We appreciate the generous assistance of the Beijing Vocational College of Agriculture, the Institute for the Application of Atomic Energy (Chinese Academy of Agricultural Science), the Bioresearch Center of Beijing Forestry University and Professor Shan-gan Zhang of the Institute of Zoology, Chinese Academy of Sciences. This research was supported by National Key R&#38;D Program of China (2017YFD0600103), the National Natural Science Foundation of China (Grant No. 31570643, 81774015), Forest Scientific Research in the Public Welfare of China (201504304), Inner Mongolia Agricultural University High-level Talent Research Startup Plan (203206038), and Inner Mongolia Autonomous Region Higher Education Research Project (NJZZ18047), Inner Mongolia Autonomous Region Linxue &#34;Double First-class&#34; Construction Project (170001).

CAUTION: Consult the material safety data sheets of reagents before using them. Several of the chemicals used during sample preparation are toxic, mutagenic, carcinogenic, and/or reprotoxic. Use personal protective equipment (gloves, lab coat, full-length pants, and closed-toe shoes) and work under a fume hood while handling the sample.
1. SEM Sample Preparation and Imaging
<ol>
	<li>Sample Fixation and Cleaning
	<ol>
		<li>Working in an area where C. caragana occur, attract adults into field traps baited with plant attractants, such as isophorone16. Preserve clean bodies of adult C. caragana in 0.1 mol L-1 phosphate-buffered saline (PBS, pH 7.2), 2.5% (wt/vol) glutaraldehyde (Anhydrous EM Grad), and 0.06% (vol/vol) Tween 20. Fix the sample at 4 &#176;C over the weekend.</li>
		<li>Remove the bodies from the preservation liquid and rinse in phosphate buffer. Using an stereomicroscope, remove the appendages and clean them ultrasonically (40 kHz) in a 0.1 mol L-1 phosphate-buffered saline (pH 7.2) with 0.06% (vol/vol) Tween 20 (PBST). After cleaning for 100 s, transfer the sample to the microscope to check if it was clean. Under normal circumstances, clean for 400s to ensure that the sample was clean enough to observe and not damaged.</li>
	</ol>
	</li>
	<li>Sample Dehydration, Mounting and Drying
	<ol>
		<li>Dehydrate the samples by using 20 min successive treatments in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (all vol/vol) ethanol. Under a stereomicroscope, use carbon double-sided adhesive tape to separately fix 3 observation surfaces (dorsal ventral and lateral) onto stubs. Note that all viewing surfaces must be kept clean and free of contamination. Place the sample stage in a petri dish containing a silica gel desiccant for 48 h.</li>
	</ol>
	</li>
	<li>Sputter-coat and Sample Insertion
	<ol>
		<li>Using Hitachi Koki (E-1010) ion sputtering instrument, rotate MAIN VALVE to OPEN position, remove the sample chamber cover, and put the sample into chamber. Put the POWER switch on, and READY light was on. Set Sputtering time as 45 seconds, and coating thickness as 70.875 &#197;. Once mechanical pump vacuum dial index dropped below 7, press DISCHARGE and start spraying platinum. At the end of experiment, turn off the power supply and take the sample out of chamber. Spray film thickness: d = KIVt (&#34;d&#34; is the thickness of the film in the unit of &#34; &#197; &#34;; &#34;K&#34; is a constant, depending on the sputtered metal and gas. For example, K of air is 0.07; &#34;I&#34; is the unit mA of plasma flow; &#34;V&#34; is the voltage applied in the unit of &#34;KV&#34;. &#34;t&#34; is time in seconds.</li>
		<li>Insert the stub containing the sample onto the stage of SEM. Make sure the sample stage with the sample stub had enough height to allow a good image. Open the SEM software and select a desired operating voltage, beginning at 20 kV.</li>
	</ol>
	</li>
</ol>
2. TEM Sample Preparation and Imaging
<ol>
	<li>Obtain and fix the sample as in steps 1.1.1 and 1.1.2.</li>
	<li>Cleaning, Secondary Fixation and Dehydration
	<ol>
		<li>Remove adult C. caragana from the preservation liquid. Using a stereomicroscope, remove the appendages, wash the samples in PBST for 3 h, and then post-fix them in 1% (wt/vol) osmium tetroxide in PBS for 1h at 25 &#176;C. Dehydrate the samples by using 20 min successive treatments in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (all vol/vol) ethanol at room temperature.</li>
	</ol>
	</li>
	<li>Resin Embedding and Polymerization
	<ol>
		<li>Embed the samples in resin in a flat embedding moulds. The sample was at the bottom of the plate and was placed as close as possible to the edge of the recessed groove. Place the label in the blank then incubate the plate containing the sample at 60 &#176;C for 72 h. Remove the capsule from the incubator and verify that the resin had polymerized.</li>
	</ol>
	</li>
	<li>Sample Sectioning and Staining
	<ol>
		<li>Once ensure the sample had been solidified, place each resin block under a fluorescence microscope and photograph them under blue light. Move the microscope&#39;s fluorescent light source so it irradiated the sample from above. Enable the sensilla in the resin block to be clearly observed. Photographed and measure distances to target the sensilla (Figure 1).</li>
		<li>Refer to SEM image of the palps (Figure 2A), and cut roughly the resin block with a razor blade to close the target receptor (Figure 2B).</li>
		<li>Next, using blue-light fluorescence microscopy, photograph the roughly cut resin block, adjusting the light source from above so that the sensilla was observed clearly. Green light excited by the blue light created a favorable observation. When imaging, the objective micrometer (DIV 0.01mm) was added to the fluorescence microscope stage, and then the distance of the target was measured by ImageJ software (U.S. National Institute of Health) (Figure 2C). The image ruler was made by Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA). Then, for ultramicrotome slicing, set the cutting distance, using 50-60 nm slice thicknesses, until the target position was reached. Use fluorescence microscopy to pinpoint the target receptor.</li>
		<li>Mount the sections on Formvar-coated, 100-mesh copper grids, double-stained with uranyl acetate and lead citrate.
		<ol>
			<li>Firstly, add 3.75 g uranyl acetate to 50 mL of 50 % methanol. Stain grids with a filtered (0.45 &#181;m) syringe of a saturated solution of uranyl acetate at room temperature for 10 min. Cover sections during staining to block light induced precipitates. Rinse 2x in 50 % methanol; 2x filtered degassed water.</li>
			<li>Secondly, add 0.02 g lead citrate to 10 mL of degassed distilled water in centrifuge tube. Add 0.1 mL of 10 N sodium hydroxide, seal and shake to dissolve. Stain grids with a solution of lead citrate for 8 min. Centrifuge before use. Staining must be done in a carbon dioxide free environment to prevent the formation of lead carbonate precipitates. Place drops of stain on squares of plastic petri dishes. Rinse in degassed filtered water and dry17. Observe them via TEM operating at 80 kV.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59251/59251fig1a.jpg" /> 
Figure 1: A fluorescent microscope photographed a resin block enclosing the appendage of the Chlorophorus caragana. (A) Antenna resin block; (B) Resin block at the end of the ovipositor. The arrow indicated the edge of the resin block; dotted circle indicates the target sensilla. <a href="https://www.jove.com/files/ftp_upload/59251/59251fig1alarge.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/59251/59251fig2.jpg" /> 
Figure 2: Procedures of the precise sensilla location method. (A) The 4th sub-segment of a maxillary palp of Chlorophorus caragana, the dotted circle showed the sensilla targeted by SEM. (B) The 4th sub-segment of a maxillary palp of C. caragana viewed by fluorescence microscopy. White arrow showed the roughly cut edge of the resin block and the dotted circle showed the accurate location. (C) The marked distance from the edge of the resin block to the maxillary palp target location (28 &#181;m in this sample). <a href="https://www.jove.com/files/ftp_upload/59251/59251fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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We have no conflict of interest to disclose.

In this article, we presented a sample preparation scheme for scanning and transmission electron microscopy for woodboring beetle. Using insect appendage as a representative study subject, we demonstrated several improvements over traditional sample preparation methods.
The liquid oil detached from the solid surface is emulsified into small droplets, which can be well dispersed and suspended in the washing medium to reduce redepositing on the surface of the object. Washing performance of surfa...

Scanning electron microscopy is an important tool in many morphology studies, that SEM shows surface structures1,2. Transmission electron microscopy&#39;s appeal is that it can be used to study a wide range of biological structures at the nanometer scale, from the architecture of cells and the ultrastructure of organelles, to the structure of macromolecular complexes and proteins. TEM shows inner structures3,4,5.
Coleoptera is the largest group of insects, including about 182 families and 350,000 species. Most of the coleopteran insects, in particular woodboring beetle, feed on plants, many of which are important pests of forests and fruit trees, causing devastating damage to trees6. At present, prevention and control population of pests based on chemical ecology theory have received increasing attention7. Efficient, low-toxic, pollution-free pheromone control methods have become an effective way8. Studying the sensilla morphology and ultrastructure of insects is an important part of insect chemical ecology research. The scanning and transmission electron microscopy (SEM and TEM, respectively) are used to great effect to study their morphology and internal anatomy. However, during preparation of insect samples for electron microscopy (EM), the objectivity and authenticity of the observation site may be affected9. In general, SEM sample preparation of insects requires cleaning, tissue fixation, dehydration, metathesis, drying, and sputter-coating10. Due to the complex environment in which woodboring beetle live, the body surface often has various pollutants and their appendages often have many fine long sensilla or bristles. In particular, some woodborers are not available from laboratory raising, which collected directly in the field, and then put into fixing fluid to ensure freshness and subsequently washed in the laboratory. If the sample is first fixed and then washed, obviously it is much more difficult to remove debris because glutaraldehyde strongly fixes it to the sample. Tween 20 is a surfactant11,12,13,14, which plays an important role in the washing process, including reducing the surface tension of water and improving the wettability of water on the surface of the laundry. In this study, Tween 20 was added to the fixing solution and PBS cleaning solution to reduce the surface tension of the liquid, and prevent the dirt from depositing on the body surface of the woodboring beetle, which made the body surface cleaner in SEM.
Using TEM, sensilla on different organs of insects can be sliced to reveal the clear structures inside them, thus providing a basis for analyzing sensilla functions. When the subject insect, such as woodboring beetle, is large, and its body wall has a substantial degree of sclerotization, so the fixative may not fully saturate organ tissues inside the insect body. Tween 20 can enhance the dispersion and suspension capacity of the dirt. In this study, Tween 20 was added to the fixative to enhance fixative fluid penetration into the insect body wall of woodboring beetle, avoiding deformation and collapse of the epidermi11,12,13. In addition, using general slicing technology, it is difficult to accurately locate different types of sensilla, in particular for some small sensilla15. Based on traditional TEM sample preparation, this study combined fluorescence microscopy and SEM to determine the position of insect sensilla in the embedded block, thus improving slicing accuracy.

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		</tr>
		<tr>
			<td>Carbon tetrachloride</td>
			<td>Sigma</td>
			<td>56-23-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>GilderGrids</td>
			<td>G300</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate</td>
			<td>Sinopharm group chemical reagent co., LTD</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T. Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat embedding molds</td>
			<td>Hyde Venture (Beijing) Biotechnology Co., Ltd.</td>
			<td>70900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>LEICA</td>
			<td>DM2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>111-30-8</td>
			<td>Anhydrous EM Grade</td>
		</tr>
		<tr>
			<td>Isophorone</td>
			<td>Sigma</td>
			<td>78-59-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Sigma</td>
			<td>512-26-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate</td>
			<td>Its group chemical reagent co., LTD</td>
			<td>7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective micrometer</td>
			<td>Olympus</td>
			<td>0-001-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma</td>
			<td>541-09-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Aldrich</td>
			<td>1998</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Gillette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resin</td>
			<td>Spurr</td>
			<td>ERL4221</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lianhui</td>
			<td>GB/T19001-2008</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>Hitachi</td>
			<td>S-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel desiccant</td>
			<td>Suzhou Longhui Desiccant Co., Ltd.</td>
			<td>112926-00-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Small brush</td>
			<td>Martol</td>
			<td>G1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter ion instrument</td>
			<td>Hitachi Koki Co. Ltd., Tokyo, Japan</td>
			<td>E-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>EZ4 HD</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>Hitachi</td>
			<td>H-7500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Tianjin Damao Chemical Reagent</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>GT Sonic</td>
			<td>GT-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Sigma</td>
			<td>6159-44-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation method of scanning and transmission electron microscope for the appendages of woodboring beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

Using cleaning and fixative solution with Tween 20, cleaner SEM image was observed than that without Tween 20 (Figure 3). Tween 20 fixing solution penetrated the glutaraldehyde fixing solution into the tissue. Microtubule structure was clearly seen. TEM image of the internal structure of the sample was blurred without Tween 20 (Figure 4).
<img alt="Figure 3" class="xfig...

Watch this Scientific Journal Video about Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle at JoVE.com

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

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18.6K Views.  Beijing Forestry University. In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

Video: Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

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 ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to follow the dynamically changing state of the ionogels, and to deliver more information and details about the subtle change of conductive properties with an increase or decrease in the temperature. Moreover, the method allows the performance of long term (i.e. days, weeks) measurements at a constant temperature to investigate the stability and durability of the system and the aging effects. The main advantage of the TSC method over classical conductometry is the ability to perform measurements during the gelation process, which was impossible with the classical method due to temperature stabilization, which usually takes a long time before the individual measurement. It is a well-known fact that to obtain the physical gel phase, the cooling stage must be fast; moreover, depending on the cooling rate, different microstructures can be achieved. The TSC method can be performed with any cooling/heating rate that can be assured by the external temperature system. In our case, we can achieve linear temperature change rates between 0.1 and approximately 10 &#176;C/min. The thermal scanning conductometry is designed to work in cycles, continuously changing between heating and cooling stages. Such an approach allows study of the reproducibility of the thermally reversible gel-sol phase transition. Moreover, it allows the performance of different experimental protocols on the same sample, which can be refreshed to initial state (if necessary) without removal from the measuring cell. Therefore, the measurements can be performed faster, in a more efficient way, and with much higher reproducibility and accuracy. Additionally, the TSC method can be also used as a tool to manufacture the ionogels with targeted properties, like microstructure, with an instant characterization of conductive properties.

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

The kinetics of the cooling process defines the properties of ionic gels based on low molecular weight gelators. This manuscript describes the usage of thermal scanning conductometry (TSC), which obtains full control over the gelation process, along with in situ measurements of the samples' temperature and conductivity.

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking.
The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink&#39;s pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.

Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

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 ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...