We kindly acknowledge Marco Albano for providing the video dealing with optical microscope and SEM and Domenico Mannetta for the rock cutting procedure. This research has been supported by the ERC Grant GLASS n&#176; 259256 and TECTONIC n&#176; 835012. This contribution was greatly improved by the comments of three anonymous reviewers and by the editorial production suggestions on the video.

1. Rock sample collection
<ol>
	<li>In a well-exposed outcrop of a natural phyllosilicate-rich fault, select the right exposure (fault rock well preserved along a plane containing fault lineation) where to collect a representative rock sample for the experiments. Take care to select a fault rock with a foliation spacing not larger than a few millimeters. This is done to capture phyllosilicate horizons in rectangular wafers up to 1.5 cm thick that will be sheared during friction experiments.</li>
	<li>Use a hammer and chisel to obtain a fault rock sample with an area of about 10 cm x 10 cm and thickness of more than 3 cm.</li>
	<li>Mark the sense of shear on the rock sample, based on kinematic indicators observed in the field (e.g., slickensides, foliations, drag folds, etc.). 
	NOTE: The area of the sample can be smaller than 10 cm x 10 cm, but it must be larger than 5 cm&#160;x 5 cm that is the dimension of the forcing blocks of the experimental apparatus. 
	&#8203;CAUTION: The foliated rock samples are very friable and therefore after the collection it may be useful to wrap the sample with some tape or a plastic film. It is mandatory that collected samples are not altered by weathering and therefore that these rocks represent the fault rock at seismogenic depth.</li>
</ol>
2. Sample preparation for friction experiments in the double direct shear configuration
<ol>
	<li>Cut the rock sample to obtain rectangular wafers that fit the forcing blocks of the rock deformation apparatus. This is usually achieved in two steps: in the first step, use a standard laboratory saw to obtain a rock sample that is slightly larger than the forcing blocks; secondly use a high precision rotary blade or a hand-grinder to shape wafers 5 cm x 5 cm in area and 0.8-1.2 cm thickness (Figure 1, left). For a standard double-direct shear test, two wafers of the same rock will be required to perform a single experiment. During the cut and shaping procedure, ensure that natural phyllosilicate-rich shear planes contained in the sample are maintained parallel to the surface of the forcing blocks. This means that the foliation is parallel to the 5 cm x 5 cm face of the wafer.</li>
	<li>Use a disk mill to crush the remaining material from the cut of the intact samples and sieve the material to obtain powders with a grain size &#60;125 &#181;m (Figure 1, right).</li>
	<li>Mount two identical wafers on stainless steel forcing blocks with nominal frictional contact area of 5 cm &#215; 5 cm and then assemble them with the central forcing block to compose the symmetric double direct shear configuration. 
	NOTE: It is important that the shear sense imposed by the machine on the sample coincides with the natural sense of shear recorded on the wafer and marked at point 1.3.</li>
	<li>Use the powders to construct two identical rock layers with thickness of about 5 mm and area of 5 cm x 5 cm2. For these powdered rock samples, the natural foliation is destroyed by the sample preparing procedure with a disk-mill. Use a precise leveling jig to obtain a uniform and reproducible rock layer composed of powdered material. Compose the symmetric double direct shear assembly.</li>
</ol>
3. Friction experiments
<ol>
	<li>In a biaxial apparatus27,28 use the horizontal servo-controlled hydraulic piston to apply and maintain a constant normal stress to the rock sample.</li>
	<li>Apply shear stress at constant displacement rate, usually 10 &#956;m/s, via the vertical servo- controlled hydraulic piston. 
	NOTE: Loads are measured via two strain gauge load cells (accuracy 0.03 kN) positioned between the ram and the sample assembly. Horizontal and vertical displacements are measured by LVDT (linear variable differential transducers), with an accuracy of 0.1 &#181;m, referenced at the load frame and the upper side of the ram27,28.</li>
	<li>Characterize all experiments by an initial strain hardening, where the shear stress increases rapidly during elastic loading, before a yield point, followed by shear at a steady-state value of frictional stress.</li>
</ol>
4. Post-experimental sample collection
<ol>
	<li>At the end of the friction test, carefully extract the experimental fault. Rubber bands or adhesive tape can be applied to the sample before load removal to maintain the integrity of the deformed rocks.</li>
	<li>Impregnate with epoxy resin the rock samples. If possible for powder experiments, avoid vacuum impregnation to prevent damage to the original microstructure by the forced flow of the resin into the sample.</li>
	<li>Cut these rock samples parallel to the experimental shear direction. There are several ways to track the experimental shear direction. In our double direct shear configuration, the surfaces of the steel sliding blocks in contact with gouge layers are machined with grooves 0.8 mm high and spaced 1 mm to avoid slip at the interface between gouges and steel and to ensure shear deformation within the gouges, therefore in our experiments the shear direction is perpendicular to grooves.</li>
	<li>Build thin sections from the cuts for microstructural studies.</li>
</ol>
5. Microstructural analysis
<ol>
	<li>Investigate with an optical microscope to characterize the bulk fault zone microstructure.</li>
	<li>Use a scanning electron microscope (SEM) to investigate the main deformation processes.</li>
	<li>Use a transmission electron microscope (TEM) to obtain details on the deformation processes at the nanoscale. Details on how to perform microstructural analysis can be found in previous publications4,5,6,7,8,9,10,29.</li>
</ol>

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The authors have nothing to disclose.

An important point worth mentioning is that with this procedure we characterize the steady state fault frictional strength, measured with experiments at low sliding velocities (i.e., 0.01 &#181;m/s &#60; v &#60; 100 &#181;m/s). The measured low values of friction demonstrate the weakness of phyllosilicate-rich faults resulting from long-term fluid assisted reaction softening and foliation development1,4,5,6,7,8,9,10,11,12,30. This low frictional strength can be used as a proxy to evaluate the fault strength at steady-state or during the pre-seismic phases of the seismic cycle. Therefore, the important dynamic weakening mechanisms that occur at high slip velocities (i.e., &#62; 10 cm/s) and induced by temperature rise33 are not considered in our analysis.
The critical steps in the protocol regard the sample collection and preparation. Since phyllosilicates are characterized by very low tensile strength in the direction perpendicular to the (001) basal planes (i.e., in the direction perpendicular to the foliation), during the work with the hammer and chisel in the field or with the hand-grinder in the lab, quite often the rock samples fall apart and the shaping process has to restart. Therefore, it is strongly recommended to collect more samples than those strictly required to run experiments and arm yourself with patience.
Before integrating mechanical with microstructural data, it is important to check that the frictional sliding along the phyllosilicate-rich foliae observed along natural fault rocks is reproduced in the lab, or in other words that the natural fault rock microstructure is similar to the one obtained from shearing the wafer (Figure 3).
In experiments on solid wafers characterized by thin networks of phyllosilicates, the continuous layers of weak mineral phases can be consumed during significant shearing (displacement &#62; 12 mm). At this stage the deformation is accommodated by a combination of cataclasis of the strong mineral phases and sliding along the phyllosilicates. This coincides with a phase of strain hardening with an increase in friction of about 0.1 or more13.
The majority of rock deformation experiments, aimed at the characterization of the frictional properties of tectonic faults, are performed on millimetric rock layers that are composed by powders obtained by crushing and sieving natural fault rocks24,27 or on fault rocks that are pre-cut34. These types of experiments are fundamental to characterize the frictional properties of faults where the deformation occurs on fault gouges35 or along sharp slipping planes of localized deformation36. For faults rich in phyllosilicates, low friction and hence fault weakness is related to the interconnectivity of the phyllosilicate-rich networks, which in the field is manifested by multiple anastomosing principal slip zones. This indicate that even a small quantity of phyllosilicates can induce significant fault weakening if their interconnectivity is very high37,38. Therefore, the final goal of our laboratory experiments on solid wafers is to preserve the natural continuity of the phyllosilicate-rich layers during friction tests.
Other laboratory experiments on powdered mixtures of strong and weak mineral phases have documented fault weakening with the addition of the weak phases18,19,20,21,22. It has been observed that amounts of 40-50% of phyllosilicates induce a significant reduction in friction because during shearing they become interconnected. This suggests that for large percentages of phyllosilicates (i.e., &#62; 40%), experiments on wafers or powders are similar25.
A compilation of friction tests conducted on a large number of natural fault rocks rich in phyllosilicates, wafers or powdered material with phyllosilicates percentages &#62; 40%, under a wide range of experimental conditions show that friction is in the range of 0.1-0.330. This implies that a significant number of crustal faults are weak.

The overall goal of this procedure is to test the frictional properties of intact phyllosilicate-rich faults sheared in their in situ geometry and to show that their friction is significantly lower than friction obtained from experiments conducted on powders of the same material.
Numerous geological studies have documented fluid-assisted reaction softening during the long-term evolution of tectonic faults. Softening occurs by the replacement of strong minerals, like quartz, feldspar, calcite, dolomite, olivine, pyroxene, with weak phyllosilicates1,2,3,4,5,6,7,8,9,10. This weakening originates at the grain-scale and is mainly due to sliding, at very low friction, along the phyllosilicate foliae that act together to produce a form of lubrication. From the grain-scale, fault weakening is transmitted to the entire fault zone via the interconnectivity of the phyllosilicate-rich zones11. To capture the role of frictional sliding along interconnected phyllosilicate foliae, intact solid wafers of natural fault-rock samples have been sheared in their in situ geometry during rock deformation experiments12,13,14. At the end of the experiment, microstructural studies on the tested samples have been performed to check if effectively the deformation was accommodated by frictional sliding along the phyllosilicate foliae.
In comparison with traditional friction tests performed on powdered materials obtained from crushing and sieving the fault rock, experiments on intact wafers can capture the frictional sliding along the interconnected phyllosilicate-rich layers formed by fluid assisted reaction softening. In fact, during the process of powder preparation, the crushing and sieving of the fault rock disrupts the connectivity of the phyllosilicate layers and when the material is sheared in the laboratory, the absence of continuous phyllosilicate horizons favors a deformation mainly consisting of grain crushing, rotation and translation resulting in high friction.
Experiments on solid wafers show a significantly lower friction in comparison to experiments on powdered material obtained from the same rock type, particularly when the percent of the phyllosilicates is &#60; 40%15. With increasing phyllosilicate abundance, a reduction in friction has been documented also for tests on powdered material, since in this case the large volume of phyllosilicates is sufficient to promote interconnectivity of the weak mineral phases through the entire experimental fault16,17,18,19,20,21,22. Alternatively, to simulate frictional sliding on the interconnected weak layers, other types of friction tests have been performed on powders composed of 100% weak mineral phases23,24,25.
Geometrical fault weakening promoted by rock fabric in deformation experiments at high temperature, and therefore representative of the ductile lithosphere, has been well known for many years26. The results obtained from the procedure presented here indicate that phyllosilicate fabric promotes fault weakening also for a large number of faults contained within the seismogenic upper crust.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>disk mill</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Standard disk mills to pulverize rocks</td>
		</tr>
		<tr>
			<td>fault rock</td>
			<td>Natural outcrops</td>
			<td>none</td>
			<td>All the outcrops rich in phyllosilicates worldwide</td>
		</tr>
		<tr>
			<td>hammer and chisel</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Standard hammer and chisel used by geologists</td>
		</tr>
		<tr>
			<td>optical microscope</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Standard microscope used for mineralogy</td>
		</tr>
		<tr>
			<td>rock deformation apparatus</td>
			<td>we use prototypes like BRAVA &#38; BRAVA2.0</td>
			<td>none</td>
			<td>Eock deformation apparatusses (Marone et al., 1998; Collettini et al., 2014)</td>
		</tr>
		<tr>
			<td>saw to cut rocks</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Standard saws to cat fault rocks</td>
		</tr>
		<tr>
			<td>SEM, scanninc electron microscope</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Microscope to investigate microstructures at the micron scale</td>
		</tr>
		<tr>
			<td>TEM, transmission electron microscope</td>
			<td>Plenty of companies</td>
			<td>none</td>
			<td>Microscope to investigate microstructures at the nano scale</td>
		</tr>
	</tbody>
</table>

the role of fabric in frictional properties of phyllosilicate-rich tectonic faults

Many rock deformation experiments used to characterize the frictional properties of tectonic faults are performed on powdered fault rocks or on bare rock surfaces. These experiments have been fundamental to document the frictional properties of granular mineral phases and provide evidence for crustal faults characterized by high friction. However, they cannot entirely capture the frictional properties of faults rich in phyllosilicates.
Numerous studies of natural faults have documented fluid-assisted reaction softening promoting the replacement of strong minerals with phyllosilicates that are distributed into continuous foliations. To study how these foliated fabrics influence the frictional properties of faults we have: 1) collected foliated phyllosilicate-rich rocks from natural faults; 2) cut the fault rock samples to obtain solid wafers 0.8-1.2 cm thick and 5 cm x 5 cm in area with the foliation parallel to the 5x5cm face of the wafer; 3) performed friction tests on both solid wafers sheared in their in situ geometry and powders, obtained by crushing and sieving and therefore disrupting the foliation of the same samples; 4) recovered the samples for microstructural studies from the post experiment rock samples; and 5) performed microstructural analyses via optical microscopy, scanning and transmission electron microscopy. 
Mechanical data show that the solid samples with well-developed foliation show significantly lower friction in comparison to their powdered equivalents. Micro- and nano-structural studies demonstrate that low friction results from sliding along the foliation surfaces composed of phyllosilicates. When the same rocks are powdered, frictional strength is high, because sliding is accommodated by fracturing, grain rotation, translation and associated dilation. Friction tests indicate that foliated fault rocks may have low friction even when phyllosilicates constitute only a small percentage of the total rock volume, implying that a significant number of crustal faults are weak.

In a diagram of normal stress vs. shear stress both solid foliated and powdered samples plot along a line consistent with a brittle failure envelope, but the solid wafers have friction values significantly lower than their powdered analogues30. For example, in the specific case of a talc-rich foliation, foliated fault rocks at each normal stress have a friction coefficient that is 0.2-0.3 lower than the powders made from them (Figure 2 and12). The lower friction is explained by microstructural studies of the tested rocks showing that the sliding surfaces of the foliated solid wafers occur along the pre-existing phyllosilicate-rich foliation. TEM images show that slip is mainly accommodated by (001) easy sliding associated with interlayer delamination. In contrast, experimental microstructures from the powdered material show that significant deformation is accommodated by grain-size reduction and localization.
Although the foliated wafers of intact fault rocks and their powders have identical mineralogical compositions, the foliated samples show friction that is significantly lower than their powdered analogues. Microstructural studies indicate that the lower friction (i.e., fault weakness) of the foliated fault rocks is due to the reactivation of the pre-existing natural phyllosilicate-rich surfaces that are absent in the powdered samples since the sample preparation steps (2.2 - 2.4) disrupt them.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62821/62821fig01.jpg" /> 
Figure 1:&#160;Representative images of the tested fault rocks: solid foliated vs. powdered material. (left) Solid foliated samples sheared parallel to the natural foliation marked by the arrows. (right)&#160;Powders obtained by crushing and sieving the solid foliated rock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62821/62821fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62821/62821fig02.jpg" /> 
Figure 2:&#160;Friction tests on the same material (talc-rich foliation) but solid foliated samples vs. powdered rock. Each dataset plot along a line consistent with a brittle failure envelope, but the solid foliated rocks are characterized by friction significantly lower than their powdered analogue, friction, &#956; = 0.3 and &#956; = 0.57 respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62821/62821fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62821/62821fig03.jpg" /> 
Figure 3:&#160;Natural vs. laboratory reactivated foliation. In the left picture an example of a natural talc-rich foliation with surrounding sigmoidal clasts of calcite31. The right picture shows the same foliation at the end of a friction test on wafers32. Note that during the friction test most of the slip occurs by frictional sliding along the phyllosilicate layers and that the original microstructure is preserved. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62821/62821fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Role of Fabric in Frictional Properties of Phyllosilicate-Rich Tectonic Faults at JoVE.com

The Role of Fabric in Frictional Properties of Phyllosilicate-Rich Tectonic Faults

Friction of phyllosilicates-rich faults sheared in their in situ geometry is significantly lower than friction of their powdered equivalents.

Many rock deformation experiments used to characterize the frictional properties of tectonic faults are performed on powdered fault rocks or on bare rock ...

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3.0K Views.  Sapienza Università di Roma. Friction of phyllosilicates-rich faults sheared in their in situ geometry is significantly lower than friction of their powdered equivalents. 

Video: The Role of Fabric in Frictional Properties of Phyllosilicate-Rich Tectonic Faults

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future energy resources and energy storage media. To develop a method for gas production from natural MH-bearing sediments and hydrate-based technologies, it is imperative to understand the thermal properties of gas hydrates.
The thermal properties' measurements of samples comprising sand, water, methane, and MH are difficult because the melting heat of MH may affect the measurements. To solve this problem, we performed thermal properties' measurements at supercooled conditions during MH formation. The measurement protocol, calculation method of the saturation change, and tips for thermal constants' analysis of the sample using transient plane source techniques are described here.
The effect of the formation heat of MH on measurement is very small because the gas hydrate formation rate is very slow. This measurement method can be applied to the thermal properties of the gas hydrate-water-guest gas system, which contains hydrogen, CO2, and ozone hydrates, because the characteristic low formation rate of gas hydrate is not unique to MH. The key point of this method is the low rate of phase transition of the target material. Hence, this method may be applied to other materials having low phase-transition rates.

We present a protocol for measuring the thermal properties of synthetic hydrate-bearing sediment samples comprising sand, water, methane, and methane hydrate.

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce stable foams and emulsions, and can act as a thickener. Recently interest in this product has been enhanced through the internet where it is proposed that this solution, now called aquafaba by a growing community, can be used a replacement for egg and milk protein. As aquafaba is both new and being developed by an internet based community little is known of its composition or properties. Aquafaba was recovered from 10 commercial canned chickpea products and correlations among aquafaba composition, density, viscosity and foaming properties were investigated. Proton NMR was used to characterize aquafaba composition before and after ultrafiltration through membranes with different molecular weight cut offs (MWCOs of 3, 10, or 50 kDa). A protocol for electrophoresis, and peptide mass fingerprinting is also presented. Those methods provided valuable information regarding components responsible for aquafaba functional properties. This information will allow the development of practices to produce standard commercial aquafaba products and may help consumers select products of superior or consistent utility.

Aquafaba is a viscous juice from canned chickpea that, when stirred vigorously, produces a relatively stable white froth or foam. The primary research goal is to identify the components of aquafaba that contribute viscosifying/thickening properties using nuclear magnetic resonance (NMR), ultrafiltration, electrophoresis, and peptide mass fingerprinting.

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for plastic materials to be re-utilized, they must be identified and separated according to their polymer composition. In this study, the identification of these materials was performed using near-infrared spectroscopy (NIR), which identified material based on their physical-chemical properties. Advantages of the NIR method are a low environmental impact and rapid measurement (within a few seconds) in the spectral range of 1600-2400 nm without special sample preparation. Limitations include its inability to analyze dark materials. The identified polymers were utilized as a component for wood-polymer composite (WPC) that consists of a polymer matrix, low cost fillers, and additives. The components were first compounded with an agglomeration apparatus, followed by production by extrusion. In the agglomeration process, the aim was to compound all materials to produce uniformly distributed and granulated materials as pellets. During the agglomeration process, the polymer (matrix) was melted and fillers and other additives were then mixed into the melted polymer, being ready for the extrusion process. In the extrusion method, heat and shear forces were applied to a material within the barrel of a conical counter-rotating twin-screw type extruder, which reduces the risk of burning the materials and lower shear mixing. The heated and sheared mixture was then conveyed through a die to give the product the desired shape. The above-described protocol proved the potential for re-utilization of CDW materials. Functional properties must be verified according to the standardized tests, such as flexural, tensile, and impact strength tests for the material.

Secondary material streams have been shown to include potential raw materials for production. Presented here is a protocol in which CDW-plastic waste as a raw material is identified, followed by various processing steps (agglomeration, extrusion). As a result, a composite material was produced, and mechanical properties were analyzed.

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A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

Simulation of Early Earth Hydrothermal Chimneys in a Thermal Gradient Environment

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the emergence of life. The growth of hydrothermal chimneys in a thermal gradient environment within an early Earth vent system was successfully simulated by using different hydrothermal simulants, such as sodium sulfide, which were injected into an early Earth ocean simulant containing dissolved ferrous iron. Moreover, an apparatus was developed to sufficiently cool the ocean simulant to near 0 &#176;C in a condenser vessel immersed in a cold water bath while injecting a sulfide solution at hot to room temperatures, effectively creating an artificial chimney structure in a temperature gradient environment over a period of a few hours. Such experiments with different chemistries and variable temperature&#160;gradients resulted in a variety of morphologies in the chimney structure. The use of ocean and hydrothermal fluid simulants at room temperature resulted in vertical chimneys, whereas the combination of a hot hydrothermal fluid and cold ocean simulant inhibited the formation of robust chimney structures. The customizable 3D printed condenser created for this study acts as a jacketed reaction vessel that can be easily modified and used by different researchers. It will allow the careful control of injection rate and chemical composition of vent and ocean simulants, which should help accurately simulate prebiotic reactions in chimney systems with thermal gradients similar to those of natural systems.

The goal of this protocol is to form simulated hydrothermal chimneys via chemical garden injection experiments and introduce a thermal gradient across the inorganic precipitate membrane, using a 3D printable condenser that can be reproduced for educational purposes.

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the ...

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Zebrafish embryos/larvae develop externally and are optically transparent. The bioaccumulation of microplastics in fish at early life stages is readily assessed with fluorescently labeled microbeads.

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The ...

Field Measurement of Effective Leaf Area Index using Optical Device in Vegetation Canopy

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between green components of plants and the atmosphere, and many physiological processes occur there, primarily photosynthetic uptake, respiration, and transpiration. LAI is also an input parameter for many models involving carbon, water, and the energy cycle. Moreover, ground-based in situ measurements serve as the calibration method for LAI obtained from remote sensing products. Therefore, straightforward indirect optical methods are necessary for making precise and rapid LAI estimates. The methodological approach, advantages, controversies, and future perspectives of the newly developed LP 110 optical device based on the relation between radiation transmitted through the vegetation canopy and canopy gaps were discussed in the protocol. Furthermore, the instrument was compared to the world standard LAI-2200 Plant Canopy Analyzer. The LP 110 enables more rapid and more straightforward processing of data acquired in the field, and it is more affordable than the Plant Canopy Analyzer. The new instrument is characterized by its ease of use for both above- and below-canopy readings due to its greater sensor sensitivity, in-built digital inclinometer, and automatic logging of readings at the correct position. Therefore, the hand-held LP 110 device is a suitable gadget for performing LAI estimation in forestry, ecology, horticulture, and agriculture based on the representative results. Moreover, the same device also enables the user to take accurate measurements of incident photosynthetically active radiation (PAR) intensity.

Fast and precise leaf area index (LAI) estimation in terrestrial ecosystems is crucial for a wide range of ecological studies and calibrating remote sensing products. Presented here is the protocol for using the new LP 110 optical device for taking ground-based in situ LAI measurements.

Leaf area index (LAI) is an essential canopy variable describing the amount of foliage in an ecosystem. The parameter serves as the interface between ...