concomitant reduction in the etch rate are noted. The utility of the technique is demonstrated with results from a study of the microstructure of a cast polypropylene film as a function of depth through the film.

An Improved Method for the Electrochemical C‐V Profiling of Indium Phosphide

C‐V analysis of n‐type using metal‐semiconductor Schottky contacts is inadequate for determining free carrier concentration vs. depth profiles due to low barrier heights, uncontrolled oxide formation, and metal semiconductor chemical reactions. Concentration vs. depth profiles can be obtained over a wide range of carrier concentrations to virtually unlimited depth using electrochemical C‐V (ECV) analysis. However, the in aqueous solution reported in the literature is not ideal for the anodic dissolution of . This work reports an improved anodic etchant for ECV profiling of n‐type and the design of a constant current etch, difference method ECV profiler utilizing a new sample mounting technique.