This protocol is an excerpt from Koon&#160;et al.,&#160;Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM),&#160;J. Vis. Exp.&#160;(2019).
1. Preparation and Fixation
<ol>
	<li>Prepare Drosophila melanogaster (fruit fly).
	<ol>
		<li>Anesthetize adult flies using 100% carbon dioxide. Place anesthetized adults (about 10 to 30 flies) in a small plastic screw cap vial or 1.5 mL centrifuge tube.</li>
		<li>Immerse anesthetized flies in 1 mL of fixative (1.25% glutaraldehyde, 0.1 M phosphate buffer pH 7.2) for 2 h (or overnight) at 4 &#176;C. If the flies float to the surface of the fixative, add a few drops of 2.5% polyethylene glycol tert-octylphenyl ether to weaken the surface tension of the fixative allowing for total submersion of the tissue. Remove the fixative using a glass pipet.</li>
	</ol>
	</li>
</ol>
2. Washing and Dehydration
<ol>
	<li>Wash and dehydrate Drosophila melanogaster (fruit fly).
	<ol>
		<li>Wash the fixed sample three times with 1 mL of 0.1 M phosphate buffer pH 7.2 at room temperature for 10 min in a 1.5 mL microcentrifuge tube. Remove each wash with a glass pipette, being careful not to remove the flies.</li>
		<li>Dehydrate the sample using a graded ethanol series, for 10 min in a volume of 1 mL in a microfuge tube. The ethanol concentrations are: 25%, 50%, 75%, 80%, 95%, 100%. Remove the ethanol with a glass pipette, being careful not to remove the flies.</li>
		<li>Retain the sample in the 1.5 mL microcentrifuge tube with just enough 100% ethanol to cover the sample before drying.</li>
	</ol>
	</li>
</ol>
3. Drying
<ol>
	<li>Perform chemical drying using hexamethyldisilazane (HMDS).
	<ol>
		<li>Replace the 100% ethanol solution with a 1:2 solution of HMDS and 100% ethanol for 20 min. Replace the 1:2 solution with a 2:1 solution of HMDS and 100% ethanol for 20 min. Replace the 2:1 solution with 100% HMDS for 20 min. Repeat once. 
		Note: HMDS is flammable and an acute toxin (dermal route). HMDS should be handled in a chemical fume hood using appropriate personal protective equipment including gloves, lab coat, and eye protection.</li>
		<li>Transfer the sample in HMDS, if in a 1.5 mL microcentrifuge tube, into a disposable aluminum weighing dish. Once in the aluminum weighing dish, replace the 100% HMDS with just enough fresh 100% HMDS to cover the sample.</li>
		<li>Transfer the sample to a plastic or glass non-vacuum desiccator with fresh desiccant (5-6 cm deep) and place into in a chemical fume hood. Alternatively, place the sample directly in a chemical fume hood to dry with a loose lid, such as a box, to prevent debris from falling on the sample. Allow the sample to dry for 12 to 24 h.</li>
	</ol>
	</li>
</ol>
4. Mounting
<ol>
	<li>Mount Drosophila melanogaster (fruit fly).
	<ol>
		<li>Label the bottom of the aluminum mounting stub to indicate what is being placed on top. 
		Place the dried flies in the desired position on adhesive or carbon adhesive tab secured to the top of a stub under a dissecting microscope with precision tweezers.</li>
		<li>Apply silver conductive adhesive, i.e., silver paint, around the outer edges of the stubs. Connect the silver paint to the flies using a toothpick to ensure conductivity. Do not allow the paint to touch the desired imaging area.</li>
		<li>Place the stubs in a stub holder box and place the open stub holder box in a desiccator. Allow the silver paint to dry at least 3 h or overnight for best results.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Silver conductive adhesive 503</td><td>Electron Microscopy Sciences</td><td>12686-15</td><td></td></tr><tr><td>aluminum weighing dish</td><td>Fisher Scientific</td><td>08-732-100</td><td></td></tr><tr><td>aluminum mounting stubs (12 mm)</td><td>Electron Microscopy Sciences</td><td>75210</td><td></td></tr><tr><td>aluminum mounting stubs (25 mm)</td><td>Electron Microscopy Sciences</td><td>75186</td><td></td></tr><tr><td>Adhesive tabs</td><td>Electron Microscopy Sciences</td><td>76760</td><td></td></tr><tr><td>conductive carbon adhesive tabs</td><td>Electron Microscopy Sciences</td><td>77825</td><td></td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td><td>VWR</td><td>97062-208</td><td></td></tr><tr><td>Hitachi 3400N-II SEM</td><td>Hitachi</td><td>&lt;a href=&quot;https://www.hitachi-hightech.com/us/product_list/?ld=sms2&amp;amp;md=sms2-1&amp;amp;sd=sms2-1-2&amp;amp;gclid=EAIaIQobChMIpq_jtJfj3AIVS7jACh2mdgkPEAAYASAAEgKAnfD_BwE&quot;&gt;https://www.hitachi-hightech.com/us/product_list/&lt;/a&gt;</td><td>The company doesn&#039;t appear to sell this model any longer</td></tr></tbody></table>

chemical drying: biological sample preparation for scanning electron microscopy (sem)

Source: Koon, M. A., et al. <a href="https://www.jove.com/video/58761/" target="_blank">Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)</a>. J. Vis. Exp. (2019).
Scanning electron microscopy (SEM) is a powerful tool to image&#8212;primarily&#8212;the surface features of a biological sample. Proper sample preparation, however, is critical to obtaining quality images. This video focuses on the dehydration and drying steps of preparing intact adult Drosophila for SEM.

Chemical Drying: Biological Sample Preparation for Scanning Electron Microscopy (SEM)

Source: Koon, M. A., et al. Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron ...

- To begin, start by dehydrating and drying a fixed sample to reduce sample distortion caused by the removal of gases and liquid water at the reduced operating pressure of conventional scanning electron microscopes, SEMs. To dehydrate the sample, wash it with increasing concentrations of ethanol in water until 100% ethanol is reached. Then repeat this process with a chemical drying agent in ethanol until the sample is washed with pure drying agent. Transfer the sample and drying agent to an evaporating dish. Cover it with minimal additional drying agent and let it dry in a fume hood overnight. Next, mount the prepared sample on the top of a microscope stub using carbon adhesive tape. Finally, apply silver paint to the edges of the stub, then from the edge of the stub to each of the samples, to reduce the buildup of excess electrons, which could result in undesirable image artifacts. In this experiment, fruit flies, Drosophila melanogaster, are prepared for SEM analysis using the drying agent HMDS. - To prepare Drosophila melanogaster, immerse anesthetized flies in 1 milliliter of fixitive for 2 hours at 4 degrees Celsius, making sure they are completely submerged. Use a glass pipette to remove the fixitive. Wash the fixed Drosophila sample in a 1.5 milliliter microcentrifuge tube three times with 1 milliliter of 0.1 molar phosphate buffer pH 7.2 at room temperature for 10 minutes. Use a glass pipette to remove each wash, being careful not to remove the sample. Then dehydrate the sample in a microfuge tube, in a volume of 1 milliliter, using a graded ethanol series for 10 minutes each. Use a glass pipette to remove each wash, being careful not to remove the sample. Keep the sample in the 1.5 milliliter microcentrifuge tube with just enough 100% ethanol to cover it. To perform chemical drying of the flies using HMDS, first, replace the 100% ethanol solution with a 1 to 2 solution of HMDS and 100% ethanol for 20 minutes. Then replace the solution with a 2 to 1 solution of HMDS and 100% ethanol for 20 minutes. Finally, replace this solution with 100% HMDS for 20 minutes, and then repeat the process once. Transfer the sample, which is in a 1.5 milliliter microcentrifuge tube and HMDS, into a disposable aluminum weighing dish. Replace the 100% HMDS with just enough fresh 100% HMDS to cover the sample. Place the sample within the aluminum dish directly in a chemical fume hood to dry with a loose lid, such as a box, to prevent debris from falling on the sample, and allow it to dry for 12 to 24 hours. To mount Drosophila label the bottom of an aluminum mounting stub. Use precision tweezers to place the dried flies in the desired position on a carbon adhesive tab secured to the top of a stub under a dissecting microscope. Apply silver conductive adhesive around the outer edges of the stubbs. Use a toothpick to connect the silver paint to the flies, ensuring conductivity, making sure that the paint doesn't touch the desired imaging area. Place the stubs in a stub holder box. And then place the open stub holder box in a desiccator and allow the silver paint to dry for at least three hours.

chemical-drying-biological-sample-preparation-for-scanning-electron

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JoVE Encyclopedia of Experiments

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Drosophila melanogaster (fruit fly)

Adult

5.2K Views. - To begin, start by dehydrating and drying a fixed sample to reduce sample distortion caused by the

Abstract

Protocol

<table><tbody><tr><td>Silver conductive adhesive 503</td><td>Electron Microscopy Sciences</td><td>12686-15</td><td></td></tr><tr><td>aluminum weighing dish</td><td>Fisher Scientific</td><td>08-732-100</td><td></td></tr><tr><td>aluminum mounting stubs (12 mm)</td><td>Electron Microscopy Sciences</td><td>75210</td><td></td></tr><tr><td>aluminum mounting stubs (25 mm)</td><td>Electron Microscopy Sciences</td><td>75186</td><td></td></tr><tr><td>Adhesive tabs</td><td>Electron Microscopy Sciences</td><td>76760</td><td></td></tr><tr><td>conductive carbon adhesive tabs</td><td>Electron Microscopy Sciences</td><td>77825</td><td></td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td><td>VWR</td><td>97062-208</td><td></td></tr><tr><td>Hitachi 3400N-II SEM</td><td>Hitachi</td><td><a href="https://www.hitachi-hightech.com/us/product_list/?ld=sms2&amp;md=sms2-1&amp;sd=sms2-1-2&amp;gclid=EAIaIQobChMIpq_jtJfj3AIVS7jACh2mdgkPEAAYASAAEgKAnfD_BwE">https://www.hitachi-hightech.com/us/product_list/</a></td><td>The company doesn't appear to sell this model any longer</td></tr></tbody></table>

Chemical Drying: Biological Sample Preparation for Scanning Electron Microscopy (SEM)

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Name	Company	Catalog Number	Comments
Silver conductive adhesive 503	Electron Microscopy Sciences	12686-15
aluminum weighing dish	Fisher Scientific	08-732-100
aluminum mounting stubs (12 mm)	Electron Microscopy Sciences	75210
aluminum mounting stubs (25 mm)	Electron Microscopy Sciences	75186
Adhesive tabs	Electron Microscopy Sciences	76760
conductive carbon adhesive tabs	Electron Microscopy Sciences	77825
Polyethylene glycol tert-octylphenyl ether (Triton X-100)	VWR	97062-208
Hitachi 3400N-II SEM	Hitachi	https://www.hitachi-hightech.com/us/product_list/	The company doesn't appear to sell this model any longer