We thank E. Arzt for his support through INM. The research was in part supported by the Leibniz Competition 2014.

<ol>
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The authors have nothing to disclose.

The described protocol enables STEM of AuNPs in a liquid, including the observation of dynamic processes. The assembly of the holder is an easy-to-learn technique. However, several aspects must be considered when working with the liquid flow TEM holder. For instance, broken edges of the Si microchip or large particles on the O-rings may result in leakage of the liquid cell. On the other hand, large particles (&#62;200 nm; e.g., dust or Si debris) on the SiN membrane may result in an increased thickness of the liquid cell, leading to a low imaging contrast or to a low spatial resolution and may even cause SiN windows to break. Importantly, residues of salt or other chemicals may influence the outcome of the experiments in an undesired way. Therefore, it is crucial that the different steps of sample preparation and holder assembly are carried out carefully and in a clean and dust-free environment.
The thickness of the liquid cell determines the achievable resolution, as well as the contrast of the obtained images17. This thickness can be adjusted via spacers located on one of the two Si microchips. Depending on the dimensions of the sample, different thicknesses of the liquid cell can be realized. For the study of AuNPs, it is possible to use small spacers (200-500 nm), while whole eukaryotic cells need larger spacers of up to 5 &#181;m. The thickness of the liquid cell is further influenced by the bulging of the SiN membrane windows resulting from the pressure difference between the liquid cell and the surrounding vacuum. This effect becomes more pronounced with larger SiN membrane windows. Thus, in order to minimize the thickness of the liquid cell, it is recommended to use small SiN membrane windows. In case it is difficult to find an overlap between two small windows, they can be assembled in a crossed configuration using a different base microchip. Alternative configurations largely prevent bulging and consist of a monolithic microchip18 or membrane windows supported by pillars19, but those exhibit disadvantages regarding sample loading. One of the most challenging aspects of the current technology is the lack of precise control over the liquid thickness. Often, the liquid is much thicker than what is expected from the spacer dimensions used, as was shown here. Several groups used closed liquid chambers4,20,21,22; these systems have some advantages regarding spatial resolution, as the liquid thickness can be reduced by inducing a bubble in the liquid. Alternatively, the SiN windows can be forced to collapse, leading to a thinner liquid layer. Thirdly, the enclosure of other thinner windows exists (e.g., graphene)23, also resulting in much thinner liquids than what is possible with the system described in this protocol. However, it is impossible to flow liquid in those systems.
As for any high&#160;resolution microscopy technique, a number of experimental aspects must be considered. The most important aspect is the interaction of the electron beam with the liquid or the sample. In addition to radiation damage, which limits the achievable spatial resolution for many solid samples24, the liquid samples are also influenced by electron beam-generated radiolysis products15,25. Since these products may influence the experiment, careful data interpretation and experimental design are essential26. The microscope settings should be chosen according to the objectives of a particular study. ADF STEM is more powerful for imaging nanoparticles of a high atomic number (Z) in larger thicknesses of the liquid cell, while TEM gives better contrast on low-Z materials and is typically faster but requires thinner liquid layers3. Instead of using the ADF detector, the Bright Field (BF) detector is sometimes used to image the liquid cell, since BF STEM is advantageous for imaging low-Z materials in thick layers27. With increasing thickness of the liquid cell, more current is needed. However, this also increases the concentrations of radiolysis products and increases radiation damage. It should also be noted that an inversion of contrast is observed in the ADF detector for very thick liquids (&#62;10 &#181;m for water).
The liquid conditions were changed between our experiments by removing the holder from the microscope and exchanging both the sample and the liquid. In addition to changing the salt concentration, it is readily possible to change other properties of the liquid by flowing in different liquids (e.g., one may use buffer solutions in order to set a specific pH16 or may introduce organic solutions or other additives). It is also possible to change the liquid while the holder is still inserted in the microscope by flowing liquids through the microfluidic system. However, in this case, it is unknown at which time point the liquid at the sample changes. It is also noteworthy that microchips supporting electrodes are available, so nanoscale electrochemistry experiments can be carried out28.
The objects of study are not limited to AuNPs in water, but a wide variety of specimens can be studied using the protocol described above, including silica, titanium oxide, and polymers. If movements of the objects are too fast to capture in an image within the acquisition, the viscosity can be reduced by an order of magnitude by using a mixture of 50% glycerol and 50% water.
From the aforementioned points, a number of advantages, possibilities, and also disadvantages become apparent. When working with liquid-phase STEM, the most important disadvantages to consider are that: 1) any experiment is influenced by the dynamic interaction of the electron beam with the entire specimen (the object under observation, the liquid, and the SiN membranes); 2) sample handling is tedious, and it is often difficult to achieve a thin liquid layer because the sample or the microchips contain some micrometer-sized particles; 3) the liquid thickness usually differs largely from the intended thickness set by the spacer; and 4) spatial resolution and contrast strongly depend upon the liquid thickness and the difference between the change density of the object under observation and the liquid.
Presently, ample methods exist for the microscopy of objects in liquid with nanometer spatial resolution. Electron microscopy in amorphous ice is a powerful technique29, but the involved experimental procedures are delicate, not all experiments allow the preparation of the sample in ice, and time-resolved experiments are impossible. X-ray microscopy30,31 could in principle be used, but it has a limited spatial resolution and is not widely available in laboratories. Atomic force microscopy in liquid has been established but is a surface technique only32,33,34,35. Light microscopy does not exhibit sufficient spatial resolution. At the present, electron microscopy in liquid seems the most powerful technique for direct microscopy of nanoscale objects and processes in liquid.
Liquid-phase TEM and STEM are not yet routine analytical techniques but are still developing. The number of parameters to take into account is considerable, and it is often difficult to reproduce experimental results. Moreover, quantitative data is difficult to obtain because the effects under investigation are intertwined with processes occurring as a result of the electron beam. The protocol described here aims to standardize the experimental protocol, thereby accounting for all relevant base aspects of the experiment. We hope that this protocol will lead to better reproducibility of experimental work in this emerging field.

Conventional Scanning Transmission Electron Microscopy (STEM) is limited by the range of specimens appropriate for analysis, specifically the dry and solid samples suitable for placement in a high vacuum. However, many scientific and technological questions concern nanoscale materials and processes in liquid environment. Samples fully embedded in liquid can now be studied with STEM using a concept that involves a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM1. This newly developed technique has become increasingly popular, as it provides new insight into important processes of various research topics, including the growth, dissolution, and aggregation processes of nanoparticles2,3,4,5,6. Not only metals, but also biominerals7 and biological systems can be studied8,9,10,11. The sample loading and image acquisition for liquid-phase STEM is different than for STEM of dry samples and involve a protocol that requires dedicated training.
The microfluidic system consists of two silicon microchips supporting Silicon Nitride (SiN) membrane windows transparent for the electron beam at 200 keV of energy12 (see Figure 1A). Details of the dimensions and of the handling of these microchips can be found elsewhere12,13. The sample usually contains nanoscale objects. In this paper we observed gold nanoparticles (AuNPs).&#160;The AuNPs are immobilized at the top window (with respect to a downward-traveling electron beam) or float in the liquid. Nanoscale spatial resolution in STEM is obtained by scanning the electron beam over the AuNPs and collecting transmitted scattered electrons using the Annular Dark Field (ADF) detector9. The two microchips are placed in a small slot in the tip of the liquid flow TEM holder1 (the holder operates for both STEM and TEM but is referred to as the TEM holder). One of the microchips contains a spacer so that a liquid compartment is formed between the microchips. O-rings on both sides of the two microchips provide vacuum sealing of the liquid compartment13 (see Figure 1B).
The aim of this article is to demonstrate the basic steps of sample loading and data acquisition so that interested users may find easy access to this emerging new technique. A system available from a specific company is used, but the protocol is also valid for systems of other companies. The technique is more complex than conventional TEM and STEM, and a number of practical aspects must be considered when working with a liquid holder system13. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. Therefore, it is highly important to work cleanly and to prevent the formation of dust during the preparation and assembly of the liquid flow TEM holder. In particular, the O-rings and the two silicon microchips need to be free from all contamination. Even small particles of dust on one of the microchips may severely increase the thickness of the assembled cell, which may prevent the achievement of a useful spatial resolution. A vacuum seal is important so that no contamination or damage will be left in the electron microscope after the experiment. This protocol describes the loading procedure and several necessary tests. The operation of the electron microscope is straightforward, but it requires some extra steps compared to microscopy of solid samples. With increasing liquid thicknesses, more electrons are absorbed and scattered by the liquid; a measurement of the liquid thickness is essential. Finally, the protocol explains how images are taken and how dynamic processes can be studied.
<img alt="Figure 1" src="/files/ftp_upload/54943/54943fig1.jpg" /> 
Figure 1: Liquid Flow Cell for Scanning Transmission Electron Microscopy (STEM). (A)Schematic illustration of the assembled liquid cell. Two silicon microchips with Silicon Nitride (SiN) membrane windows are positioned between two O-rings. The liquid is enclosed between the SiN membrane and is thus separated from the vacuum in the electron microscope. A focused electron beam scans over the sample. Contrast is obtained from scattered electrons. Gold nanoparticles (AuNPs) are immobilized within the liquid at the SiN membrane but can also move in the liquid. (B)Schematic side view cross section of the stack of two microchips with O-rings. <a href="http://cloudflare.jove.com/files/ftp_upload/54943/54943fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="800">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Binocular light microscope</td>
			<td style="width:141px;">Leica</td>
			<td style="width:163px;">M60 CMO</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanning transmission electron mircoscope with spherical aberration corrector</td>
			<td>JEOL</td>
			<td>ARM200F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid flow TEM specimen holder</td>
			<td>DENS Solutions</td>
			<td>Ocean</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfluidic syringe pump</td>
			<td>Harvard Scientific</td>
			<td>PicoPlus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma cleaner</td>
			<td>Gatan</td>
			<td>Solarus950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone, Rotisolv&#160; Plus for HPLC</td>
			<td>Sigma-Aldrich</td>
			<td>7328.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water, chromasolv Plus for HPLC</td>
			<td>Sigma-Aldrich</td>
			<td>34877-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, Rotisolv HPLC grade</td>
			<td>Carl Roth</td>
			<td>P076.2</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td>DENS Solutions</td>
			<td>for Ocean system</td>
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			<td>for Ocean system</td>
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			<td>Electron Microscopy Sciences</td>
			<td>78322-7Te</td>
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			<td>Electron Microscopy Sciences</td>
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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.

The liquid flow TEM holder was used to study the behavior of AuNPs in liquid. AuNPs were stably immobilized on the SiN membrane in pure water and were imaged with nanoscale resolution using liquid-phase STEM (Figure 6). Excellent contrast was obtained on the strongly-scattering gold. The current density on the phosphor screen measured for a dry test sample was 20 pA/cm&#178;, while it amounted to 8 pA/cm&#178; with the liquid flow TEM holder inserted. Using Equation 1, twater = 2.4 &#177;0.5 &#181;m, much larger than what was expected based on the spacer thickness of 200 nm. Nevertheless, the thickness is not too large for the imaging of the AuNPs with nanometer spatial resolution. The liquid thickness was thicker than the 200 nm set by the spacer due to bulging of the SiN membranes, non-flatness of the microchips, and debris residing on the microchips.
For pure water, the AuNPs maintain their shape during imaging16, although reactive radiolysis products (e-aq, H&#8226;, H+, OH&#8226;) originating from the interaction of the electron beam with water may oxidize single gold atoms, leading to a change of shape of the AuNPs15. However, when the liquid flow system was used to introduce chloride ions in a second experiment, the stability of the AuNPs changed. Chloride ions are capable of stabilizing oxidized gold atoms in the form of tetrachloroaureat, AuCl4-. Figure 7 shows that the AuNPs slowly dissolved during a STEM imaging time-lapse series, similar to results reported earlier16. For the used electron dose rate, it took ~300 s to dissolve the 30 nm-sized AuNPs.
The movements of AuNPs in water were studied in a third experiment (Figure 8). Prior to the experiment, the liquid flow TEM holder was cleaned in order to remove any traces of salt. Differing from the first experiment, an alternative sample preparation approach was used to achieve a weaker attachment of the AuNPs to the SiN membrane14. In this experiment, the AuNP solution was placed on the silicon microchip and assembled in the liquid flow TEM holder without letting the solution dry out. In this way, the AuNPs easily detached from the SiN membrane upon imaging at the dose rate used. Some of the AuNPs moved away from the field of view into the bulk solutions, while the remaining AuNPs remained within the field of view in close proximity to the SiN window. Movements of these AuNPs were observed, and eventually they agglomerated. After a while, these agglomerates also detached from the SiN membrane and moved out of the field of view and into the solution.
<img alt="Figure 6" src="/files/ftp_upload/54943/54943fig6.jpg" /> 
Figure 6: Scanning Transmission Electron Microscopy (STEM) Micrograph of AuNPs 30 nm in Diameter at the Top of a Pure Water Layer. The image shown is a selected area of the original image. The image size was 1,024 x 1,024 pixels, the pixel dwell time was 19 &#181;s, the pixel size was 0.73 nm, and the magnification was 400,000X. The electron dose was thus 7.1 x 104 e-/nm&#178;. The current density measured on the phosphor screen was 8 pA/cm2, so the liquid thickness was calculated to amount to 2.4 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54943/54943fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54943/54943fig7.jpg" /> 
Figure 7: Time-lapse Series of STEM Micrographs of AuNPs in Saline. (A-D) Images extracted from the time-lapse series of STEM images at 30&#160;s intervals. The AuNPs gradually dissolve in liquid as a consequence of the presence of chloride ions. The pixel dwell time was 2 &#181;s, the frame time of the time lapse series was 1.75 s, the pixel size was 0.44 nm, and the magnification was 500,000X. The electron dose per image was 1.2 x 104 e-/nm&#178;. The liquid thickness was 2.4 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54943/54943fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54943/54943fig8.jpg" /> 
Figure 8: STEM Micrograph of AuNPs Moving in Pure Water. (A) SiN membrane with AuNPs, of which several are selected with arrows. (B) Motion tracks of the selected AuNPs (see A). Some AuNPs move away from the field of view during the time of imaging. The remaining AuNPs move laterally along the SiN membrane and start agglomerating. Upon reaching a critical cluster size, they dispatch from the membrane and move away from the field of view.The pixel dwell time was 1 &#181;s, the frame time was 0.52 s, the pixel size was 1.8 nm, and the magnification was 120,000X. The electron dose per image was 3.5 x 102 e-/nm&#178; and the liquid thickness was 2.4 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54943/54943fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy at JoVE.com

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

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 ...

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12.6K Views.  INM-Leibniz Institute for New Materials. 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.

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

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 ...

Scanning-probe Single-electron Capacitance Spectroscopy

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the electronic quantum structure of small systems - including individual atomic dopants in semiconductors. Here we present a capacitance-based method, known as Subsurface Charge Accumulation (SCA) imaging, which is capable of resolving single-electron charging while achieving sufficient spatial resolution to image individual atomic dopants. The use of a capacitance technique enables observation of subsurface features, such as dopants buried many nanometers beneath the surface of a semiconductor material1,2,3. In principle, this technique can be applied to any system to resolve electron motion below an insulating surface.
As in other electric-field-sensitive scanned-probe techniques4, the lateral spatial resolution of the measurement depends in part on the radius of curvature of the probe tip. Using tips with a small radius of curvature can enable spatial resolution of a few tens of nanometers. This fine spatial resolution allows investigations of small numbers (down to one) of subsurface dopants1,2. The charge resolution depends greatly on the sensitivity of the charge detection circuitry; using high electron mobility transistors (HEMT) in such circuits at cryogenic temperatures enables a sensitivity of approximately 0.01 electrons/Hz&frac12; at 0.3 K 5.

Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

The integration of low-temperature scanning-probe techniques and single-electron capacitance spectroscopy represents a powerful tool to study the ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

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 ...

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared.

The optical, electrical, and structural properties of dislocations and of grain boundaries in semiconductor materials can be determined by experiments performed in a scanning electron microscope. Electron microscopy has been used to investigate cathodoluminescence, electron beam induced current, and diffraction of backscattered electrons.

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications ...

Precision Milling of Carbon Nanotube Forests Using Low Pressure Scanning Electron Microscopy

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning electron microscope (ESEM) operating with a low pressure water vapor ambient. In this technique, a portion of the electron beam interacts with the water vapor in the vicinity of the CNT sample, dissociating the water molecules into hydroxyl radicals and other species by radiolysis. The remainder of the electron beam interacts with the CNT forest sample, making it susceptible to oxidation from the chemical products of radiolysis. This technique may be used to trim a selected region of an individual CNT, or it may be used to remove hundreds of cubic microns of material by adjusting ESEM parameters. The machining resolution is similar to the imaging resolution of the ESEM itself. The technique produces only small quantities of carbon residue along the boundaries of the cutting zone, with minimal effect on the native structural morphology of the CNT forest.

Low pressure scanning electron microscopy in a water vapor ambient is used to machine nanoscale to microscale features in carbon nanotube forests.

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning ...

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 ...

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.

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 ...

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 ...

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.