Name: liquid-cell transmission electron microscopy for tracking self-assembly of nanoparticles
Uploaded: 2017-10-16T13:00:00
Description: Watch this Scientific Journal Video about Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles at JoVE.com

We thank Prof. A. Paul Alivisatos at the University of California, Berkeley and Prof. Taeghwan Hyeon at Seoul National University for the helpful discussion. This work was supported by IBS-R006-D1. W.C.L. gratefully acknowledges support from the research fund of Hanyang University (HY-2015-N).

1. Synthesis of Nanoparticles
<ol>
	<li>Synthesis of platinum nanoparticles
	<ol>
		<li>Combine 17.75 mg of ammonium hexachloroplatinate(IV) ((NH4)2Pt(IV)Cl6), 3.72 mg of ammonium tetrachloroplatinate(II) ((NH4)2Pt(II)Cl4), 115.5 mg of tetramethylammonium bromide, 109 mg of poly(vinylpyrrolidone) (MW: 29,000), and 10 mL of ethylene glycol with a stir bar in a 100 mL 3-neck round bottom flask equipped with a rubber septum.</li>
		<li>Equi...

<ol>
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H., Choi, S., Takeuchi, S., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Cell+Electron+Microscopy+of+Nanoparticle+Self-Assembly+Driven+by+Solvent+Drying.">Liquid Cell Electron Microscopy of Nanoparticle Self-Assembly Driven by Solvent Drying.</a> J. Phys. Chem. Lett. 8, 647-654 (2017).</li><li>Park, J., 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=3D+Structure+of+Individual+Nanocrystals+in+Solution+by+Electron+Microscopy.">3D Structure of Individual Nanocrystals in Solution by Electron Microscopy.</a> Science. 349, 290-295 (2015).</li><li>Chee, S. W., Baraissov, Z., Loh, N. D., Matsudaira, P. 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Platinum nanoparticles with a size of 7 nm were synthesized via the reduction of ammonium hexachloroplatinate (IV) and ammonium tetrachloroplatinate (II) using poly (vinylpyrrolidone) (PVP) as a ligand and ethylene glycol as a solvent and a reducing agent27. A ligand-exchange reaction with oleylamine was performed to disperse the particles in a hydrophobic solvent. Lead selenide nanoparticles were synthesized via the thermal decomposition of lead-oleate complexes using TOP-Se as a selenium source<...

The self-assembly of colloidal nanoparticles is of interest because it provides an opportunity to access collective physical properties of individual nanoparticles11. One of the most effective methods of self-assembly used in practical device-scale applications is self-organization of nanoparticles on a substrate through evaporation of a volatile solvent6,7,8,9,10,11. This solvent evaporation method is a nonequilibrium process, which is largely influenced by kinetic factors such as evaporation rate and changes in nanoparticle-substrate interactions. However, since it is difficult to estimate and control the kinetic factors, the mechanistic understanding of nanoparticle self-assembly by solvent evaporation is not fully mature. Although in situ X-ray scattering studies have provided ensemble-averaged information of the nonequilibrium nanoparticle self-assembly process12,13,14, this technique cannot determine the motion of individual nanoparticles, and their association with the overall trajectory cannot be easily accessed.
Liquid-cell TEM is an emerging tool for tracking the trajectory of individual nanoparticles, enabling us to understand the inhomogeneity of nanoparticle motions and their contribution to ensemble behaviors15,16,17,18,19,20,21,22,23,24,25,26. We have previously used liquid-cell TEM to track the motion of individual nanoparticles during solvent evaporation, showing that the movement of the solvent boundary is a major driving force for inducing nanoparticle self-assembly on a substrate18,19. Herein, we introduce experiments where we can observe the process of nanoparticle self-assembly using liquid-cell TEM. First, we provide protocols for the synthesis of platinum and lead selenide nanoparticles, before introducing the fabrication procedures of liquid-cells for TEM and how to load nanoparticles into the liquid-cell. As representative results, we show snapshot images from TEM movies of nanoparticle self-assembly driven by solvent drying. By tracking individual particles in these movies, we can understand the detailed mechanisms of solvent-drying-mediated self-assembly at a single nanoparticle level. During self-assembly, the platinum nanoparticles on the silicon nitride window mainly follow the movement of the evaporating solvent front because of the strong capillary forces acting on the thin solvent layer. Similar phenomena were also observed for other nanoparticles (lead selenide) and substrates (silicon), indicating that the capillary force of the solvent front is an important factor in particle migration near a substrate.

<table>
	<tbody>
		<tr>
			<td>ammonium hexachloroplatinate (IV)</td>
			<td>Sigma-Aldrich</td>
			<td>204021</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium tetrachloroplatinate (II)</td>
			<td>Sigma-Aldrich</td>
			<td>206105</td>
			<td></td>
		</tr>
		<tr>
			<td>tetramethylammonium bromide, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>195758</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(vinylpyrrolidone) powder</td>
			<td>Sigma-Aldrich</td>
			<td>234257</td>
			<td>Mw ~29,000</td>
		</tr>
		<tr>
			<td>ethylene glycol, anhydrous, 99.8%</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane, anhydrous, 95%</td>
			<td>Samchun Chem.</td>
			<td>H0114</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol, anhydrous, 99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>oleylamine, 70%</td>
			<td>Sigma-Aldrich</td>
			<td>O7805</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>lead(II) acetate trihydrate, 99.99%</td>
			<td>Sigma-Aldrich</td>
			<td>467863</td>
			<td></td>
		</tr>
		<tr>
			<td>oleic acid, 90%</td>
			<td>Sigma-Aldrich</td>
			<td>364525</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>diphenyl ether, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>P24101</td>
			<td>ReagentPlus</td>
		</tr>
		<tr>
			<td>selenium powder, 99.99%</td>
			<td>Sigma-Aldrich</td>
			<td>229865</td>
			<td></td>
		</tr>
		<tr>
			<td>tri-n-octylphosphine, 97%</td>
			<td>Strem</td>
			<td>15-6655</td>
			<td>Air sensistive</td>
		</tr>
		<tr>
			<td>Toluene, anhydrous, 99.9%</td>
			<td>Samchun Chem.</td>
			<td>T2419</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone 99.8%</td>
			<td>Daejung Chem.</td>
			<td>1009-2304</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide, 95%</td>
			<td>Samchun Chem.</td>
			<td>P0925</td>
			<td></td>
		</tr>
		<tr>
			<td>p-type silicon-on-insulator wafers</td>
			<td>Soitec</td>
			<td>Power-SOI</td>
			<td>for liquid cells with silicon windows</td>
		</tr>
		<tr>
			<td>tetramethylammonium hydroxide, 25% in H2O</td>
			<td>J.T.Baker</td>
			<td>02-002-109</td>
			<td></td>
		</tr>
		<tr>
			<td>AZ 5214 E</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 5214 E</td>
			<td>Positive photorest</td>
		</tr>
		<tr>
			<td>AZ-327</td>
			<td>AZ Electronic Materials</td>
			<td>AZ-327</td>
			<td>AZ 5214 develper</td>
		</tr>
		<tr>
			<td>indium pellets 99.98-99.99%</td>
			<td>Kurt J. Lesker Company</td>
			<td>EVMIN40EXEB</td>
			<td>thermal evaporator target</td>
		</tr>
		<tr>
			<td>1,2-dichlorobenzene, &#62;99%</td>
			<td>TCI</td>
			<td>D1116</td>
			<td></td>
		</tr>
		<tr>
			<td>pentadecane, &#62;99%</td>
			<td>Sigma-Aldrich</td>
			<td>P3406</td>
			<td></td>
		</tr>
		<tr>
			<td>buffered oxide etch 7:1</td>
			<td>microchemicals</td>
			<td>BOE 7-1 VLSI</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoric acid, 85%</td>
			<td>Samchun Chem.</td>
			<td>P0449</td>
			<td></td>
		</tr>
	</tbody>
</table>

liquid-cell transmission electron microscopy for tracking self-assembly of nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

The liquid-cell is composed of a top chip and a bottom chip, which are equipped with silicon nitride windows that are transparent to an electron beam with a thickness of 25 nm. The top chip has a reservoir for storing the sample solution and evaporated solvent. The chips are made via conventional microfabrication processing25. The masks used for the top and bottom chips are shown in Figure 1a and 1b, r...

Watch this Scientific Journal Video about Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles at JoVE.com

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

The overall goal of this procedure is to use liquid-cell transmission electron microscropy to investigate the motion of nanoparticles in the solution phase in real time. This method can help answer key questions in the field of nanoscience, for example, about how nanoparticles form self-assembled structures during solvent drying. The main advantage of this technique is that it makes checking nanoparticle motion in real space and real time possible.The implications of this liquid cell technique is tends towards tracking individual motions of nanoparticles that are not shown by conventional methods. Though this method can provide insight into self-assembly of nanoparticles it can be also applied into other models such as oriented attachment of nanoparticles. To begin the procedure, place in a 100 milliliter three neck round bottom flask 17.75 milligrams of ammonium hexacholoroplatinate, 3.72 milligrams of ammonium tetrachloroplatinate and 115.5 milligrams of tetramethylammonium bromide.Add to the flask 109 milligrams of polyvinylpyrrolidone and 10 milliliters of anhydrous ethylene glycol. Equip the flask with a stir bar, a rubber septum and a reflux condenser. Start the stir motor and while stirring at 1000 rpm, degas the reaction flask under vacuum for one hour.Then, under a flow of argon, heat the reaction mixture to 180 degrees Celsius at 10 degrees per minute. Stir the mixture at 180 degrees Celsius for 20 minutes then allow the mixture to cool to room temperature. Transfer the cooled mixture to a 50 milliliter centrifuge tube.Add to the tube 30 milliliters of acetone to precipitate the platinum nanoparticles. Centrifuge the mixture at 2400 times G for 10 minutes. Discard the supernatant and disperse the precipitate in 10 milliliters of ethanol.Obtain a four inch 100 micron silicon wafer coated with about 25 nanometers of silicon nitride. Load photo resist using a spin coater. Then use photo resist to mount the ultra thin wafer on a 500 micron thick silicon wafer.Spin coat the wafer with 10 milliliters of positive photo resist at 3000 rpm for 30 seconds. Bake the wafer at 85 degrees Celsius for 60 seconds. Then cover the wafer with a chromium mask and expose the wafer to 365 nanometer light for 10 seconds.Immerse the wafer in 50 milliliters of the appropriate developer solution for 40 seconds and then in 50 milliliters of deionized water for one minute. Immerse the wafer in 50 milliliters of deionized water for one minute, then place the patterned wafer in a reactive ion etcher. Etch the exposed silicon nitride for one minute.Use a water bath to evenly heat a container of a 30 milligram per liter aqueous solution of potassium hydroxide to 85 degrees Celsius. Soak the ultra thin wafer in the hot potassium hydroxide for two hours to etch the exposed silicon. When the exposed silicon appears to have been completely etched away, carefully remove the wafer from the solution at an angle to avoid rupturing the silicon nitride window.Repeat this process with the second chromium mask to obtain the top and bottom chips. Use the third chromium mask to pattern indium spacers onto the bottom chip. Align the top and bottom chips and bond the chips together at 100 degrees Celsius.Transfer 20 microliters of the prepared nanoparticle dispersion to a five milliliter vial. Allow the solvent to evaporate under ambient conditions for 10 minutes. Inspect the liquid cell under an optical microscope to verify that the silicon nitride windows are intact.Then disperse the nanoparticles in a mixture of one milliliter of orthodichlorobenzene, 250 microliters of pentadecane and 10 microliters of oleylamine. With the liquid cell under the optical microscope use an injector with an ultra thin capillary to load 100 nanoliters of the dispersion into the liquid cell reservoirs. Use filter paper to absorb the excess dispersion outside the reservoirs.Allow the cell to sit in ambient air for 10 minutes to evaporate the orthodichlorobenzene. Then apply vacuum grease to one side of a two millimeter copper aperture grid with a 600 micron hole. Carefully cover the liquid cell with the grid, being careful to align the aperture with the liquid cell window.Mount the cell in a standard TEM holder and load the cell into the instrument. Acquire images in continuous image acquisition mode as the solvent dries. Use image processing software to calculate the radial distribution function for the particles in each acquired image.TEM images of a platinum nanoparticle suspension drying in a silicon nitride liquid cell showed the nanoparticles being pulled inward by the receding solvent front. This behavior was attributed to the strong capillary forces of the thin layer of solvent and the reduced free energy of the nanoparticles at the solvent interface. The nanoparticles initially formed amorphous multilayer agglomerates as they were drawn together.As the solvent dried, the agglomerates flattened into an ordered monolayer. This ordering is reflected in the radial distribution functions derived from the TEM images. The radial distribution function of the image taken after 90 seconds had a large peak at 8.3 nanometers.The oleylamine capped platinum nanoparticles are about 8.3 nanometers in diameter, suggesting that a significant number of particles were assembled as closely as possible. Once mastered, this technique can be done in two days if it is performed properly. Generally individuals who are new to this method may struggle because fabricating and working with the liquid cell require different levels of optimization for different nanoparticles or liquid cell compositions.While attempting this procedure, remember to protect the windows of the liquid cell from breaking. Following this procedure, other methods like applying voltages to the liquid cell can be performed to answer additional questions about the self-assembly of nanoparticles in the presence of external forces. After its development, this technique paved the way for researchers in the field of nanoscience to explore the assembly process of nanoparticles in the overall dry mechanism.After watching this video, you should have a good understanding of how to prepare liquid cells and measure the motions of nanoparticles in TEM experiment. Don't forget that working with KUH agent can be extremely hazardous. Precautions such as wearing safety glasses should always be taken while performing this experiment.

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Research

JoVE Journal

Chemistry

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

This paper describes a protocol to achieve 3D chemical maps combining energy filtered imaging and electron tomography. The chemical distribution of two catalyst supports formed by elements that are difficult to distinguish by other imaging techniques was studied. Each application consists of mapping overlapped chemical elements - respectively spaced-ionization edges.

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...