Name: preparation of prokaryotic and eukaryotic organisms using chemical drying for morphological analysis in scanning electron microscopy (sem)
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM at JoVE.com

This work was funded by a grant to MLS and a Summer Scholar Award to MAK from the Office of Research and Graduate Studies at Central Michigan University. Cyanobacteria were supplied by the Zimba and plankton lab, part of the Center for Coastal Studies, Texas A&#38;M University at Corpus Christi. Euglenoids were supplied by the Triemer lab, Michigan State University.

1. Preparation and Fixation
<ol>
	<li>Prepare cyanobacteria.
	<ol>
		<li>Grow unialgal cultures in F/2 media24,25 at a temperature of 28 &#176;C on a 14:10 h light dark cycle. Transfer sufficient culture to a 1.5 mL microcentrifuge tube such that after allowing 15 min to settle, the bacterial pellet is approximately 0.05 mL in size. Remove the media and replace with 1.5 mL of fixative (1.25% glutaraldehyde, 0.1 M phosphate buffer pH 7.0), gently invert several ...

<ol>
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Here we described a protocol using SEM to obtain detailed information about external morphological characteristics of three types of organisms that others can apply to examine features of many types of organisms or tissues. Within each step of the protocol, there are potential points of error that may arise and are discussed in detail below.
While the volumes for fixative and washes given here are specific, in general the fixative and washes should be 5-10x the volume of the specimen. All fixa...

A scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate an image from secondary electrons that shows the morphology and topography of a sample1. SEM can be used to directly determine the physical size of a sample, the surface structure, and the three-dimensional shape, and offers greater resolution and larger depth of field compared to light microscopy. Another form of electron microscopy (EM), transmission electron microscopy (TEM) uses focused electrons that pass through the sample, generating images with fine details of internal structure. While TEM has higher resolution than light or SEM and can be used to resolve structures as small as single atoms, it has three major disadvantages: extensive sample preparation, a small field of view, and a shallow depth of field2,3. Although other visualized protocols exist using SEM to examine specific cells, organelles, or tissues4,5,6,7,8,9,10 , this protocol is unique in that we describe methods that can be broadly applied to a large range of organisms for morphological assessment.
SEM has found broad applications for examining inorganic materials including nanoparticles11,12, polymers13, and numerous applications in geological, industrial, and material sciences14,15,16. In biology, SEM has long been used as a method for examining biological samples ranging from individual proteins to whole organisms17,18. SEM is of particular value because morphological surface details can be used to inform scientific discovery. SEM analysis is a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of a wide range of biological samples.
Because an SEM normally operates under high vacuum (10-6 Torr minimum) to support a coherent beam of high-speed electrons, no liquids (water, oils, alcohols) are permitted in the sample chamber, as liquids prevent a vacuum from forming. Thus, all samples examined using SEM must be dehydrated, typically using a graded ethanol series followed by a drying process to remove the ethanol. There are several methods of drying biological tissues for use in the SEM, including air drying, lyophilization, use of a critical point drying (CPD) device, or chemical drying using t-butyl alcohol (TBA) or hexamethyldisilazane (HMDS)19,20,21,22. Most often, selection of a drying method is empirical since each biological sample may react differently to each drying method. For any given sample, all of these methods may be appropriate, so comparing the advantages and disadvantages of each is useful in selecting the appropriate method.
While air drying a sample at room temperature or in a drying oven (60 &#176;C) is the simplest method, most biological samples show drying-induced damage such as shriveling and collapse, resulting in distortion of the specimen. The process of lyophilization also removes water (or ice) from a sample, but requires samples to be flash-frozen and placed under vacuum to remove the ice via the process of sublimation, potentially damaging the sample. In addition, the user must have access to a lyophilizer. The most commonly used method for dehydrating samples for SEM is critical point drying (CPD). In CPD, the ethanol in a sample is replaced with liquid carbon dioxide (CO2) and, under specific temperature and pressure conditions known as the critical point (31.1 &#176;C and 1,073 psi), CO2 vaporizes without creating surface tension, thereby effectively maintaining the morphological and structural features of the sample. While CPD is generally the standard method, it has several drawbacks. First, the process requires access to a critical point dryer, which is not only expensive, but also necessitates the use of liquid carbon dioxide. Second, the size of the sample that can be dried is limited to the chamber size of the critical point dryer. Third, the exchange of liquids during CPD can cause turbulence that can damage the sample.
Chemical drying offers many advantages over CPD and serves as a suitable alternative that is becoming widely used in SEM sample preparation. The use of chemical dehydrants such as TBA and HMDS offers a fast, inexpensive, and simple alternative to other methods, while still maintaining the structural integrity of the sample. We recently showed that there was no difference in the integrity of the tissue or the quality of the final image captured when using CPD or TBA as the drying method in adult Drosophila retinal tissue23. Unlike CPD, TBA and HMDS do not require a drying instrument or liquid CO2 and there is no limitation on the size of the sample to be dried. In addition to obtaining the chemicals, only a standard chemical fume hood and appropriate personal protective equipment (gloves, lab coat, and safety goggles) are required to complete the drying process. While both TBA and HMDS are flammable, TBA is less toxic and less expensive (approximately 1/3 the cost of HMDS) than HMDS.
In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). These organisms represent a wide range in size (0.5 &#181;m to 4 mm) and cellular diversity (single-celled to multicellular), yet all are easily amenable to SEM analysis with only small variations needed for specimen preparation. This protocol describes the methods for using chemical dehydration and SEM analysis to examine morphological details of three types of organisms and would be broadly applicable to examining many organismal and tissue types.

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			<td height="40" style="height:40px;width:351px;">&#160;gold&#8211;palladium</td>
			<td style="width:373px;">Ted Pella, Inc.</td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 8 &#956;m, polycarbonate</td>
			<td>Sigma</td>
			<td>WHA110614</td>
			<td></td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.8 &#956;m, polycarbonate, black</td>
			<td>Sigma</td>
			<td>WHA110659</td>
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			<td height="40" style="height:40px;width:351px;">Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.2 &#956;m, polycarbonate</td>
			<td>Sigma</td>
			<td>WHA110606</td>
			<td></td>
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			<td height="40" style="height:40px;">aluminum weighing dish&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-732-100</td>
			<td></td>
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			<td height="40" style="height:40px;">aluminum mounting stubs (12 mm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>75210</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">aluminum mounting stubs (25 mm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>75186</td>
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			<td>Electron Microscopy Sciences</td>
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			<td style="width:373px;">Culture Collection of Algae at the University of Texas at Austin</td>
			<td style="width:395px;">https://utex.org/products/f_2-medium</td>
			<td>No Catalog number given - see link&#160;</td>
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			<td height="40" style="height:40px;">AF6 media</td>
			<td style="width:373px;">Bigelow - National Center for Marin Algae and Microbiota</td>
			<td style="width:395px;">https://ncma.bigelow.org/media/wysiwyg/Algal_recipes/NCMA_algal_medium_AF6_1.pdf</td>
			<td>No Catalog number given - see link&#160;</td>
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			<td height="40" style="height:40px;">Soil water medium</td>
			<td>Carolina Biological Supply Company</td>
			<td>153785</td>
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			<td height="40" style="height:40px;">Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td>
			<td>VWR</td>
			<td>97062-208</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hummer 6.2 Sputter Coater</td>
			<td>Anatech USA</td>
			<td>http://www.anatechusa.com/hummer-sputter-systems/4</td>
			<td>No Catalog number given - see link&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hitachi 3400N-II SEM&#160;</td>
			<td>Hitachi</td>
			<td style="width:395px;">https://www.hitachi-hightech.com/us/product_list/?ld=sms2&#38;md=sms2-1&#38;sd=sms2-1-2&#38;gclid=EAIaIQobChMIpq_jtJfj3AIVS7jACh2mdgkPEAAYASAAEgKAnfD_BwE</td>
			<td>The company doesn&#39;t appear to sell this model any longer&#160;</td>
		</tr>
	</tbody>
</table>

preparation of prokaryotic and eukaryotic organisms using chemical drying for morphological analysis in scanning electron microscopy (sem)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Cyanobacteria are a prokaryotic group of organisms that are critical to the global carbon, oxygen, and nitrogen cycles28,29. Of the estimated 6000 species of cyanobacteria30, most have a mucilaginous sheath that cover and connect the cells together and to other structures31, which along with the shape, can be resolved microscopically32. Cell size, shape, and p...

Watch this Scientific Journal Video about Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM at JoVE.com

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...