Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy
July 15th, 2019
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.
Liquid cell electron microscopy is a powerful technique to investigate nano features in C2 in liquid media at high resolution and provides unique, real time insights into dynamic processes at the nano scale. The graphene-supported, microwell liquid cell combines the advantages of both graphene and silicon technology based cell architectures. It allows for correlation with analytical methods, such as EDX spectroscopy, on sight.
The technique enables us to directly follow a material's processes in liquids, while they happen. This in situ observation is at the very core of our research training group. In situ microscopy with electrons, X-rays, and scanning probes funded by the German Research Foundation.
Demonstrating the procedure will be Diplom-Chemist, Robert Branscheid, a staff member from my laboratory. To begin, transfer the graphene onto TEM grids, first wetting the tissue supporting the six to eight layers of CVD graphene on PMMA. Take care that the water is not directly applied to the PMMA membrane.
Fully immerse the PMMA-coated graphene in a Petri dish filled with DI water, and then use filter paper to scoop up the graphene layer. Take care that the graphene side of the graphene PMMA stack stays on top during the whole procedure. Cut the graphene layer into pieces that are large enough to cover all of the fabricated wells.
Then, re-immerse the cut pieces into the Petri dish. Next, using a pair of anti-capillary tweezers, pick up a TEM grid coated with a support layer of holey-carbon. Carefully dive the grid into the water, and catch the graphene floating on the surface.
Let the sheets dry for a few hours. Then, remove the PMMA protection layer by transferring it into an acetone bath for 30 minutes. Following the acetone bath, immediately immerse the sample in ethanol and DI water without drying the sample in between solutions.
Use a flat vessel to easily remove the specimen afterwards. When finished, remove the sample from the DI water, and dry it afterwards for 30 minutes at ambient conditions. Create a liquid cell template with micro-patterned microwells by following along in the text protocol.
Rinse the fabricated liquid cell template with acetone, followed by ethanol. Then, apply an ambient 20%oxygen, 80%nitrogen plasma for five minutes to enhance the wettability of the membrane. Dispense 0.5 microliters of the specimen solution onto the template or the graphene layer.
Ensure a smooth working procedure to minimize changes in concentration due to evaporation. Next, place the TEM grid onto the micro-patterned silicon nitride layer with the graphene facing the template. Carefully press the graphene-coated TEM grid onto the template, making sure not to destroy the bottom silicon nitride membrane.
Remove excess solution with a tissue to accelerate the cell drying and mitigate concentration changes. After approximately two to three minutes, observe a contrast change as the graphene silicon nitride Van der Waals interactions seal the liquid cell. Then, place the sample under an optical microscope.
Use a pair of tweezers to carefully remove the TEM grid by pushing the tip between the grid and the graphene-supported microwell liquid cell frame. To reduce sheer force damage, start from the grid site parallel to the smaller window edge. Ensure that at least one membrane of the graphene-supported microwell liquid cells are still intact.
Using a standard TEM holder, load the sample into the holder. Directly after its preparation, load the holder and sample into the scanning transmission electron microscope. Image the sample appropriately with regards to both sample and microscope characteristics.
Use a low dose to minimize pre-induced artifact, and a short exposure time to avoid movement-related blurring. For long time experiments, block the beam to reduce radiation damage. After acquiring images, use a suitable image processing platform to extract features of interest.
For particle tracking and analysis, use the open source ImageJ distribution, Fiji. After loading an image and converting it to a binary image, utilize the analyze particles function to gain precise information regarding the projected area and the particle's barycenter for every particle in each frame. Invert the original image so the particles appear as bright spots.
Then, connect the particles between the frames with the help of the plugin, TrackMate. By default, TrackMate searches for bright particles on a dark background. Finally, combine the results of TrackMate and analyze particles with a suitable script, utilizing the Python-based open source ecosystem, SciPy.
A successful encapsulation of the specimen solution can be verified during electron microscopy. This video shows the dissolution of an ensemble of nano-particles, and the growth of a dendritic structure. In order to gain insights into particle growth and dissolution kinetics, it is important to investigate each particle individually, rather than to analyze the development of average parameters.
By estimating the growth exponent, alpha, of the equivalent radius variation of individual particles over time, information of the underlying reaction kinetics can be obtained. Here, the distribution of alpha, based on 73 dissolving particles, is displayed. Only particles where an allometric model explains the radius decline to at least 50%are regarded.
At the end of the video, a dendrite structure emerges. Dendrite formation is another typical, well-documented process in liquid cells. To quantify dendrite growth, the structural outlines are analyzed.
The evolution of the tip radius and the velocity over time reveals the expected hyperbolic relationship. Dendrite growth is caused by local super saturation of gold ions due to the aforementioned particle etching. At this part in the video, it is clearly visible that particles are still dissolving, whilst the oversaturated system relaxes into dendrite growth.
This may be caused by local concentration variations in both the gold ions and the oxidative species. The liquid loading and suhrf is highly dependent on the specimen of interest because the graphene adhesion and the required drying times may vary if different specimen solutions are applied. The graphene-supported microwell liquid cell architecture also allows for complementary in situ methods such as yields in EDSX.
In addition, SEM in transmission-mode and tomography experiments have been performed successfully. After development of this technique, it became possible to unravel the growth mechanism of gold, silver, kawsh-en, and other particles down to atomic resolution. The results are an excellent agreement with the plasma resonance measurements acquired by our colleagues in the physics department who also participate in the research training group.
Although not shown here, the carrier frame production employs corrosives and toxic species. Please be careful during operation and take the required precautions.
Overview
This article presents a protocol for preparing graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals. The study includes an analysis routine to quantify etching and growth dynamics of the nanocrystals.
Key Study Components
Area of Science
- Electron microscopy
- Nanomaterials
- In situ observation
Background
- Liquid cell electron microscopy allows for high-resolution imaging in liquid media.
- Graphene-supported microwell cells combine benefits of graphene and silicon technologies.
- This technique provides real-time insights into dynamic processes at the nanoscale.
- It is essential for correlating with analytical methods like EDX spectroscopy.
Purpose of Study
- To demonstrate a protocol for preparing liquid cells for electron microscopy.
- To analyze the etching and growth dynamics of gold nanocrystals.
- To provide insights into particle growth and dissolution kinetics.
Methods Used
- Transfer of graphene onto TEM grids and preparation of liquid cell templates.
- Use of anti-capillary tweezers for precise handling of samples.
- Application of plasma treatment to enhance wettability.
- Image processing using ImageJ and TrackMate for particle analysis.
Main Results
- Successful encapsulation of specimen solutions verified during microscopy.
- Observation of dissolution and growth of dendritic structures in real-time.
- Quantification of particle growth kinetics and analysis of structural outlines.
- Identification of hyperbolic relationships in dendrite growth dynamics.
Conclusions
- The protocol enables detailed observation of nanocrystal behavior in liquid environments.
- Insights gained can inform future studies on particle dynamics.
- The methodology can be applied to various nanomaterials for enhanced understanding of their properties.
Frequently Asked Questions
Executive Industry Relevance
Graphene-supported microwell liquid cells enable high-resolution, real-time observation of nanomaterial dynamics in liquid environments, supporting target validation through direct visualization of etching and growth processes. This approach enhances predictive confidence in early discovery by providing quantitative, single-particle tracking data that informs mechanistic understanding of nanomaterial behavior. The method’s reproducibility and compatibility with correlative techniques like EDX spectroscopy position it as a scalable tool for de-risking nanomaterial-based therapeutic candidates.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables direct interrogation of nanomaterial dissolution and growth kinetics at single-particle resolution.
- Operational Value: Provides reproducible well depths and standardized liquid volumes for consistent experimental conditions.
- Strategic Value: Supports hypothesis testing of nanomaterial stability and reactivity in physiologically relevant environments.
Screening & Assay Development
- Scientific Value: Facilitates preparation of validated biological-nanomaterial interfaces for downstream screening workflows.
- Operational Value: Allows tailoring of enclosed liquid volume via silicon nitride thickness to minimize window bulging and improve imaging fidelity.
- Strategic Value: Enables reliable compound evaluation by correlating structural changes with elemental composition via in situ EDX.
Translational & Preclinical Research
- Scientific Value: Offers disease-relevant system insights by visualizing nanomaterial transformations under liquid conditions mimicking biological milieus.
- Operational Value: Ensures continuity from discovery through preclinical validation via correlative imaging and spectroscopy.
- Strategic Value: Supports risk-adjusted advancement decisions by quantifying growth exponents and dendrite formation dynamics.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early nanomaterial screening to lead optimization by enabling real-time, quantitative analysis of particle behavior in liquid environments.
- Discovery Biology: Supports hypothesis testing of nanomaterial stability and interaction dynamics through direct visualization of etching and growth.
- Screening: Delivers assay readiness via reproducible microwell fabrication and graphene sealing without bonding or alignment steps.
- Analytics: Provides quantitative dependent variable measurements including particle projected area, barycenter, tip radius, and velocity over time for kinetic modeling.
- Translational Research: Connects to preclinical continuity by enabling analysis of structural outlines and growth dynamics relevant to nanomaterial fate in biological systems.
- Enterprise Reuse: Establishes a reusable platform for nanomaterial characterization across multiple projects due to facile manufacturing and handling.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence through single-particle tracking and allometric modeling of radius decline.
- Operational Value: Standardization and scalability via single-substrate fabrication eliminating wafer bonding.
- Strategic Value: Reduced late-stage biological risk by informing go/no-go decisions based on quantified dissolution and growth kinetics.
- Portfolio Impact: Risk-adjusted prioritization of nanomaterial candidates using dendrite growth velocity and tip radius evolution as decision points.
Implementation Considerations
- Requires expertise in graphene transfer, plasma treatment, and liquid cell assembly.
- Depends on access to scanning transmission electron microscopy and anti-capillary tweezers for precise handling.
- Necessitates cross-team standardization of plasma conditions and liquid loading protocols across model systems.
- Involves adaptation considerations for varying specimen solutions affecting graphene adhesion and drying times.
- Includes practical limitations such as liquid loading dependency on specimen-specific properties and potential radiation damage requiring beam blocking for long experiments.
