Exploiting the full potential of the advanced two-hexapole corrector for STEM exemplified at 60kV.

Ultimate resolution in scanning transmission electron microscopy (STEM) with state-of-the-art aberration correctors requires careful tuning of the experimental parameters. The optimum aperture semi-angle depends on the chosen high tension, the chromatic aberration and the energy width of the source as well as on potentially limiting intrinsic residual aberrations. In this paper we derive simple expressions and criteria for choosing the aperture semi-angle and for counterbalancing the intrinsic sixth-order three-lobe aberration of two-hexapole aberration correctors by means of the fourth-order three-lobe aberration. It is noteworthy that for such an optimally adjusted electron probe the so-called flat area of the Ronchigram is explicitly not maximized. The above considerations are validated by experiments with a CEOS ASCOR in a C-FEG-equipped JEOL NEOARM operated at 60 kV. Sub-Angstrom resolution is demonstrated for a Si[112] single crystal as well as for a single-layered MoS2 crystalline film. Lattice reflections of 73 pm for silicon and 93 pm for molybdenum disulfide are visible in the Fourier transform of the images, respectively. Moreover, single sulfur vacancies can be clearly identified in the MoS2.

Chromatic Aberration Correction for Atomic Resolution TEM Imaging from 20 to 80 kV.

Atomic resolution in transmission electron microscopy of thin and light-atom materials requires a rigorous reduction of the beam energy to reduce knockon damage. However, at the same time, the chromatic aberration deteriorates the resolution of the TEM image dramatically. Within the framework of the SALVE project, we introduce a newly developed C_{c}/C_{s} corrector that is capable of correcting both the chromatic and the spherical aberration in the range of accelerating voltages from 20 to 80 kV. The corrector allows correcting axial aberrations up to fifth order as well as the dominating off-axial aberrations. Over the entire voltage range, optimum phase-contrast imaging conditions for weak signals from light atoms can be adjusted for an optical aperture of at least 55 mrad. The information transfer within this aperture is no longer limited by chromatic aberrations. We demonstrate the performance of the microscope using the examples of 30 kV phase-contrast TEM images of graphene and molybdenum disulfide, showing unprecedented contrast and resolution that matches image calculations.

Thermal magnetic field noise limits resolution in transmission electron microscopy.

The resolving power of an electron microscope is determined by the optics and the stability of the instrument. Recently, progress has been obtained towards subångström resolution at beam energies of 80 kV and below but a discrepancy between the expected and achieved instrumental information limit has been observed. Here we show that magnetic field noise from thermally driven currents in the conductive parts of the instrument is the root cause for this hitherto unexplained decoherence phenomenon. We demonstrate that the deleterious effect depends on temperature and at least weakly on the type of material.

17-ß estradiol and testosterone mineralization and incorporation into organic matter in broiler litter-amended soils.

The presence of the hormones estradiol and testosterone in the environment is of concern because they adversely affect vertebrate sexual characteristics. Land spreading broiler litter introduces these hormones into the environment. We conducted two studies. The first study determined the mineralization of C-labeled estradiol and testosterone at three water potentials and three temperatures in four broiler litter-amended soils. With a few exceptions, the mineralization of each hormone either stayed the same or increased with increasing water content (both hormones) and increasing (estradiol) or decreasing (testosterone) temperature. Mineralization was dependent on soil type. The second study determined the incorporation of C-labeled estradiol and testosterone into (i) three soil organic matter (SOM) fractions (fulvic acid, humic acid, and humin) at two water potentials, two temperatures, and one sampling time, and (ii) at one water potential, one temperature, and seven sampling times. As time increased, higher temperature and water potential decreased percentages of C estradiol and testosterone in water- and acetone-soluble fractions and increased percentages in SOM fractions. However, the distribution of the two hormones in SOM fractions differed. For estradiol, higher temperature and water potential increased the percentage in all three SOM fractions. For testosterone, higher temperature and water potential increased the percentage of hormone in fulvic acid and humin. Although the mineralization studies suggest the potential for these hormones to still have environmental effects, the incorporation of the two hormones into SOM suggest that land spreading these hormones may actually be less of an environmental concern.

Probing the out-of-plane distortion of single point defects in atomically thin hexagonal boron nitride at the picometer scale.

Crystalline systems often lower their energy by atom displacements from regular high-symmetry lattice sites. We demonstrate that such symmetry lowering distortions can be visualized by ultrahigh resolution transmission electron microscopy even at single point defects. Experimental investigation of structural distortions at the monovacancy defects in suspended bilayers of hexagonal boron nitride (h-BN) accompanied by first-principles calculations reveals a characteristic charge-induced pm symmetry configuration of boron vacancies. This symmetry breaking is caused by interlayer bond reconstruction across the bilayer h-BN at the negatively charged boron vacancy defects and results in local membrane bending at the defect site. This study confirms that boron vacancies are dominantly present in the h-BN membrane.

First application of Cc-corrected imaging for high-resolution and energy-filtered TEM.

Contrast-transfer calculations indicate that C(c) correction should be highly beneficial for high-resolution and energy-filtered transmission electron microscopy. A prototype of an electron optical system capable of correcting spherical and chromatic aberration has been used to verify these calculations. A strong improvement in resolution at an acceleration voltage of 80 kV has been measured. Our first C(c)-corrected energy-filtered experiments examining a (LaAlO(3))(0.3)(Sr(2)AlTaO(6))(0.7)/LaCoO(3) interface demonstrated a significant gain for the spatial resolution in elemental maps of La.

17beta-Estradiol and testosterone in drainage and runoff from poultry litter applications to tilled and no-till crop land under irrigation.

Thirteen million [corrected] metric tons of poultry litter are produced annually by poultry producers in the U.S. Poultry litter contains the sex hormones estradiol and testosterone, endocrine disruptors that have been detected in surface waters. The objective of this study was to evaluate the potential impact of poultry litter applications on estradiol and testosterone concentrations in subsurface drainage and surface runoff in irrigated crop land under no-till and conventional-till management. We conducted an irrigation study in fall of 2001 and spring of 2002. Four treatments, no-till plus poultry litter, conventional-till plus poultry litter, no-till plus conventional fertilizer, and conventional-till plus conventional fertilizer, were evaluated. Flow-weighted concentration and load ha(-1) of the two hormones were measured in drainage and runoff. Soil concentrations of estradiol and testosterone were measured. Based on comparisons to the conventional fertilizer (and control) treatments, poultry litter did not add to the flow-weighted concentration or load ha(-1) of either estradiol or testosterone in subsurface drainage or surface runoff. Significant differences were, however, observed between tillage treatments: flow-weighted concentrations of estradiol were greater for no-till than conventional-till plots of the June irrigation; and runoff loads of both estradiol and testosterone were less from no-till than conventional-till plots for the November irrigation. Although the differences between no-till and conventional-tillage appeared to affect the hydrologic transport of both hormones, the differences appeared to have inconsequential environmental impact.

Combining targeted sampling and fluorometry to identify human fecal contamination in a freshwater creek.

Many bacterial source tracking (BST) methods are too expensive for most communities to afford. In an effort to develop an inexpensive method of detecting human sources of fecal contamination in a freshwater creek during baseflow and stormflow conditions, we combined targeted sampling with fluorometry. Targeted sampling is a prelude to BST and finds sources of fecal contamination by continued sampling and resampling over ever-decreasing distances. Fluorometry identifies human fecal contamination in water by detecting fluorescing compounds, optical brighteners, from laundry detergents. Potato Creek, a freshwater creek in Georgia (U.S.A.), had three reaches identified as containing high numbers of fecal bacteria, and these reaches were sampled by targeted sampling and fluorometry. Targeted sampling quickly and inexpensively identified humans, cattle, and dogs as the major sources of fecal contamination in the first, second, and third reaches, respectively. Fluorometric values were consistent with these identifications, but high fluorometric values were sometimes observed in areas with no fecal contamination. One likely cause of these false-positive signals was fluorescence from organic matter. For targeted sampling, the cost of each sample was $6, with a one-time equipment cost of $3,650; for fluorometry, the cost of each sample was negligible, with a one-time equipment cost of $14,250. This was the first study of this relatively inexpensive combination in freshwater during both baseflow and stormflow conditions.

Geographic sharing of ribotype patterns in Enterococcus faecalis for bacterial source tracking.

The limited host range of Enterococcus faecalis may reduce its clonal diversity and thereby increase its geographic sharing of ribotype patterns. Such sharing would be advantageous for bacterial source tracking (BST). We determined the geographic sharing of ribotype patterns in 752 Ent. faecalis isolates obtained primarily from wastewater treatment plants in Delaware (15 locations; 490 isolates), Georgia (2 locations; 48 isolates), Idaho (1 location; 118 isolates), New York (2 locations; 48 isolates), and Puerto Rico (2 locations; 48 isolates). Isolates were ribotyped with a RiboPrinter. When pooled across all locations and analyzed at a similarity index of 100% and a tolerance level of 1.00%, the 752 Ent. faecalis isolates yielded 652 different ribotypes, of which 429 (66%) were unshared. Even when the matching criterion was relaxed by decreasing the tolerance level from 1% to 10% or lowering the similarity cutoff from 100% to 90%, half or almost half of the ribotypes were unshared. A Mantel test of zero correlation showed no statistically significant correlation between ribotype patterns and geographic distance among the 32 samples (one location at one time) at either the 1.00% (P = 0.91) or 10.00% (P = 0.83) tolerance levels. Therefore, the percentage of ribotype patterns shared between two locations did not increase as the distance between locations decreased. In the case of BST, a permanent host origin database sufficiently large to encompass these ribotype patterns would be time-consuming and expensive to construct.

Exposing water samples to ultraviolet light improves fluorometry for detecting human fecal contamination.

Fluorometry identifies human fecal contamination by detecting optical brighteners in environmental waters. Because optical brighteners are sensitive to sunlight, we determined if we could improve fluorometry by exposing water samples to ultraviolet (UV) light to differentiate between optical brighteners and other fluorescing organic compounds. Optical brighteners were likely present when the relative percentage difference in fluorometric value of the water before and after UV light exposure was >30% (glass cuvettes, 30 min exposure) or >15% (polymethacrylate cuvettes, 5 min exposure). In a blind study, we correctly identified the presence or absence of optical brighteners in 178 of 180 (99%) of the samples tested with a more expensive field fluorometer and in 175 of 180 (97%) of the samples tested with a less expensive handheld fluorometer. In the field, the method correctly identified two negative and three positive locations for human fecal contamination. When combined with counts of fecal bacteria, the new fluorometric method may be a simple, quick, and easy way to identify human fecal contamination in environmental waters.

Identifying sources of fecal contamination inexpensively with targeted sampling and bacterial source tracking.

Most bacterial source tracking (BST) methods are too expensive for most communities to afford. We developed targeted sampling as a prelude to BST to reduce these costs. We combined targeted sampling with three inexpensive BST methods, Enterococcus speciation, detection of the esp gene, and fluorometry, to confirm the sources of fecal contamination to beaches on Georgia's Jekyll and Sea Islands during calm and stormy weather conditions. For Jekyll Island, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. For the Sea Island beach during calm conditions, the most likely sources of fecal contamination were leaking sewer lines and wildlife feces. The leaking sewer lines were confirmed with fluorometry and detection of the esp gene. For the Sea Island beach during stormflow conditions, the most likely sources of fecal contamination were wildlife feces and runoff discharging from two county-maintained pipes. For the pipes, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. Sediments were also a reservoir of fecal enterococci for both Jekyll and Sea Islands. Combining targeted sampling with two or more BST methods identified sources of fecal contamination quickly, easily, and inexpensively. This combination was the first time targeted sampling was conducted during stormy conditions, and the first time targeted sampling was combined with enterococcal speciation, detection of the esp gene, and fluorometry.

Mineralization of hormones in breeder and broiler litters at different water potentials and temperatures.

When poultry litter is landspread, steroidal hormones present in the litter may reach surface waters, where they may have undesirable biological effects. In a laboratory study, we determined the mineralization of [4-14C]-labeled 17beta-estradiol, estrone, and testosterone in breeder litter at three different water potentials (-56, -24, and -12 MPa) and temperatures (25, 35, and 45 degrees C), and in broiler litter at two different water potentials (-24 and -12 MPa) and temperatures (25 and 35 degrees C). Mineralization was similar in both litters and generally increased with increasing water content and decreasing temperature. After 23 wk at -24 MPa, an average of 27, 11, and <2% of the radiolabeled testosterone applied to breeder litter was mineralized to 14CO2 at 25, 35, and 45 degrees C, respectively. In contrast, mineralization of the radiolabeled estradiol and estrone was <2% after 25 wk at all water potentials, except after 17 wk at 25 degrees C and -12 MPa, where up to 5.9% of the estradiol and 7.8% of the estrone was mineralized. The minimal mineralization suggests that the litters may still be potential sources of hormones to surface and subsurface waters.

Advancing the hexapole Cs-corrector for the scanning transmission electron microscope.

Aberration correctors using hexapole fields have proven useful to correct for the spherical aberration in electron microscopy. We investigate the limits of the present design for the hexapole corrector with respect to minimum probe size for the scanning transmission electron microscope and discuss several ways in which the design could be improved by rather small and incremental design changes for the next generation of advanced probe-forming systems equipped with a gun monochromator.

Atomic scale analysis of planar defects in polycrystalline diamond.

Planar defects in a polycrystalline diamond film were studied by high-resolution transmission electron microscopy (HRTEM) and high-resolution scanning transmission electron microscopy (STEM). In both modes, sub-Angstrom resolution was achieved by making use of two aberration-corrected systems; a TEM and a STEM C(s)-corrected microscope, each operated at 300 kV. For the first time, diamond in (110) zone-axis orientation was imaged in STEM mode at a resolution that allows for resolving the atomic dumbbells of carbon at a projected interatomic distance of 89 pm. Twin boundaries that show approximately the sigma3 CSL structure reveal at sub-Angstrom resolution imperfections; that is, local distortions, which break the symmetry of the ideal sigma3 type twin boundary, are likely present. In addition to these imperfect twin boundaries, voids on the atomic level were observed. It is proposed that both local distortions and small voids enhance the mechanical toughness of the film by locally increasing the critical stress intensity factor.

Experimental evaluation of a spherical aberration-corrected TEM and STEM.

We have successfully developed a spherical aberration (Cs)-corrected electron microscope for probe- and image-forming systems using hexapole correctors. The performance of the microscope has been evaluated experimentally. The point resolution attained using the image-forming Cs-corrector is better than 0.12 nm. For scanning transmission electron microscopy, the Ronchigram flat area was >40 mrad in half-angle using the probe-forming Cs-corrector.

Presence of Enterococcus faecalis in broiler litter and wild bird feces for bacterial source tracking.

When Enterococcus faecalis is isolated from fresh feces, its host range appears to be limited to humans and birds. Although E. faecalis is found in human sewage, the extent to which the bacterium is found in broiler litter and in the feces of wild birds is unclear. These results have implications for bacterial source tracking. We determined if media designed for the isolation of fecal enterococci affected this host range, and if E. faecalis was routinely found in broiler litter and in the feces of wild birds. Of five different isolation media, none affected the isolation of E. faecalis. Enterococcus faecalis was routinely found in fresh broiler feces (522 of 1092 isolates; 48%), but rarely in broiler litter (12 of 1452 isolates; <2%). Therefore, broiler litter selects against this bacterium, and broiler litter is an unlikely environmental source of this bacterium. The presence of E. faecalis in eight wild bird species was highly variable. Unless the fecal loading rate from migratory or resident wild birds is high, water samples collected during baseflow conditions with high numbers of E. faecalis may indicate human fecal contamination.

Targeted sampling protocol as prelude to bacterial source tracking with Enterococcus faecalis.

Recent studies suggest that host origin databases for bacterial source tracking (BST) must contain a large number of isolates because bacterial subspecies change with geography and time. A new targeted sampling protocol was developed as a prelude to BST to minimize these changes. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. A general sampling of the river showed fecal enterococcal numbers ranging from <10 (below the limit of detection) to 990 colony-forming units (CFU) per 100 mL. Locations with high enterococcal numbers were combined with local knowledge to determine targeted sampling sites. Fecal enterococcal numbers around one site ranged from <10 to 24,000 CFU per 100 mL. Bacterial source tracking was conducted to determine if a wastewater treatment facility at the site was responsible for this contamination. The fecal indicator bacterium was Enterococcus faecalis. Ribotyping, automated with a RiboPrinter (DuPont Qualicon, Wilmington, DE), was the BST method. Thirty-seven ribotypes were observed among 83 Ent. faecalis isolates obtained from the Sapelo River and the wastewater lagoon. Sixteen ribotypes were associated with either the river or the lagoon, and only five ribotypes (14%) were shared. Nevertheless, these five ribotypes represented 39 of the 83 Ent. faecalis isolates, almost a majority (47%). These results suggest that the fecal contamination in the river came from the wastewater treatment facility. As a prelude to BST, targeted sampling minimized subspecies changes with geography and time, and eliminated the need for a permanent host origin database by restricting BST to a small geographic area and requiring sampling to be completed in one day.

Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking.

Ribotyping is one of a number of genotypic methods for bacterial source tracking. This method requires a host origin database of one bacterial species be established in order to identify environmental isolates. Researchers establishing these databases have observed considerable ribotype diversity within a specific bacterial species. One source of this diversity may be diet. We determined the effect of diet on ribotype diversity for Escherichia coli in penned and wild deer (Odocoileus virginianus) in a 13-ha forested watershed. A total of 298 E. coli isolates was obtained, 100 from penned deer, 100 from wild deer, and 98 from the stream in the watershed to which all deer had access. The wild deer had significantly more ribotypes (35) than the penned deer (11 ribotypes, p = 0.05). This result suggests that diet affected ribotype diversity, and that a host origin database for bacterial source tracking should contain bacterial isolates from wild rather than from captive animals. Also, 42 of 98 (42.9%) environmental isolates matched penned and wild deer ribotypes. If bacterial source tracking determines that fecal contamination is predominantly from wildlife, then it may be unnecessary to monitor these watersheds because control over wildlife is difficult.

A spherical aberration-corrected 200 kV TEM.

A spherical aberration (Cs)-corrected 200 kV TEM was newly developed. The column of the microscope was extended by 25 cm and the inner yoke of the objective lens was modified to insert some parts of the corrector elements. The corrector has two hexapole elements that play a main role in Cs correction and they are placed at a position equivalent to the coma-free point of the objective lens by using two transfer doublet lenses. The Cs correction was successfully carried out by means of the third-order aberration that was generated in the two extended hexapoles. The Cs can be corrected to the desired value and also can be overcompensated in order to produce a negative Cs, as with the corrected Cs of -23 microm shown in this work. The optical system of the corrector does not produce second- and fourth-order aberrations, and can correct residual aberrations up to the third order. All of the corrector elements are computer-controlled and the third-order aberrations are quite stable after they are properly corrected. The resolution of 0.135 nm was experimentally confirmed by the Young's fringe method. Image simulations of a silicon [110] single crystal were made with various Cs and defocus values to demonstrate the effectiveness of arbitral control of Cs.

Comparison of genotypic-based microbial source tracking methods requiring a host origin database.

Microbial source tracking (MST) results, obtained using identical sample sets and pulsed field gel electrophoresis (PFGE), repetitive element PCR (rep-PCR) and ribotyping techniques were compared. These methods were performed by six investigators in analysis of duplicate, blind sets of water samples spiked with feces from five possible sources (sewage, human, dog, cow and seagull). Investigators were provided with samples of the fecal material used to inoculate the water samples for host origin database construction. All methods correctly identified the dominant source in the majority of the samples. Modifications of some of these methods correctly identified the dominant sources in over 90% of the samples; however, false positive rates were as high as 57%. The high false positive rates appeared to be indirectly proportional to the levels of stringency applied in pattern analysis. All the methods produced useful data but the results highlighted the need to modify and optimize these methods in order to minimize sources of error.