Zheng thanks Prof. A. Paul Alivisatos and Dr. Ulrich Dahmen for helpful
discussions during the early development of EM liquid cells. She is grateful to the support of
DOE Office of Science Early Career Research Program.

1. Microfabrication of Liquid Cells
<ol>
	<li>Prepare silicon wafers (p-doped, 100 &mu;m in thickness and 4 inches in diameter) and clean the wafers using a standard wafer bath cleaning procedure.</li>
	<li>Deposit low-stress silicon nitride thin films (20 nm in thickness) on both sides of the silicon wafers by low-pressure chemical vapor deposition (LPCVD) method. A custom developed recipe is used for the deposition, which allows the growth of silicon-rich nitride (SiNx, x&lt;4/3).</li>
	<li>Fabricate the bottom chip (2.6 &times; 2.6 mm; 3 mm in diameter) with a viewing window (1 &times; 50 &mu;m) and the top chip (2.6 &times; 2.6 mm; 3 mm in diameter) with a viewing window (1 &times; 50 &mu;m) and two reservoirs (0.6 &times; 1.2 &times; 0.1 mm) by following a sequence of standard fabrication processes, including photolithographic patterning, plasma etching of the SiNx membrane (using SF6 as the active gas), KOH wet etching of the exposed silicon wafer, etc. We use the most common photolithographic process, such as spin coating of a photoresist (positive photoresist with spin speed of 3,000 rpm for 1 min; the thickness of the photoresist is roughly 1 &mu;m), UV exposure under the Cr mask of liquid cells, lithographic patterning using developer and fixer (deionized water), etc. There are different choices of the photoresist for spin coating and the developer for patterning. And, the corresponding parameters for the processes may also vary. Since features of the pattern are relatively large (hundreds of &mu;m or larger), the process is easy to be accomplished. The KOH solution is prepared by dissolving potassium hydroxide power into deionized water with the potassium hydroxide:water weight ratio of 1:2. The KOH solution is maintained at 80 &deg;C during etching. An etching rate of 1 &mu;m per min can be achieved. SiNx membrane is an ideal protective mask for KOH etching of silicon. Since etching lines are used, individual chips are connected with thin lines of etched wafer after KOH etching. Pieces of chips can be easily separated from the wafer using sharp tweezers for subsequent processes. No dicing process is needed.</li>
	<li>Deposit indium spacer on the flat side of the bottom chip. First, do lithographic patterning of the chips by following the similar process in 1.3. To assist handling of the chips, stick individual chips (can be a piece of several chips) on a thin glass sheet using photoresist and let it air dry for 5 mins before spin coating, UV exposure, etc. Second, clean the patterned chips by O2 plasma cleaning at 50 Watts for 1 min; Third, deposit indium thin film with the thickness of 100 nm on the chip by using an evaporator; Third, lift-off process is carried out to generate the indium spacer.</li>
	<li>Bond the bottom and top chips together. We first align two silicon nitride viewing windows of the bottom and top chips under an optical microscope and apply a pressure of about 0.1 MPa by using a clamp. It requires practice in order to precisely align the windows on top of each other. Subsequently, liquids cells are baked in a vacuum oven at 120 &deg;C for 1 hr. Lastly, we collect the cells and store the as-prepared cells in a vacuum desiccator for future usage.</li>
</ol>
The whole fabrication process is shown in Figure 1. We conduct all the fabrication processes at the Nanofabrication Lab of the University of California, Berkeley.
2. Preparation of Reaction Solutions
We prepare the reaction solutions for the growth Pt3Fe nanorods as an example. Platinum (II) acetylacetonate (20 mg/ml) and Iron (II) acetylacetonate (20 mg/ml) were dissolved in a solvent mixture of pentadecane and oleylamine (7:3 vol/vol) or a mixture of pentadecane, oleylamine, and oleic acid (6:3:1 vol/vol/vol) is used for the comparison of surfactant effects.
3. Load Reaction Solutions
<ol>
	<li>About 50 nl of reaction solution is loaded into one of the reservoirs in a liquid cell by using a syringe and Teflon nanotubes (purchased from Cole-Parmer, IL). Then, the other reservoir is filled in the same manner.</li>
	<li>About 30 pl of the reaction solution is drawn into the cell by capillary force and forms a liquid layer (~100 nm) sandwiched between two silicon nitride membranes in the viewing window.</li>
	<li>The liquid cell is subsequently sealed using a thin copper cover (~50 &mu;m TEM grid with single slot 0.6 mm diameter hole, which was purchased from Tedd Pella, Inc.). Vacuum grease was applied on one-side of the cover and epoxy was used to seal the edge of the liquid cell. The total thickness of the final liquid cell is about 250-300 &mu;m.</li>
</ol>
4. Load Liquid Cells into TEM
<ol>
	<li>A JEOL 3010 TEM operated at 300 kV and a FEI monochromated F20 UT Tecnai operated at 200 kV are used for in situ imaging.</li>
	<li>The liquid cell is loaded into the microscope as a standard TEM sample for imaging.</li>
</ol>
5. Real Time TEM Imaging
<ol>
	<li>Tune the microscope to a perfect high resolution TEM imaging condition, and a beam current density of 1-8 &times; 105 A/m2 is maintained during the real-time imaging.</li>
	<li>For PtFe system, nucleation and growth of the nanoparticles can be initiated by casting the electron beam on the liquid layer.</li>
	<li>VirtualDub software combined with Gatan DigitalMicrograph software is utilized to record the nanoparticle dynamics.</li>
</ol>

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No conflicts of interest declared.

All fabrication processes have been done in the clean room, where semiconductor devices are made.
Before the deposition of indium, O2 plasma cleaning of the chips is necessary to eliminate the organic residue on the surface. Thus, a high quality indium spacer can be achieved, which can improve the bonding of top and bottom chips and the yield of leakage free liquid cells.
The silicon nitride viewing windows with ultrathin membrane of about 13 nm thick is a key to achieve high spatial resolution. When handling such liquid cells, special care is needed to avoid breaking the membrane during fabrication as well as experiments. For example, tweezers with a flat front are recommended. And, during the membrane cleaning process, low power and dose of O2 plasma can be incorporated (i.e., 30 Walt for 20-30 sec). Since growth kinetics can be highly dependent on the electron beam current density, maintaining the same electron beam current density while imaging is important. The liquid cell TEM method not only enables the study of growth dynamics of nanocrystals in solution in real time, but also allows revealing other dynamic processes (i.e., diffusion of nanoparticles in liquids, liquid droplet dynamics, etc.). In addition, it provides a promising pathway to visualize biological processes in the native environment.

The study of chemical reactions in liquids in real time and imaging biological materials in their native environment have been of significant interests across the research fields1-5. Due to the high spatial resolution of transmission electron microscopy (TEM), imaging through liquids using TEM has attracted a lot of attention4,5. However, it has been a great challenge to image liquid samples using TEM, since the conventional microscope is operated in a high vacuum environment. In addition, liquid samples have to be thin enough to allow the electron beam to go through. Williamson et al.6 reported that imaging of electrochemical deposition of Cu can be achieved with 5 nm resolution using an electrochemical liquid cell operated in a TEM. De Jonge et al.1 was able to image biological samples through serveral micrometer thick water using a scanning(S) TEM. The low contrast of the biological samples was not raised as an issue since gold nanoparticles were used as markers for imaging. The thick liquid sample was not a problem either since STEM imaging mode was used and nanometer resolution was achieved. We recently developed a self-contained liquid cell, which allows real time TEM imaging of colloidal nanoparticles in liquids with subnanometer resolution5,7. These newly developed liquid cells, which offer improved resolution and faster TEM imaging (30 frames per second that has not been achieved by high resolution STEM imaging), made it possible to study colloidal nanoparticle dynamics in liquids. The liquid cells fit in a standard TEM holder and can be operated as regular TEM samples. A small amount of liquid (about 30 picoliters) can be examined in situ under an extended chemical reaction. Various imaging and analytical (i.e., energy-dispersive X-ray spectroscopy) techniques can be applied. Since the total thickness of the viewing window (including membranes and the liquid layer) can be controlled to 100 nm or below, direct imaging of biological samples (i.e. proteins) in liquid water without gold nanoparticle markers has also been achieved 8.
In the past two decades, there have been significant achievements on the syntheses and applications of colloidal nanocrystals9-11. However, the understanding of how nanoparticles nucleate, grow and interact with each other in liquids is largely empirical and mostly based on ex situ analyses11-13. Our development of liquid cell TEM provides a unique platform to study the dynamic processes of nanoparticles in liquids in situ5,7,14,15.
We fabricate a self-contained liquid cell using ultra thin silicon wafers (100 &mu;m) by a sequential microfabrication process. It includes deposition of silicon nitride membrane, photolithographic patterning, wafer etching, spacer deposition, and cell bonding, etc. About 50 nanoliters of the reaction solution is loaded into a reservoir, which is drawn into the cell by capillary force. We fill the other reservoir with another 50 nanoliters of the liquid. Subsequently, the cell is sealed and loaded into the microscope for in situ imaging. Inside the microscope, the liquid sandwiched between two silicon nitride membranes (total about 30 picoliters) can be examined. When the electron beam passes through the thin liquid layer, dynamic processes of nanoparticles in liquids can be monitored in real time. Nucleation and growth of nanoparticles can be induced by the electron beam in some cases5,7 or reactions can be triggered by an external heating source14,16. When the electron beam damage is of a concern, low electron beam current (dose) should be used.
Since liquid cells are fabricated from silicon microfabrication processes and in large batches, variations in membrane or liquid thickness among individual liquid cells can be small6. Any researcher who has basic microfabrication training can successfully make liquid cells. The liquid handling technique and in situ TEM operation can also be mastered after practice. It is noted that besides using silicon nitride membranes as the viewing windows, other materials such as silicon dioxide, silicon or carbon (including graphene) can be used as the membrane window as well17-19. Since our liquid cells using small viewing windows, i.e., 1 x 50 &mu;m, no bulging of the membranes has been observed. And, the liquid cell is also robust to operate, i.e. below 1% of liquid cells have broken windows during the experiments. In addition, thickness of the liquid layer can also be flexibly tuned by changing the thickness of the deposited indium spacer. During sample preparation, a sealed liquid cell can maintain liquids for several days with no leakage. The small amount of liquid can be examined for several hours under the electron beam, which allows the study of an extended chemical reaction in real time.
So far, we have visualized many unique dynamic processes of nanoparticles in liquids, for example, growth and coalescence of Pt nanoparticles5,15, diffusion of nanoparticles in thin liquids20,21, growth fluctuation of Bi nanoparticles14, and growth of Pt3Fe nanorods from nanoparticle building blocks7, etc. In addition, we have also applied this method to other areas, e.g., imaging proteins in liquid water with 2.7 nm resolution8. In summary, our liquid cell TEM technique has been proven to be a very valuable development for the study of a wide range of fundamental issues in materials science, physics, chemistry and biology. We believe there is still large room for future technical advances and applications of the liquid TEM and it will certainly be a high impact on a broad spectrum of scientific research.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Platinum(II) acetylacetonate</td><td>Aldrich</td><td>523038</td><td></td></tr><tr><td>Iron(II) acetylacetonate</td><td>Aldrich</td><td>413402</td><td></td></tr><tr><td>pentadecane</td><td>Aldrich</td><td>P3406</td><td></td></tr><tr><td>oleylamine</td><td>Aldrich</td><td>O7805</td><td></td></tr><tr><td>oleic acid</td><td>Sigma</td><td>O4137</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>TEM</td><td>JEOL</td><td>JEOL 3010</td><td></td></tr><tr><td>Monochromated TEM</td><td>FEI</td><td>F20 UT Tecnai</td><td></td></tr></tbody></table>

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.

By using liquid cell TEM method, we have visualized the solution growth of Pt3Fe nanorods from nanoparticle building blocks. Figure 2 shows sequential images depicting the growth trajectory of a Pt3Fe nanorod in different solution conditions. False coloring process using Photoshop was employed to highlight the nanoparticles.
When the solvent mixture of pentadecane and oleylamine (7:3 vol/vol) was used, three distinct stages of growth can be identified (Figure 2A). Firstly, many small nanoparticles are formed when the Pt and Fe precursors are reduced by electron beam irradiation. Some of them grow by monomer attachment; others undergo coalescence. Secondly, short nanoparticle chains are formed via nanopaticle interactions. Thirdly, the as-formed short nanoparticles chains act as building blocks to form relatively long winding nanoparticle chains. When a mixture of pentadecane, oleylamine, and oleic acid (6:3:1 vol/vol/vol) was used, winding nanoparticle chains are formed first, and then the nanoparticle chains straighten and form single-crystalline nanorods within a short period of time (Figure 2B).
In summary, we have shown the formation of single crystal nanorods via the growth of winding polycrystalline nanoparticle chains from shape-directed nanoparticle attachment followed by the straightening, orientation and shape corrections of the building blocks. Statistics and quantification of nanoparticle dynamics from the real-time imaging are of great significance to the understanding and control of hierarchical nanomaterials growth and self-assembly for functional devices7.
<img alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50122/50122fig1highres.jpg" src="/files/ftp_upload/50122/50122fig1.jpg" /> 
Figure 1. Overview of the microfabrication process of liquid cells. (I) Deposit silicon nitride membranes on both sides of the silicon wafer; (II) Fabricate the viewing window and reservoirs by photolithographic patterning and etching; (III) Deposit indium spacer on the flat side of the bottom chip; (IV) Bond the bottom and top chips together with a clamp.
<img alt="Figure 2" fo:content-width="5.5in" fo:src="/files/ftp_upload/50122/50122fig2highres.jpg" src="/files/ftp_upload/50122/50122fig2.jpg" /> 
Figure 2. The growth of Pt3Fe nanorods in a liquid cell during the exposure to electron beam. (A) Sequential TEM images showing the evolution from the initial nucleation and growth in the molecular precursor solution to a later stage of nanowire formation by shape-directed nanoparticle attachment. A solvent mixture of pentadecane and oleylamine (7:3 vol/vol) was used. (B) Formation of twisted Pt3Fe nanorods and the subsequent straightening process. (a) Sequential TEM images of the growth of a short Pt3Fe nanorod. (b) Sequential TEM images showing the growth of a long Pt3Fe nanorod. A solvent mixture of pentadecane, oleylamine, and oleic acid (6:3:1 vol/vol/vol) was used. In both (A) and (B), time is displayed as min:sec, and the initial time is arbitrary7.

Watch this Scientific Journal Video about Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy at JoVE.com

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

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 ...

revealing-dynamic-processes-materials-liquids-using-liquid-cell

Nanyang Technological University

Research

JoVE Journal

Engineering

12.6K Views.  Lawrence Berkeley National Laboratory. 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.

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

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Bioengineering

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

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

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

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

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.

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 ...

Chemistry

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 ...

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. ...

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

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

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Chapters in this video

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

In situ TEM of Biological Assemblies in Liquid

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

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

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

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

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System