Name: sample preparation method of scanning and transmission electron microscope for the appendages of woodboring beetle
Uploaded: 2020-02-03T12:00:00
Description: Watch this Scientific Journal Video about Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle at JoVE.com

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 ...

<ol>
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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anatomical lens</td>
			<td>Chongqing Auto Optical 
			limited liability company</td>
			<td>1425277</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon adhesive tape</td>
			<td>SPI Supplies, Division of Structure Probes, Inc.</td>
			<td>7311</td>
			<td></td>
		</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>
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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...

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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 ...

Woodboring beetle their body surface are always hard in SEM. The Tween-20 is added into fixative and washing solution. The body surface of woodboring beetle In TEM, the Tween-20 can enhance fixative penetration into the body wall and better fix tissue and organ in the body.The fluorescence microscope is helpful for improving slicing accuracy. The technique is helpful for washing body surface and penetrating into the body wall in the woodboring beetle. Thus, it would clear SEM and TEM vision.Fluorescence microscope is helpful for improving slicing accuracy in TEM. Demonstrating the procedure will be Zhang Yanru, a graduate student from our laboratory. In an area where Chlorophorus caragana live, place field traps baited with plant attractants such as isophorone on a funnel trap.Attract woodboring beetle adults into the traps. Preserve the clean bodies of adult Chlorophorus caragana in 0.1 mole per liter PBS at pH 7.2, 2.5%weight by volume glutaraldehyde, and 0.06%volume by volume Tween-20. Fix the sample at four degrees Celsius for three days.Remove the bodies from the preservation liquid and rinse in PBS. Under a stereo microscope, use forceps to remove the appendages and clean them ultrasonically at 40 kilohertz in PBST. After cleaning for 100 seconds, transfer the sample to the microscope to check if it is clean.Do not clean longer than 400 seconds to avoid damage. Then immerse the samples in ethanol at a series of concentrations for 20 minutes each to perform dehydration. After the dehydration, under a stereo microscope, use carbon double-sided adhesive tape to separately fix three observation surfaces, dorsal, ventral, and lateral, onto stubs.Ensure that all viewing surfaces are kept clean and free of contamination. Place the sample stage in a Petri dish containing a silica gel desiccant for 48 hours. Next, on an ion sputtering instrument, rotate the main valve to the open position, remove the sample chamber cover and put the sample into the chamber.Turn the power switch on and ensure the ready light is on. Set the sputtering time as 45 seconds and the coating thickness as 70.875 angstrom. Once the mechanical pump vacuum dial index drops below seven, press discharge and start spraying platinum.At the end of the procedure, turn off the power supply and take the sample out of the chamber. Calculate the spray film thickness D in angstrom equaling K times I times V times T.K is a constant determined by the sputtered metal and gas. I is the plasma flow in milliamps while V is the voltage applied in kilovolts and T is the time in seconds.Insert the stub containing the sample onto the stage of the SEM. Make sure the sample stage with the sample stub has enough height to allow a good image. Open the SEM software and select the desired operating voltage beginning at 20 kilovolts.After obtaining the appendages from adult Chlorophorus caragana, wash the samples in PBST in an ultrasonic bath for three hours and then post-fix them in 1%weight by volume osmium tetroxide in PBS for one hour at 25 degrees Celsius. Dehydrate the samples by using 20-minute successive treatments in 50%60%70%80%85%90%95%100%and 100%volume by volume ethanol at room temperature. With forceps, apply heated resin in a flat embedding mold and embed the samples in the resin.Ensure that the sample is at the bottom of the plate and placed as close as possible to the edge of the recessed groove. Label and then incubate the plate containing the sample at 60 degrees Celsius for 72 hours. After incubation, remove the plate from the incubator and verify that the resin has polymerized.Once the sample has solidified, place each resin block under a fluorescence microscope, move the microscope's fluorescent light source to irradiate the sample with blue light from above and photograph them. Make sure the sensilla in the resin block are clearly visible. Measure the distance between the edge of the resin block and the target sensor to target the sensilla.Refer to the SEM image of the palps and roughly cut the resin block with a razor blade close to the target receptor. Next, under a blue light fluorescence microscopy, adjust the light source from above so that the sensilla are observed clearly. Add the objective micrometer to the fluorescence microscope stage.Photograph the roughly cut resin block and then using ImageJ measure the distance between the target and the edge of the resin. Place the resin sample on an ultramicrotome and cut 50 to 60 nanometer thick sections until the target position has been reached. Mount the sections on formvar-coated 100 mesh copper grids in Petri dishes.Stain the grids with a saturated filtered solution of uranyl acetate at room temperature. Cover the sections during staining to block light-induced precipitates. Stain for 10 minutes.In a fume hood, rinse twice in 50%methanol, then twice in filtered degassed water. Place drops of prepared lead citrate stain on the squares of the plastic Petri dishes and let them sit for eight minutes. Rinse in degassed filtered water and dry.Mount the grids onto a sample stage within the TEM machine. Set the voltage to 80 kilovolts and examine the sections. In this protocol, using cleaning and fixative solution with Tween-20, a clear SEM image was observed than that without Tween-20.With the Tween-20 fixing solution allowing the glutaraldehyde fixing solution to penetrate the tissue, microtubule structures were clearly seen in the TEM image while the internal structure of the sample prepared without Tween-20 was blurry. Continuous cross-sectional views of the peg of the type one sensilla twig basiconica on the maxillary palps showed that the dendritic sheath surrounded the outer dendritic segments and extended to the tip pore. Seven unbranched outer dendritic segments existed inside the inner receptor lymph cavity which was surrounded by an outer cavity.The tubular body was separated by a dendritic sheath from other outer dendritic segments at each sensillar socket base. In the ciliary region, eight dendrites of different diameters were identified indicating the presence of eight bipolar neurons. The technique can be used to further study the function of nerves such as single cell recording or in situ hybridization.

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