Focused ion beam fabrication of solidified ferritin into nanoscale volumes for compositional analysis using atom probe tomography.

The purpose of this investigation was to conduct atom probe tomography (APT) analyses on ferritin specimens prepared with focused ion beam (FIB) to assess whether this approach can be used to effectively characterize biomaterials. Soft matter is particularly sensitive to ion beam exposure which can induce physical and chemical changes. We employ protective metal films and low-energy ion fluence to mitigate potential problems that may be introduced by FIB. This study had two major objectives: (1) to qualitatively assess the viability of the specimens when subjected to the unique physical conditions of APT analysis, namely ultrahigh vacuum, high electric field, and thermal pulsing using a laser and (2) to quantitatively assess the data from such specimens under various experimental parameters and compare the results with appropriate control specimens. For the first objective, a range of experimental parameters were determined that met the basic criteria necessary to validate that ferritin-based specimens prepared with FIB can retain structural integrity during APT analysis. Initial field evaporation time-of-flight mass spectrometry (TOF-MS) data show that the specimens fabricated with FIB are capable of emitting ions under various laser pulsing conditions with a high electric field applied. For the second objective, the experimental parameter space was narrowed to a range that yielded data quality sufficient to produce meaningful comparison between the ferritin-based specimens and the salt-only controls.

Improvements in planar feature reconstructions in atom probe tomography.

Standard atom probe tomography spatial reconstruction techniques have been reasonably successful in reproducing single crystal datasets. However, artefacts persist in the reconstructions that can be attributed to the incorrect assumption of a spherical evaporation surface. Using simulated and experimental field evaporation, we examine the expected shape of the evaporating surface and propose the use of a variable point projection position to mitigate to some degree these reconstruction artefacts. We show initial results from an implementation of a variable projection position, illustrating the effect on simulated and experimental data, while still maintaining a spherical projection surface. Specimen shapes during evaporation of model structures with interfaces between regions of low- and high-evaporation-field material are presented. Use of two-and three-dimensional projection-point maps in the reconstruction of more complicated datasets is discussed.

Atom probe tomography analysis of poly(3-alkylthiophene)s.

Pulsed-laser atom-probe tomography is used to compare the field-evaporation mass spectrum and spatial distribution of molecular fragments from various poly(3-alkylthiophene) films deposited on sharpened aluminium specimen carriers using two different deposition methods. Films deposited via a modified solution-cast methodology yield small fragments with a uniform structural morphology whereas films deposited via an electrospray ionization methodology yield a wide range of fragments with a very non-uniform structural morphology. The main field-evaporated chemical species identified for both deposition types were, in order of typical relative abundance, C(2)H(5) (+), CH(3) (+), C(2)H(4) (+), followed by C(3)H(7,8) (+)/SC(+) and SCH(+). Thick electrospray depositions allowed investigation of the influence of laser-pulse energy on the analysis. Evidence is presented supporting the presence of a critical laser-pulse energy whereby changes in film morphology are signalled by the appearance of a new mass fragment at 190 Da.

Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues.

The hepatocyte nuclear factor 3alpha (HNF-3alpha) and 3beta proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3alpha and HNF-3beta mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.

In situ site-specific specimen preparation for atom probe tomography.

Techniques for the rapid preparation of atom-probe samples extracted directly from a Si wafer are presented and discussed. A systematic mounting process to a standardized microtip array allows approximately 12 samples to be extracted from a near-surface region and mounted for subsequent focused-ion-beam sharpening in a short period of time, about 2h. In addition, site-specific annular mill extraction techniques are demonstrated that allow specific devices or structures to be removed from a Si wafer and analyzed in the atom-probe. The challenges presented by Ga-induced implantation and damage, particularly at a standard ion-beam accelerating voltage of 30 keV, are shown and discussed. A significant reduction in the extent of the damaged regions through the application of a low-energy "clean-up" ion beam is confirmed by atom-probe analysis of the damaged regions. The Ga+ penetration depth into {100} Si at 30 keV is approximately 40 nm. Clean-up with either a 5 or 2 keV beam reduces the depth of damaged Si to approximately 5 nm and <1 nm, respectively. Finally, a NiSi sample was extracted from a Si wafer, mounted to a microtip array, sharpened, cleaned up with a 5 keV beam and analyzed in the atom probe. The current results demonstrate that specific regions of interest can be accessed and preserved throughout the sample-preparation process and that this preparation method leads to high-quality atom probe analysis of such nano-structures.

Encapsulation method for atom probe tomography analysis of nanoparticles.

Open-space nanomaterials are a widespread class of technologically important materials that are generally incompatible with analysis by atom probe tomography (APT) due to issues with specimen preparation, field evaporation and data reconstruction. The feasibility of encapsulating such non-compact matter in a matrix to enable APT measurements is investigated using nanoparticles as an example. Simulations of field evaporation of a void, and the resulting artifacts in ion trajectory, underpin the requirement that no voids remain after encapsulation. The approach is demonstrated by encapsulating Pt nanoparticles in an ZnO:Al matrix created by atomic layer deposition, a growth technique which offers very high surface coverage and conformality. APT measurements of the Pt nanoparticles are correlated with transmission electron microscopy images and numerical simulations in order to evaluate the accuracy of the APT reconstruction.

Intimo-intimal intussusception and other unusual manifestations of aortic dissection.

Four cases with unusual aortic dissection found unexpectantly at the time of operation, and their aortographic and anatomic findings and surgical treatment are described. One of the patients had a type I aortic dissection with intimo-intimal intussusception and another had a type I dissection with one portion of the intimal wall unidentifiable up to the origin of the innominate artery. One patient had a type III dissecting aneurysm with retrograde dissection into the aortic arch and ascending aorta, and the fourth patient had a dissecting aneurysm involving the ascending aorta and the innominate, right common carotid, and right vertebral arteries with a re-entry at the aortic arch.

Technique and value of operative arteriography in coronary artery bypass operations.

In an attempt to make evaluation of coronary artery bypass more meaningful, operative coronary arteriography has been used to aid in defining more precisely the technical aspects of the operation at a time when corrective measures can be carried out. Arteriography is performed following the completion of distal bypass graft anastomoses, whether saphenous vein or internal mammary artery (IMA) bypasses are used. Cut films are exposed at the rate of 2 to 4 frames per second and are evaluated for showing (1) status of anastomosis, (2) status of runoff, (3) lesions undetected preoperatively, and (4) any problems with the bypass conduit. A total of 934 vessels (including 107 IMA grafts) have been visualized in 535 patients. Some form of technical revision was necessary in nine instances. The use of this technique during intraoperative balloon dilatation, in conjunction with and to extend the benefits of conventional bypass, is currently being evaluated.

"Redo" surgery after operations for aneurysm and occlusion of the abdominal aorta.

During a 20 year period, 1,287 patients were submitted to arterial reconstruction for aneurysm and occlusion of the aorta and iliac arteries. Good results were obtained in 1,230 (95.6 percent). Of these, 83 (6.4 percent) returned for reoperation. Reoperation was performed also in 18 patients operated upon elsewhere. Thus this series of cases of reoperation consists of 101 patients. The most common cause for reoperation and interval after initial procedure were recurrent obstruction--44 to 90 months, false aneurysm--94 months, and infection--25 months. The most common complication in patients treated initially for aneurysm was false aneurysm and recurrent obstruction in the patients treated for aortoiliac artery occlusion. Infection was rare and occurred in both groups. False aneurysm was due to fragmentation of suture in most cases and recurrent obstruction was related to progression of the disease, the type of original procedure employed, and technical factors. Although various methods of treatment were employed, the preferred are (1) aneurysm replacement for false aneurysm, (2) bilateral aortofemoral or aortapopliteal bypass for recurrent obstruction with profundaplasty when necessary, and (3) bilateral axillofemoral or axillopopliteal bypass and graft removal for infection. The results of reoperation were considered to be satifactory with survival in 89 (88 percent) and good functional results in 84 (94 percent) of survivors. Amputation was necessary in only five (6 percent) patients.

Arterial fibromuscular disease. Observations on pathogenesis and surgical management.

Fibromuscular dysplasia is being recognized more often as a significant pathologic vascular disease separate from atherosclerosis. The pathogenesis and pathology are still being investigated. This disease involves mainly three areas--renal, carotid and iliac arteries--with the renal area being the most common. Surgery has been used almost exclusively for symptomatic lesions, and the indications and procedures have been refined since the original procedures of resection and grafting. In patients with renal involvement, we first attempt to treat hypertension medically, followed by surgery if this is unsuccessful. In those with carotid involvement, we are very aggressive with symptomatic lesions since there is a 22 percent incidence of a significant preceding event in the patients operated on. In patients with iliac involvement, we treat according to symptoms. Graduated internal dilatation has been used very successfully alone and in combination with other methods. Postoperative follow-up and arteriography have shown the effects of surgery to be lasting and rewarding, with minimal morbidity and mortality and no recurrence.

Stabilization of carbon-fiber cold field-emission cathodes with a dielectric coating.

A comprehensive investigation has been carried out to determine the source of an inherent temporal instability in the spatial distribution and the electron emission current obtained from field-emitting carbon fiber tips. These instability effects were successfully overcome by coating the tip with a sub-micron layer of dielectric epoxy resin coating. The influence of the coating thickness was studied and an optimum thickness of 0.2-0.3 microm that produced high emission stability was found. A large reduction in the intensity fluctuations of the emission image, at this coating thickness is demonstrated by using chart recorder traces in addition to slow scans of an optically monitored screen signal. The current-voltage (I-V) characteristics were obtained at a threshold field that is a few times lower than that of the uncoated tip. At low emission current levels linear F-N plots were obtained with a slope value lower than that of the uncoated emitter. The spatial distribution consisted of a very bright spot without any internal structure. The total energy distribution of the emitted electrons demonstrated a non-metallic behavior. The spectra obtained consisted of a single peak for low currents and a double peak for higher currents. The electron energy was measured relative to the Fermi level of tungsten and a spectral shift was shown to be a function of the current. Experiments have shown that the coated tips are not affected by the variations of pressure conditions down to 10(-6) mbar. These results suggest that a resin coated fiber tip offers superior performance to tungsten as a cold field emission electron source. Numerous improvements in the performance are underway. This includes a variety of polymeric coatings and more emissive carbon fibers.

Fabrication of specimens of metamorphic magnetite crystals for field ion microscopy and atom probe microanalysis.

Field ion specimens have been successfully fabricated from samples of metamorphic magnetite crystals (Fe3O4) extracted from a polymetamorphosed, granulite-facies marble with the use of a focused ion beam. These magnetite crystals contain nanometer-scale, disk-shaped inclusions making this magnetite particularly attractive for investigating the capabilities of atom probe field ion microscopy (APFIM) for geological materials. Field ion microscope images of these magnetite crystals were obtained in which the observed size and morphology of the precipitates agree with previous results. Samples were analyzed in the energy compensated optical position-sensitive atom probe. Mass spectra were obtained in which peaks for singly ionized 16O, 56Fe and 56FeO and doubly ionized 54Fe, 56Fe and 57Fe peaks were fully resolved. Manganese and aluminum were observed in a limited analysis of a precipitate in an energy compensated position sensitive atom probe.

On the many advantages of local-electrode atom probes.

Local extraction electrodes offer several crucial advantages for operation of atom probes. Because of the proximity of the local extraction electrode to the specimen, the electric field produced at the specimen apex by a given voltage is enhanced and the voltage required for field evaporation is reduced. In a voltage-pulsed atom probe, the absolute magnitude of the energy uncertainty is correspondingly reduced. High mass resolution (m/deltam > 1000) may therefore be obtained by accelerating the evaporated ions to a greater total potential after the local extraction electrode. The low extraction voltage may also be pulsed rapidly (100 ps rise time) and at high repetition rates (up to 10(5) pulses per second) using currently available solid-state pulsers. Furthermore, a local electrode and intermediate electrodes may be used as optical elements to control the image magnification. All of these benefits may be applied to any type of atom probe. Local-electrode atom probes (LEAP) should be especially advantageous for developing three-dimensional atom probes with high mass resolution and a large field of view. A sample has been developed that consists of many microtips formed on a planar sample using ion beam mask etching. Microtip samples are especially suited to LEAP. Analysis of electrically insulating samples may also be possible with microtip samples in a LEAP. This combination of features suggests flexible, high speed, high mass resolution atom probes that can work with either conventional needle-shaped specimens or the new style of planar microtip specimens.

Evaluation of chicken infectious anemia virus and associated risk factors with disease and production losses in broilers.

A case-control study was performed to determine the significance of chicken infectious anemia virus (CIAV) as a risk factor associated with secondary disease in commercial broilers and to identify the significance of production losses associated with CIAV. The study also examined the relationship between bursal and thymic atrophy and the presence of CIAV. Cases were defined as submissions to the Alabama Veterinary Diagnostic Laboratories with a history of clinical disease and with a diagnosis of coccidiosis, gangrenous dermatitis, or respiratory disease. Controls were selected from submissions with neither a history of disease nor evidence of disease on necropsy. CIAV was detected in fresh tissues by polymerase chain reaction. Both thymic atrophy and the detection of CIAV were significantly associated with a disease case (P < 0.05). Bursal atrophy was a significant risk factor associated with the detection of CIAV in a submission (P < 0.05). Whereas CIAV was associated with disease cases that showed production losses in both percentage of livability and percentage of condemnations (P < 0.05), detection of CIAV alone was not associated with detectable losses in production or flock performance.

Advances in Atom Probe Specimen Fabrication from Planar Multilayer Thin Film Structures.

A sample preparation method has been developed whereby sharp needle-shaped specimens for atom probe analysis are fabricated from multilayer thin films deposited onto silicon substrates. The specimens are fabricated in an orientation such that atom probe composition profiles across the layer interfaces can be determined with atomic-layer spatial resolution, i.e., the layer normals are parallel to the needle axis. The method uses standard silicon etching techniques and focused ion-beam milling. The feasibility and utility of this technique are shown through its application to a NiFe/CoFe/Cu/CoFe-based thin film structure.