Name: a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy
Uploaded: 2018-01-17T10:45:00
Description: Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

We sincerely thank Shigeo Kita for his valuable suggestions and discussion. We also thank John and Sumire Eckstein for their critical reading of the manuscript.

NOTE: The specimens used in this study were microorganisms, rapidly frozen with propane in liquid nitrogen, freeze substituted in acetone containing 2% osmium tetroxide, and embedded in epoxy resin1,2,3,4,5,6,7,8,9,</...

<ol>
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K., Suzuki, Y., Furukawa, H., Takeo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+a+pathogenic+yeast+Exophiala+dermatitidis+cell+by+freeze-substitution+and+serial+sectioning+electron+microscopy.">Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy.</a> FEMS Microbiol. Lett. 219, 17-21 (2003).</li><li>Kozubowski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+kinetochore+assembly+in+the+human+pathogenic+basidiomycetous+yeast,+Cryptococcus+neoformans.">Ordered kinetochore assembly in the human pathogenic basidiomycetous yeast, Cryptococcus neoformans.</a> mBio. 4, e00614-e00613 (2013).</li><li>Yamaguchi, M., Adachi, K., The Japanese Society of Microscopy, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specimen+support.">Specimen support.</a> Guide Book for Electron Microscopy. , 52-57 (2011).</li><li>Hayat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+films.">Support films.</a> Principles and Techniques of Electron Microscopy: Biological Application. , 216 (2000).</li><li>Yamaguchi, M., Kita, S., The Japanese Society of Microscopy, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+sectioning.">Ultrathin sectioning.</a> Guide Book for Electron Microscopy. , 40-52 (2011).</li><li>Yamaguchi, M., Aoyama, T., Yamada, N., Chibana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+hydrophilicity/hydrophobicity+of+the+plasma-polymerized+naphthalene+film+(Super+support+film)+and+other+support+films+and+grids+in+electron+microscopy.">Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super support film) and other support films and grids in electron microscopy.</a> Microscopy. 65, 444-450 (2016).</li><li>Kita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+method+for+obtaining+serial+ultrathin+sections+using+new+small+tools.">Reliable method for obtaining serial ultrathin sections using new small tools.</a> Kenbikyo. 46, 253-257 (2011).</li><li>Yamaguchi, M., Shimizu, M., Yamaguchi, T., Ohkusu, M., Kawamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+use+of+uranyl+acetate+solution+in+section+staining+in+transmission+electron+microscopy.">Repeated use of uranyl acetate solution in section staining in transmission electron microscopy.</a> Plant Morphology. 17, 57-59 (2005).</li><li>Hayworth, K. 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The method presented here requires no expensive equipment. It requires only an aluminum rack (Figure 3), three-slit grids (Figure 6c), section-holding loops (Figure 10a), water-surface-raising loop (Figure 11a), and a staining tube (Figure 13). There are many features of the present method. The large part of the specimen is used to adjust the specimen surface and knife edge...

Proper serial ultrathin sectioning technique is indispensable to study cells and cell components three-dimensionally at the electron microscopic level. We have studied the dynamics of spindle pole body in the cell cycle of yeast cells, and revealed morphological changes of their ultrastructure during the cell cycle and the time of duplication1,2,3,4,5. In 2006, we coined a new word &#39;structome&#39; by combining &#39;structure&#39; and &#39;-ome&#39;, and defined it as the &#39;quantitative and three-dimensional structural information of a whole cell at the electron microscopic level&#39; 6, 7.
By structome analysis, which requires serial ultrathin sectioning technique, it was found that a yeast cell of Saccharomyces cerevisiae and Exophiala dermatitidis had about 200,000 ribosomes7, 8, an Escherichia coli cell had 26,000 ribosomes9, a Mycobacterium tuberculosis cell had 1,700 ribosomes10 and Myojin spiral bacteria had only 300 ribosomes11. This information is useful in not only estimating the growth rate in each organism, but also in identification of species9.
Further, structome analysis led to the discovery of a new organism; Parakaryon myojinensis was found in the deep sea off the coast of Japan, whose cell structure were an intermediate between those of prokaryotes and eukaryotes12,13,14,15. At present, serial ultrathin sectioning technique is considered to be so difficult that it would take a long time to master. In this study, we have developed a reliable method in which anybody can perform serial ultrathin sectioning without difficulty.

<table>
	<tbody>
		<tr>
			<td>Formvar making apparatus</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>652</td>
			<td>W 180 x D 180 x H 300 mm</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Matsunami Co. Ltd., Osaka</td>
			<td>-</td>
			<td>76 x 26 x 1.3 mm</td>
		</tr>
		<tr>
			<td>Aluminum rack with 4-mm holes</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>658</td>
			<td>W 30 x D 25 x H 3 mm, Refer to this paper</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>SMZ 645</td>
		</tr>
		<tr>
			<td>LED illumination for stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>SM-LW 61 Ji</td>
		</tr>
		<tr>
			<td>Trimming stage</td>
			<td>Sunmag Co.Ltd., Tokyo</td>
			<td>-</td>
			<td>Tilting mechanism equipped, Refer to this paper</td>
		</tr>
		<tr>
			<td>LED illumination for trimming stage</td>
			<td>Sunmag Co.Ltd., Tokyo</td>
			<td>-</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Ultrasonic trimming blade</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>5240</td>
			<td>EM-240, Refer to this paper</td>
		</tr>
		<tr>
			<td>Diamond knife for trimming</td>
			<td>Diatome Co. Ltd., Switzerland</td>
			<td>-</td>
			<td>45&#176;</td>
		</tr>
		<tr>
			<td>Diamond knife for ultrathin sectioning</td>
			<td>Diatome Co. Ltd., Switzerland</td>
			<td>-</td>
			<td>45&#176;</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems, Vienna</td>
			<td>-</td>
			<td>Ultracut S</td>
		</tr>
		<tr>
			<td>Mesa cut</td>
			<td>Leica Microsystems, Vienna</td>
			<td>-</td>
			<td>Mirror</td>
		</tr>
		<tr>
			<td>0.5% Neoprene W solution</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>605</td>
			<td></td>
		</tr>
		<tr>
			<td>Special 3-slit nickel grid</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2458</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Special 3-slit copper grid</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2459</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Section-holding loop</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>526</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Water-surface-raising loop</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>527</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Staining tube</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>463</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Multi-specimen holder</td>
			<td>JEOL Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>EM-11170</td>
		</tr>
		<tr>
			<td>JEM-1400</td>
			<td>JEOL Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Transmission electron microscope</td>
		</tr>
	</tbody>
</table>

a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

In this protocol, three-slit grids were used for picking up serial sections. The grids are made of nickel or copper. The serial sections are placed on the middle slit. The slits on both sides are necessary to view the sections when picking them up with the grid. To keep the grids parallel with the serial sections when picking them up with tweezers (Figure 11d), the handle is bent (Figure 6c, right). A small handle is advantageous...

Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

The overall goal of this procedure is to make serial ultrathin sections safely and easily. This method can help answer key questions in the cell biology field, providing quantitative and three-dimensional structural information of a whole cell at the electron microscopic level. The main advantage of this technique is that serial ultrathin sectioning can be done without an expensive machine, and high resolution images can be obtained with this method.To begin this procedure, clean the surface of a glass slide. Then dip one half of the glass slide in 1.5%Formvar solution in the upper column of the Formvar support filmmaking apparatus by pressing the solution with air through the three-way stopcock using a rubber ball. Drain the solution from the column by opening the three-way stopcock and releasing air to reduce the pressure.Then take out the glass slide from the apparatus, and dry in air to form a film on the surface of the glass slide. Scrape off the four edges of the film on the glass slide with a razor blade. After breathing onto the slide to facilitate the separation of the film from the slide, float off the film by immersing the glass slide into water slowly at a low horizontal angle.Then scoop up the Formvar film from the water using an aluminum rack with holes. To trim the specimen block with an ultrasonic trimming blade and a razor blade under a stereo microscope, first make sure that there are cells on the surface of the block by observing the tip of the block with a light microscope. Then mount the specimen block in the chuck, and mount the chuck on a trimming stage.Trim the blocks to a size of 0.7 by 1.0 millimeters. If the epoxy resin is very hard, trim the box first using an ultrasonic trimming blade, then trim the block further with a razor blade. Cut one shoulder to mark the direction of cutting.To trim the specimen block with a diamond knife using the microtome, set the specimen block chuck in the specimen holder of the ultra microtome. Cut the surface of the block with a diamond trimming knife. Set the diamond knife edge parallel to the specimen block face, and cut the minimum amount of specimen surface so as not to lose any specimen.Cut the left edge of the block using the trimming knife by rotating the knife stage 30 degrees to the left. This edge become the upper side of the sample in serial sectioning. Then cut the block face at about 100 microns from the left edge of the specimen by rotating the knife stage 30 degrees to the right.This edge becomes the lower side of the specimen in serial sectioning. Remove the trimming knife, and rotate the specimen block 90 degrees clockwise. Set an ultrathin sectioning knife to the knife stage.Adjust the specimen surface and the knife edge so that they face parallel to each other using the large part of the specimen surface. Next, apply tape on the chuck and the chuck holder of the microtome, and cut the tape at the boundary. Take the specimen chuck out from the microtome and place it under the stereo microscope.Cut off the large part of the specimen with a razor blade leaving the slim part from which serial sections will be obtained Make the section sides adhesive by using a Pasteur pipette to drop about one microliter of 0.5%of Neoprene solution onto the specimen to be used for serial sectioning. Then cover the whole specimen with Neoprene solution. Absorb excess Neoprene solution immediately with a piece of filter paper placed near the specimen.Next, prepare three slit grids for picking up serial sections. To do so, bend the handle of the grids to 60 degrees, and treat the grids with 0.5%Neoprene solution. Then make the grids hydrophylic by glow discharge.Place the specimen block chuck back in the microtome at the same position by aligning the taped parts. This keeps the block face and the knife edge perfectly parallel to each other. Then bring the knife close to the specimen.After filling the knife boat with water, cover the microtome with a plastic cover to prevent air flow. Start cutting the specimen at a section thickness of 200 nanometers. After the first section is cut, set the section thickness to 70 nanometers.When the number of serial sections reaches 20, set the section thickness to 10 nanometers. Since the microtome cannot cut 10 nanometer thick sections, no new section appears, and the previously cut sections become separated from the knife edge. Next, set the section thickness to 60 nanometers.This will produce 70 nanometer sections because the machine will add the previous 10 nanometer thickness. Finally, set the section thickness back to 70 nanometers, and continuing cutting until 1.8 millimeter-long sections are obtained. To pick up the serial sections, first place the section holding loop on the third section group.Then place water surface raising loop on the knife stage. Place the loop of the WSRL just above the serial sections by moving its shaft and handle. Lower the loop of the WSRL onto the water surface so that the loop encircles the sections.Push the loop down by turning the screw downward so that the water surface is raised by surface tension. Move the section to the center of the loop. Position it on the apex of the water and adjust the direction of the section by using a hair strand.Pick up section groups by touching it with a bare slit grid, which is helped by tweezers and kept parallel to the water surface. Place the grids with sections along with a tiny drop of water onto the Formvar support film. Remove excess water using filter paper.After the sections are completely dried, tear the Formvar film around the grids;and remove the grids bearing the sections. Set the grids in the groove of the staining tube in the proper order, and stain sections with uranyl acetate and lead citrate. Set the five grids in the multi-specimen holder in the proper order, orienting the long axis of the grid slit perpendicular to the specimen holder axis.Fix the grid with grid fixers, and insert the specimen holder into the transmission electron microscope. Proceed to take pictures of the target cells and all the cell sections at high magnification. Shown here is a low-magnification view of five grids that carry serial sections.18-25 sections were mounted on one grid. Note that the sections are attached together and have the same thickness. Serial sections of pericarion myogenesis are shown here.The figure shows 12 out of 67 complete sections. This cell was found to have a large nucleoid consisting of naked DNA fibers with a single nucleoid membrane and endosymbionts that resemble bacteria but no mitochondria. Thus, this organism appears to be an intermediate life form evolving from prokaryote to eukaryote.Shown here are serial sections of Escherichia coli. The figure shows 12 complete sections. Interestingly, this cell was found to contain 21, 700 ribosomes.After watching this video, you should have a good understanding of how to make serial ultrathin sections. Once mastered, this technique can be done in three hours if it is performed properly.

a-method-for-obtaining-serial-ultrathin-sections-microorganisms

Research

JoVE Journal

Engineering

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography (CT) and Light Microscopy (LM) Correlated with Scanning Electron Microscopy (SEM)

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of micro-characterization, plays an important role. Commonly, different techniques like X-ray computed tomography (CT), light microscopy (LM) and scanning electron microscopy (SEM) are used separately. Similarly, the results have to be treated for each technique independently. Here a comprehensive study is shown which demonstrates the potentials leveraged by linking CT, LM and SEM. In depth characterization is performed on a white emitting LED, which can be operated throughout all characterization steps. Major advantages are: planned preparation of defined cross sections, correlation of optical properties to structural and compositional information, as well as reliable identification of different functional regions. This results from the breadth of information available from identical regions of interest (ROIs): polarization contrast, bright and dark-field LM images, as well as optical images of the LED cross section in operation. This is supplemented by SEM imaging techniques and micro-analysis using energy dispersive X-ray spectroscopy.

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography CT and Light Microscopy LM Correlated with Scanning Electron Microscopy SEM

A workflow for comprehensive micro-characterization of active optical devices is outlined. It contains structural as well as functional investigations by means of CT, LM and SEM. The method is demonstrated for a white LED which can be still be operated during characterization.

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of ...

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

Fabrication of Magnetic Nanostructures on Silicon Nitride Membranes for Magnetic Vortex Studies Using Transmission Microscopy Techniques

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared on transparent membranes which are very fragile and difficult to manipulate. We present processes for the fabrication of samples with magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for Lorentz transmission electron microscopy and magnetic transmission x-ray microscopy studies. The samples are prepared on silicon nitride membranes and the fabrication consists of a spin coating, UV and electron-beam lithography, the chemical development of the resist, and the evaporation of the magnetic material followed by a lift-off process forming the final magnetic structures. The samples for the Lorentz transmission electron microscopy consist of magnetic nanodiscs prepared in a single lithography step. The samples for the magnetic x-ray transmission microscopy are used for time-resolved magnetization dynamic experiments, and magnetic nanodiscs are placed on a waveguide which is used for the generation of repeatable magnetic field pulses by passing an electric current through the waveguide. The waveguide is created in an extra lithography step.

A protocol for the fabrication of magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for transmission electron microscopy (TEM) and magnetic transmission x-ray microscopy (MTXM) studies is presented.

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers are directly grown on NPSS using catalyst-free atmospheric-pressure chemical vapor deposition (APCVD). By applying nitrogen reactive ion etching (RIE) plasma treatment, defects are introduced into the graphene film to enhance chemical reactivity. During metal-organic chemical vapor deposition (MOCVD) growth of AlN, this N-plasma treated graphene buffer enables AlN quick growth, and coalescence on NPSS is confirmed by cross-sectional scanning electron microscopy (SEM). The high quality of AlN on graphene-NPSS is then evaluated by X-ray rocking curves (XRCs) with narrow (0002) and (10-12) full width at half-maximum (FWHM) as 267.2 arcsec and 503.4 arcsec, respectively. Compared to bare NPSS, AlN growth on graphene-NPSS shows significant reduction of residual stress from 0.87 GPa to 0.25 Gpa, based on Raman measurements. Followed by AlGaN multiple quantum wells (MQWS) growth on graphene-NPSS, AlGaN-based deep ultraviolet light-emitting-diodes (DUV LEDs) are fabricated. The fabricated DUV-LEDs also demonstrate obvious, enhanced luminescence performance. This work provides a new solution for the growth of high quality AlN and fabrication of high performance DUV-LEDs using a shorter process and less costs.

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers ...

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different materials show variability in resistive switching properties due to the intrinsic nature of the material used, leading to discrepancies in the field because of underlying operation mechanisms. This highlights a need for a reliable technique to understand mechanisms using nanostructural observations. This protocol explains a detailed process and methodology of in&#160;situ nanostructural analysis as a result of electrical biasing using transmission electron microscopy (TEM). It provides visual and reliable evidence of underlying nanostructural changes in real time memory operations. Also included is the methodology of fabrication and electrical characterizations for asymmetric crossbar structures incorporating amorphous vanadium oxide. The protocol explained here for vanadium oxide films can be easily extended to any other materials in a metal-dielectric-metal sandwiched structure. Resistive switching crossbars are predicted to serve the programmable logic and neuromorphic circuits for next-generation memory devices, given the understanding of the operation mechanisms. This protocol reveals the switching mechanism in a reliable, timely, and cost-effective way in any type of resistive switching materials, and thereby predicts the device&#39;s applicability.

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Presented here is a protocol for analyzing nanostructural changes during in&#160;situ biasing with transmission electron microscopy (TEM) for a stacked metal-insulator-metal structure. It has significant applications in resistive switching crossbars for the next generation of programmable logic circuits and neuromimicking hardware, to reveal their underlying operation mechanisms and practical applicability.

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...