Name: studying dynamic processes of nano-sized objects in liquid using scanning transmission electron microscopy
Uploaded: 2017-02-05T14:00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy at JoVE.com

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>

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	<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">
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			<td>DENS Solutions</td>
			<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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</table>

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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