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    Policy, drought and fires combine to affect biodiversity in the Amazon basin

    NEWS AND VIEWS
    01 September 2021

    Policy, drought and fires combine to affect biodiversity in the Amazon basin

    Analysis of the ranges of nearly 15,000 plant and vertebrate species in the Amazon basin reveals that, from 2001 to 2019, a majority were affected by fire. Drought and forest policy were the best predictors of fire outcomes.

    Thomas W. Gillespie

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    Thomas W. Gillespie

    Thomas W. Gillespie is in the Department of Geography and at the Institute of the Environment and Sustainability at the University of California, Los Angeles, Los Angeles, California 90095, USA.

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    The Amazon basin contains the largest continuous area of tropical rainforests in the world, and has a crucial role in regulating Earth’s climate1. Rates of tropical-rainforest deforestation and the impacts of fire and drought there are well established2,3. Less is known, however, about how these factors might interact to affect biodiversity, and about the role that forest policy and its enforcement have had over time. Writing in Nature, Feng et al.4 address these issues.

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    doi: https://doi.org/10.1038/d41586-021-02320-0

    References1.Marengo, J. A., Tomasella, J., Soares, W. R., Alves, L. M. & Nobre, C. A. Theor. Appl. Climatol. 107, 73–85 (2012).Article 

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    The author declares no competing interests.

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    The homogenous alternative to biomineralization: Zn- and Mn-rich materials enable sharp organismal “tools” that reduce force requirements

    Because biological materials are often viscoelastic composites, with properties dependent on orientation as well as spatial and temporal scales, all tests were designed to mimic natural conditions of tool use. For example, because of possible anisotropies, indentations were made on contact surfaces instead of cross sections of the “tools”; abrasion resistance was measured in the contact direction. Fracture tests were designed to mimic the tension on the side of a tooth or sting subjected to lateral forces; the velocities in our impact tests were between 0.1 and 1 m/s, typical for tool interactions, though 1/10 as fast as for some organisms48,49.OrganismsAll organisms, except the salmon, were housed alive in our laboratory until just before testing. Samples were measured within 24 h of removal from the organism, and were maintained in high-humidity environments during the preparation period. This is particularly important because mechanical properties can depend strongly on water content and this dependence can differ between materials. For example, non-HEBs can harden more than HEBs with drying30,50,51.

    1.

    Leafcutter ants, Atta cephalotes, were obtained from colonies we collected in Flores, Guatemala and Arena Forest Reserve, Trinidad. They were maintained on Himalayan blackberry leaves, Rubus armeniacus, Portugese laurel, Prunus lusitanica, and Japanese spurge, Pachysandra terminalis.

    2.

    Nereid worms, Neanthes brandti (synonymous with Alitta brandti), were collected from trenches dug in bars near the mouth of the Coos River, Charleston, Oregon. They were kept in sea-water in a laboratory refrigerator at about 5 °C.

    3.

    Scorpions, Hadrurus arizonensis, from Arizona, were obtained from commercial suppliers (e.g. Bugs of America, http://bugsofamerica.com) and fed House crickets, Acheta domesticus, and mealworms, Tenebrio molitor larvae.

    4.

    Spiders, Araneus diadematus, were collected seasonally around the University of Oregon campus, and kept alive in a laboratory refrigerator at about 5 °C. Tarantulas, Aphonopelma hentzi were obtained from Carolina (https://www.carolina.com).

    5.

    Chitons, Katharina tunicata, and Cryptochiton stelleri were collected in rocky intertidal regions along the coast near Charleston, Oregon and kept alive in a laboratory refrigerator.

    6.

    Salmon heads, Oncorhynchus sp., were obtained fresh from seafood stores.

    7.

    Leaf cutter bees, Megachile rotundata, were obtained from Mason Bees for Sale (www.masonbeesforsale.com).

    Hardness, modulus of elasticity and damping measurementsHardness, modulus of elasticity and dynamic mechanical property measurements were made by pressing a sharp diamond probe into specimens and measuring the resulting indentation as it changed in time. A higher modulus of elasticity indicates that a structure is stiffer and suffers less elastic (quickly recovered) deformation. A higher hardness value indicates that the material will undergo less plastic (non-recovered) deformation and thus will have a smaller pit left behind after the indentation. A material with a higher loss tangent will absorb more energy of vibration (higher damping), and is characterized by lagging surface deformation and recovery (viscoelasticity) as the indention force changes. Damping can reduce damage because the energy absorbed and converted to heat is not available for breaking bonds in the material.We used an Atomic Force Microsocpe (AFM; NanoScope IIIa, Digital Instruments, Santa Barbara, CA) with an add-on force/displacement transducer (TriboScope, Hysitron Inc., Minneapolis, Minnesota). The Hysitron transducer held a polished diamond probe in place with capacitors that were used to sense the position of the probe and to impart vertical forces for indenting and imaging the specimen. Measurement regions were selected for minimal slope and surface topography as evidenced by the depth variation in AFM scans and the symmetry of the residual indents.In order to make the most biologically relevant measurements, indentations were made on regions of the external surface of structures that directly contact the environment. However, we also made measurements on cross-sections of the arthropod “tools” as part of preliminary SEM and indentation investigations to ensure that the thickness of epicuticle or other surface material would not distort the results for HEBs. Figure 2A shows an indentation on an un-polished surface—the original surface topography is visible as linear scratches that are small compared to the pyramidal indentation. When we could not avoid indentation-scale topography, we hand-polished the surface with 2000–12,000 grit sandpaper (Micro-mesh sheets, http://micro-surface.com), to smooth the surface on the scale of the test indents.Figure 2Images of testing samples for each of the measured properties. (A) AFM image of a residual indentation on the natural surface of the Mn-HEB region of the sting of a scorpion, Hadrurus arizonensis. Indentations were used in measuring hardness, modulus of elasticity and damping properties. Natural “scratches” are visible on the original surface around the triangular indentation made by the cube-cornered indenter. (B) Mandible of an ant, Atta cephalotes, before and after an abrasion testing session. The tip of the zinc-rich distal tooth in the “before” image has been flattened during a preliminary abrasion session. (C) Images from before and after energy of fracture testing of a 12 μm thick Zn-HEB test piece made from the nearly-flat side-surface of the fang of a spider, Araneus diadematus. The original fang shape is evident with the tip of the fang towards the top and the proximal side to the left. (D) Images from before and after impact resistance testing of a 12 μm thick Zn-HEB test piece made from the fang of a spider, Araneus diadematus. The test piece covers a 50 μm diameter backing-pit milled (using FIB-SEM) into a (reflective) silicon chip. The piece has been shattered from the impact in the image on the right.Full size imageSpecimens were mounted on atomic force microscopy (AFM) specimen disks (TedPella Inc., Redding, CA) in a mound of epoxy composite. The composite was prepared by mixing approximately 0.45 g of 400-grit aluminum oxide powder (Buehler, Evanston, Ill., Ted Pella Inc., Redding CA, or Kramer Industries, Piscataway, NJ—the later preferred because it was less reflective) with 0.075 mL each of resin and hardener (Quick Set Epoxy; Loctite, Rocky Hill, CT, and 5 m Quik-Cure Epoxy, Bob Smith Industries, Atascadero CA). The composite was stiff enough that the unpolished specimen had to be pressed in and could be oriented before curing so that the desired indentation region would retro-reflect a light beam sent through the eyepiece of a dissecting microscope back into the microscope, ensuring that the desired region would be flat for AFM scanning and indentation. The mounted specimens were placed in an oven at 39 °C for at least 1 h to cure the epoxy composite. Samples that were not tested immediately were kept on moist paper in a container in a refrigerator and were tested within 24 h to avoid dehydration and other changes. Additionally, the epoxy composite served as a barrier to reduce loss of water through the cut surfaces.The epoxy composite mounting technique was tested to ensure that small (about the same size as the biological specimens) “floating” glass cover-slip pieces would yield the same hardness and modulus of elasticity values as large, flat-mounted cover-slip pieces, and re-checked when relevant products were changed.In order to test whether there were any rapid changes in hardness or modulus of elasticity of HEBs, we tested a H. arizonensis sting, that was mounted for AFM measurements while still attached to an anaesthetized scorpion. We did not find a significant trend with time for 14 measurements made between 10 or 20 min (Zn-HEB and Mn-HEB respectively) after separating the live scorpion from the sting, and 5 h (largest R2: 0.006). These fast results were also comparable to results made using the standard technique, indicating that the technique described above was sufficient to prevent significant changes from dehydration. We also made preliminary measurements on scorpion joint cuticle, armour teeth, and other non-HEB regions of the cuticle, and found none that were harder than the region at the base of the sting, used here to represent non-HEB cuticle.We used a pyramid-shaped diamond probe with cubic corner facets (90° between the three faces)14,31. The steeper angle of the cubic tip, relative to a more commonly used Berkovich tip, made it easier to avoid surface features such as hairs. The diamond probe was positioned on the specimen using a 30× extra short focus monocular (M1030, Specwell Corporation, Tokyo, Japan). The indentation sequence began with the force being ramped linearly from 0 to 2 milliNewtons (mN) in 0.1 s, maintained at 2 mN for 10 s. The force was then ramped down to 1.5 mN over 0.1 s and then the force was varied sinusoidally at 10 Hz (for 25 cycles) with a peak-to-peak amplitude of 1.0 mN, in order to measure dynamic properties. The force was then ramped to 0 in 0.1 s.Probe-extension (Oliver–Pharr) and image-based measurementsTwo methods of obtaining the modulus of elasticity and hardness were employed14. In the first method, values were obtained only from force–displacement curves using the Oliver–Pharr technique52,53. The modulus of elasticity was obtained from the slope of the force–displacement curve at the beginning of withdrawal of the indenter. Oliver–Pharr hardness values were calculated from the intercept of this sloped line with the line of zero force.In addition to the hardness value obtained from the force–displacement curves, we also obtained a hardness value based on measurements of the size of the residual indentation. These image-based hardness values were calculated as H = F/A, where “F” is the maximum force applied to the probe, and “A” is the projected area of the residual indentation, obtained from the perimeter of the indentation measured on an AFM image (e.g. Fig. 2A) made by scanning the indenting probe itself minutes after indenting the specimen.The Oliver–Pharr method is inaccurate if the indentation force causes the surface of the specimen to move, such as for improperly backed specimens, because it assumes probe extension is a measure of indentation depth. In contrast, the image-based method is nearly insensitive to global displacements of the specimen because it is based only on the applied force and measurements of the residual indentation. We found it useful to obtain both values to check each other: on several occasions differences between the two measured values indicated support problems. This is important for biological specimens with multiple layers, voids, etc. For example, if there is a lumen under the shaft but not the tip, the tip may appear harder because it displaces less. In addition, calibration problems, such as from fractured silica, were quickly identified by differences in the image and Oliver–Pharr values. Finally, the image method is not sensitive to other artifacts, such as “pile-up”, that are associated with estimating contact region from probe extension53,54,55,56.There is a potential difference between the Oliver–Pharr and image hardness values associated with the different time scales. The Oliver–Pharr hardness is measured during the probe withdrawal while the image of the residual indent is obtained a couple of minutes after indentation. If the indent partially recovers in the interim, the image technique would be based on a smaller residual indentation resulting in a higher hardness value. We prefer the image method not only because it is robust to imperfectly supported specimens, but also because we would like our hardness measurement to reflect the long-term indentation damage done to the tips and blades. To test that the indent size had stabilized by the time we measured it, we re-measured an indentation in the zinc-region of a scorpion sting after more than 6 months and found that the indentation diameter had decreased little, by about 15%.We also calculated an image-based modulus of elasticity value that has been suggested for materials that produce “pile-up” artifacts53. The area measured from the image of the residual indentation (used for the image-based hardness), was substituted for the contact area calculated from probe extension in Eq. (6) of Oliver & Farr, 2004 53.Notwithstanding the differences in image-based and probe-extension based (Oliver–Pharr) measurements, there was little practical difference in results, as shown in Fig. 3A,B. The metals and plastics that we measured for Fig. 3 tended to have slightly higher Oliver–Pharr hardness values than image-based values, possibly because of “pile-up” artifacts.Figure 3Comparison of probe-extension (Oliver–Pharr) and image-based techniques for hardness (A) and reduced modulus of elasticity (B) for our indentation data. Pile-up may account for higher (above the line) Oliver–Pharr values of some of the metals and plastics.Full size imageIn the “Results” section, we plot the values obtained using the images, but the Oliver–Pharr values are included along with image-based values in the results table, Table 1.Loss tangentDynamic mechanical properties were measured from the sinusoidal segments of the indentation sequence by comparing the amplitude and phase of the displacement to the applied sinusoidal force57. Phase lags associated with the transducer and electronics were determined assuming zero true-lag from a fused silica standard obtained using the same indentation sequence and force. The loss tangents obtained in this way were for the high-stress regimes associated with indentation, as compared to low-stress tests involving bending without plastic deformation.Calibration for Oliver–Pharr measurementsIn order to obtain the contact area from the indent depth, the shape of the indenting tip must be known. We characterized the indenting tip shape directly using scanning electron microscope images, so that we could make deep, micron-scale indents that would be evident on un-polished biological surfaces. We could not make indents in silica as deep as the indents desired for our biological specimens without fracturing the silica (fracture for the cubic cornered tip began at about 3 mN) so we could not use the usual technique of estimating the tip shape at depth by calibrating with fused silica. The tip shape was characterized by three measurements: first, the angle of the three-sided pyramidal tip (α), second, a measure of the bluntness of the tip (B), the distance between the apex of the tip if it were an ideal pyramid and the actual blunt tip, and third, a measure of the distance from the blunt tip beyond which the shape of the tip was not distinguishable from an ideal pyramid (I). The value “I” was used as a limit: only indents with greater depth than “I” were used to calculate mechanical properties. For these deeper indents, the following description of the projected area (A) of the contact region between the tip and the specimen was used:$$A= frac{(0.433)(4){(D+B)}^{2}}{frac{1}{{mathrm{tan}}^{2}left(frac{alpha }{2}right)}-0.3333},$$where D is the depth of the indent, determined by the extension of the indenting probe, 0.433 is the ratio of the area of an equilateral triangle to the square of the length of a side, and 0.3333 is tan2 (30°). As an example, the tip used for the majority of measurements was characterized by α = 89.9°, B = 115 nm, I = 100 nm. Thus, for indents with a depth greater than 100 nm, for our tip, A = 2.58 (D + 115 nm)2.Measurement of residual indentation areaThe area of the residual indent was measured using an AFM image, obtained minutes after the indentation, using the indenting tip as the imaging tip. To minimize inaccuracies in indent perimeter determination, caused by finite size of the imaging probe or other systematic errors, we calibrated our area measurements so that we obtained a median value of 70 GPa for measurements of the modulus of elasticity on fused silica. Because there is some subjectivity in measuring the size of the indentation, operator-specific calibrations, based on each operator’s measurements of fused silica, were used for most of the measurements.Test piece preparation for impact resistance and energy of fracture measurementsWe measured resistance to impact and fracture using custom miniature versions of testing devices that fracture or damage standardized “test pieces” of materials. We prepared test pieces as follows: the fresh (usually immediately after removal from the organism) specimens were adhered to one end of a glass slide using a marine epoxy (Loctite, Rocky Hill, CT) which required a curing time of 2 h at 39 °C, or with cyanoacrylate adhesive (Krazy Glue, all purpose, Elmer’s Products)14, which required no extra curing time. A flat region ( > 100 μm diameter, but not wide enough to reduce the thickness of the HEB region in the center to less than 12 μm) was polished on a specimen by grinding the slide with the specimen against a sequence of flat 2000, 6000 and 12,000 grit sandpaper (Micro-mesh sheets, http://micro-surface.com). The specimen was removed from the adhesive using a scalpel, inverted, and the polished-flat region was adhered to the glass with a thin film of water and surrounded by a small bead of marine epoxy. The water film kept the epoxy from getting pulled under the specimen by capillary action. The epoxy was cured and the specimen polished to a thickness of 12 ± 2 μm as determined with a digital micrometer, using the same sandpaper sequence. The resulting test piece was then freed by scraping the epoxy from around the edges using a scalpel blade. The area of the pieces varied according to the size of flat regions and was, typically, hundreds of microns on a side.Maintaining hydration was especially important for these test pieces because they were only 12 μm thick and so they could dry quickly. To reduce artifacts from drying or other changes in the tested material, all preparation and testing took place in a ~ 15 m3 enclosure maintained at greater than 90% relative humidity.Although we used FIB-SEM (Focused Ion Beam-Scanning Electron Microscope) to shape specimens of the materials for molecular fragment analysis, we did not use this technique for preparing our micron-scale test pieces for several reasons: potential material property changes caused by beam damage, subjection to vacuum, and because some of the test pieces needed to be large enough that they would be difficult to make with FIB-SEM.Impact resistance measurementsA custom testing device was built to compare the energy required for a swinging pendulum to shatter test pieces of the different materials (Fig. 2D). A 12 ± 2 μm thick test piece was adhered by the moisture in the high-humidity enclosure and held in place with an adhesive (spots of cyanoacrylate, Krazy Glue, all purpose, Elmer’s Products, www.elmers.com, or 5-min epoxy gel) over a 50 μm-diameter circular pit milled in a silicon wafer using a FIB-SEM apparatus. The pendulum, made of carbon fiber and aluminum (length: 0.2 m, moment of inertia: 4.25 x 10−6 kg m2) with a diamond impactor tip polished to a diameter of 20 μm, was held by miniature bearings and electronically released from increasing heights until the test piece fractured. The energy required to fracture the specimen was calculated from the release height from which the pendulum fractured the test piece. This energy was normalized by the measured thickness of the specimens to give joules required per meter of thickness. Nevertheless, we consider this test to be a relative test that is not expected to be generalizable to all impacts, as the energy to fracture is likely to depend not only on thickness but also on variables such as the diameter of the impactor tip and the diameter of the backing hole.This impact test differs from Charpy and Izod tests in that the energy required to fracture the specimen was measured by releasing the pendulum from increasing heights until the specimen fractured, rather than by releasing it from a height sufficient to fracture all specimens, and measuring the residual energy of the pendulum after impact. The advantage of our threshold technique is that the threshold of fracture is likely the biologically important quantity, and direct determination of the fracture threshold avoids the possibility that the energy deposited in a single highly-energetic impact might be partially expended in plastic deformation, leading to an overestimate of the threshold energy. A drawback of our technique is that impacts that do not break the specimen may produce damage that weakens the specimen for subsequent impacts. Nevertheless, all specimens were subjected to the same series of increasingly energetic impacts, until fracture, and were thus comparable.Energy of fracture measurementsWe measured the energy or work required to slowly break a test piece in two (Fig. 2c) using a custom fracture toughness measuring device14. The device drove apart two microscope cover slips bridged by the test piece until it split in two, while recording the required force and the displacement (work is the product of force and incremental distance). This work, divided by the area of the new post-fracture surfaces, is the energy of fracture, reported in Joules per meter squared. It is a measure of the energy required, per unit area, to break the bonds that originally held the two pieces together (as long as the kinetic energy is relatively small—the pieces do not fly away), and is one indication of the resistance of a material to fracture.The length of the fracture was measured using a microscope and multiplied by the thickness of the test piece to obtain the fracture area. This area was used to normalize force–displacement curves. The work of fracture per unit area of the fracture was obtained by numerically integrating these normalized force–displacement curves. The load cell was designed to be stiff in order to minimize storage of energy within the apparatus as the specimen underwent tension58. Fracture planes were perpendicular to the original surface and approximately perpendicular to the long axis of the “tool” (Fig. 1C).The test protocol was altered from that used previously14 because the test pieces used here were smaller. Test pieces were not notched in order to avoid fractures from the notching process, and the specimens were adhered to the test apparatus in place (Krazy Glue, all purpose, Elmer’s Products, www.elmers.com) to avoid premature fracture. To improve the bonding of the cyanoacrylate adhesive to the glass cover slips in the high humidity atmosphere, we treating the cover slips with a 10-s dip in a 2% (by volume) 3-aminopropyltriethoxysilane (Sigma Chemical Co., www.sigmaaldrich.com), 98% acetone solution, followed by rinses in deionized water and air drying. The test protocol for the ant mandibular teeth varied from the others in that whole teeth were fractured instead of 12μ polished test pieces.A consistency test with AFM data was developed to identify cases of imperfect bonding to the cover slips, when part of the measured energy was expended in partially pulling the test piece out of the cyanoacrylate adhesive. For samples subject to this problem (usually specimens with small adhesive contact areas, such as the fang specimen in Fig. 1C), we required that the force–displacement curve be consistent with the stiffness of the test piece, expected from a model based on the shape of the individual test piece and the slope of the force–displacement curve for the insertion portion of the nano-indentation sequence for that material. When a piece partially pulled out and failed this test, the apparent stiffness was much lower than the expected stiffness (from nano-indentation) and slight stretch marks were often visible in the adhesive on close inspection.This stiffness consistency test was also found to be useful in identifying cases where part of the fracture was pre-existing but had not been visible in the test piece. The pre-existing fracture would tend to reduce the effective width of the specimen and thus could be identified by a lower than expected stiffness under tension.Abrasion resistance measurementsWe measured the energy required to abrade away a volume of material from our specimens by holding them against a rotating abrasive disk. The energy used in eroding the material is given by the force of friction multiplied by the distance traveled over the abrasive paper (work is the product of the force and incremental displacement), with units of Joules per meter cubed of volume worn away.The “pin on disk” type testing device, developed for testing pieces of crab cuticle14, was used with modified procedures for the smaller specimens here. Instead of cylindrical core samples, whole, approximately conical tips of teeth, fangs or stings were used (Fig. 2B). The samples were affixed with cyanoacrylate gel adhesive (Maxi-Cure, Bob Smith Industries, Atascadero CA) to a steel pin held in the head of the wear tester. This head was mounted on a custom-made load cell that measured the horizontal force produced by friction between the specimen pin and the abrasive turntable. During the wear test, the specimen pin was held against the turntable with adjustable weights that, for the standard test, produced a downward force of 0.019 Newtons. The surface of the turntable was covered with 600 grit abrasive paper (#413Q, 3 M Corporation, www.3M.com). The turntable rotation period was usually set to about 4 s, resulting in an interaction velocity of 0.027 m/s.The volume worn away was calculated from “before” and “after” measurements of microscope images (image J software) taken from the side (e.g. Fig. 2B) to measure the height of the approximate cone of worn material, and from face-on in order to measure the area of the base and top of the frustum of worn-away material. The horizontal force was recorded continuously during the wear period. The wear rate (w), defined as the volume worn away per unit energy expended, was approximated as follows:$$w= frac{V}{Fd} ,$$where F is the average force of friction measured during the wear period by the load cell, d is the distance traveled by the pin over the abrasive paper and “V” is the worn volume, approximated as a frustum:$$V = 1{/}3,Delta L , (A1 + left( {A1*A2} right)^{1/2} + A2)$$where “A1” and “A2” are the areas of the worn surface before and after the wear sessions (the area of any voids or internal lumens was subtracted from the area of the cross sections) and ΔL, the change in the length of the specimen due to wear. We defined wear resistance as the inverse of the wear rate, 1/w. While we expect this test to be most useful for relative comparisons, and the value is expected to vary somewhat with abrasive properties and normal forces, we found no statistically distinguishable difference in values from 4 samples that were re-run using a ten-times greater force to press them against the abrasive paper14.Molecular composition and nanometer-scale structureIn order to better understand the composition and structure of the HEBs—down to an atomic scale—we examined a representative HEB using Atom Probe Tomography (APT). We checked the APT results and studied their generality using Time-of-Flight–Secondary Ion Mass Spectrometry (ToF–SIMS). Both of these techniques use a pulsed beam (laser and ion respectively) to break the specimen into molecular fragments that are accelerated to a detector; for a particular charge, heavier fragments travel more slowly and arrive later at the detector. The arrival time differences are used to identify the fragments by their mass, giving information about, for example, the atoms attached to zinc atoms in the specimen and, from APT, the spatial distribution of zinc atoms on a nanometer scale.Atom probe tomography (APT)APT is a 3D nanoscale characterization method in which field evaporated ions from a sharpened needle specimen are analyzed by a position-sensitive single-particle detector, in order to provide an isotopically resolved three-dimensional representation of the real-space specimen elemental distribution59. The field evaporation of non-conductive samples is achieved using a pulsed laser focused on the needle specimen apex.A FIB-SEM based lift-out procedure was used to prepare needle-shaped APT specimens using FEI Helios 600i at the University of Oregon CAMCOR facility, and a Helios Dual Beam Nanolab 600 FIB-SEM housed at Environmental Molecular Sciences Laboratory, PNNL.The APT analysis was carried out using a CAMECA LEAP (local electrode atom probe) 4000X HR system equipped with a 355 nm wavelength picosecond pulsed UV laser. A 30 K sample base temperature and a 100 or 200 kHz laser pulse repetition rate was used. Atom probe data reconstruction and analysis was performed using Cameca IVAS software.
    Development of APT techniques for these organic materials.APT has not typically been used to examine organic materials, so we began by examining standards (such as zinc picolinate) and adjusting beam current densities and other parameters in both the FIB-SEM preparation of APT samples and in APT itself in order to minimize physical damage detected with SEM and to minimize differences between the chemical formulae and APT results for standards60. We found that current densities often employed in FIB-SEM milling were much too high for our organic specimens, resulting in beam damage visible in SEM.Based on the standards and SEM evidence of damage, we used FIB-SEM currents for producing the sample needles, and APT laser pulses for promoting evaporation, that were similar to or smaller than those used in other investigations of organic materials61,62,63,64,65,66,67,68. We used ≤ 21 pA for the electron beam, ≤ 80 pA for ion beam “trenching”, 7.7 pA for ion beam imaging and for cutting the cantilever “liftout” piece, and ≤ 24 pA for sharpening needles. The results reported here are based on samples analyzed using 10, 20 or 100 pJ laser pulses.Identification of molecular fragments from mass-to-charge ratios is particularly difficult for organic materials because of the many possible fragments of similar mass, and several techniques have been developed to aid in this analysis61,62,63,64,68,69,70,71,72,73.Our identification of zinc-containing fragments was simplified by the pattern of the 3 or 4 main zinc isotopes (Fig. 6A). In addition, we used resources with lists of fragments as a function of mass (e.g. https://webbook.nist.gov/chemistry/mw-ser/). We also cross-checked fragment identification using a Time-of-Flight–Secondary Ion Mass Spectrometry (ToF–SIMS) system.Time of flight–secondary ion mass spectrometry (ToF–SIMS)We used a ToF–SIMS system that had a higher mass-to-charge resolution than the APT system (although it had micron- instead of nanometer-scale spatial resolution) in order to check APT fragment identification. The higher mass sensitivity of the ToF–SIMS system provided additional evidence that, for example, the fragments identified as ZnCN were not actually ZnC2H2, which is only about 0.02% lighter. We also used ToF–SIMS to study the larger-scale spatial distribution of fragments, and similarities with HEBs from other species.We used an ION-TOF ToF–SIMS IV, manufactured by ION-TOF GmbH, Muenster, Germany. The primary ion beam was Bi3+ (25 kV, 10 kHz, 0.4 pA); the static limit (2 × 1012 ions/cm2) was not exceeded. The dimensions of the analysis area varied, but were between 100 × 100 μm and 300 × 300 μm. A low energy electron beam was used for charge neutralization. The spectra were analyzed using the vendor’s software. Chemical maps of peaks of interest were created from the total spectra and used as a basis for retrospective analysis—i.e., pixel-specific extraction of spectra in order to determine the chemical makeup of features of interest. More

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    Dead trees play an under-appreciated role in climate change

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    01 September 2021

    Dead trees play an under-appreciated role in climate change

    How insects help release carbon stored in forests, and the upcoming biodiversity summit COP 15.

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    Nick Petrić Howe

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    In this episode:00:44 Fungi, insects, dead trees and the carbon cycleAcross the world forests play a huge role in the carbon cycle, removing enormous amounts of carbon dioxide from the atmosphere. But when those trees die, some of that carbon goes back into the air. A new project studies how fast dead wood breaks down in different conditions, and the important role played by insects.Research Article: Seibold et al.09:37 Research HighlightsMassive stars make bigger planets, and melting ice moves continents.Research Highlight: Why gassy planets are bigger around more-massive starsResearch Highlight: So much ice is melting that Earth’s crust is moving12:04 The UN’s Convention on Biological DiversityAfter several delays, the fifteenth Conference of the Parties (COP 15) to the United Nations Convention on Biological Diversity, is now slated to take place next year. Even communicating the issues surrounding biodiversity loss has been a challenge, and reaching the targets due to be set at the upcoming meeting will be an even bigger one.Editorial: The scientific panel on biodiversity needs a bigger role 19:32 Briefing ChatWe discuss some highlights from the Nature Briefing. This time, cannibal cane toads and a pterosaur fossil rescued from smugglers.News: Australia’s cane toads evolved as cannibals with frightening speedResearch Highlight: A plundered pterosaur reveals the extinct flyer’s extreme headgearNational Geographic: Stunning fossil seized in police raid reveals prehistoric flying reptile’s secretsSubscribe to Nature Briefing, an unmissable daily round-up of science news, opinion and analysis free in your inbox every weekday.Never miss an episode: Subscribe to the Nature Podcast on Apple Podcasts, Google Podcasts, Spotify or your favourite podcast app. Head here for the Nature Podcast RSS feed.

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