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

The overall goal of this procedure is to encapsulate small pockets of liquid between graphene sheets for imaging nanomaterials using transmission electron microscopy, or TEM. This method can help answer key questions in the nanomaterials field, such as the growth and etching of nanocrystals with the high spatial resolution of electron microscopy. The main advantage of this technique is the ability for research groups to perform liquid cell TEM experiments using an existing holder with low start-up costs and high spatial resolution.

Generally, individuals new to this method will struggle, because handling graphene coated TM grids is difficult and requires a lot of practice. To begin, cut out a roughly two square centimeter piece of pre-made graphene on copper, which fits around six to eight TEM grids. After cleaning the graphene as detailed in the text protocol, smooth out the graphene on copper piece to remove any microscopic wrinkles.

To do so, take two clean glass slides and place a folded wipe on the bottom glass slide. On top of the wipe, place the graphene on copper piece. Finally, place the second glass slide on top.

Press down on the top slide, gradually smoothing out any wrinkles in the graphene on copper piece. Reduce the number of folds in the tissue and repeat the pressing process. Continue the process until a final pressing between the two glass slides with no tissue wipe.

Lay down the TEM grids on the graphene on copper piece by first placing the holey amorphous carbon support foil TEM grids down on the graphene with the amorphous carbon in contact with the graphene. Place a couple droplets of isopropanol on the grids. Let the grids dry for over two hours to make sure the grids are properly bonded.

This drying process brings the holey amorphous carbon into better contact with the graphene. Next, etch the copper using a sodium persulfate solution. Using tweezers, carefully place the graphene on copper piece on the sodium persulfate solution with the copper side down.

Let the piece float on the top of the sodium persulfate solution. Keep the solution with graphene coated grids sitting overnight. Note that the solution will become blue as the copper etches and there will be no visible copper behind the graphene sheet when etching has finished.

Wash the grids to clean off the sodium persulfate by removing the floating grids from the solution and placing them on top of clean, deionized water in a second Petri dish. Repeat this process three times to remove all sodium persulfate residue from the graphene coated grids. Pick up the grids with tweezers.

Place the grids, graphene side up on a filter paper and let them dry. Take two graphene coated TEM grids and place them graphene side up on a glass slide. Using a small surgical scalpel blade, cut off the edge of one of the graphene coated TEM grids, approximately one-fourth to one-eighth of the area of the grid.

Next, place approximately 0.5 microliters of a droplet of solution to be encapsulated on the non-cut graphene coated TEM grid. Use tweezers to hold the edge of the TEM grid down while placing the droplet so that the capillary forces do not pick up the TEM grid. It is critical to put the droplet in the center of the TEM grid, preferably as small a droplet as possible.

If the TEM grids are correctly made, the liquid will form a bead-like shape on the hydrophobic graphene surface. Quickly and carefully place the graphene coated TEM grid with the cut corner on top of the droplet. The goal is to have the second grid come to rest on top of the first grid with no liquid getting squeezed out.

Wait five minutes to let graphene liquid cell pockets form. Placing the second TEM grid on top of the liquid droplet requires careful hand control. Usually it works best to place the edge of the top grid down first and then gradually remove the tweezers.

Place the graphene liquid cell in a traditional TEM single tilt holder. Load the TEM holder into the TEM column. After preparing the TEM as described in the text protocol, begin searching for nanoparticles in liquid pockets while keeping the dose rate low.

When a nanoparticle is found in a liquid pocket, fine tune the focus on the nanoparticle while maintaining a low dose rate. Use the calibration curve to set the condenser lens current for the desired dose rate. Begin collecting a time series of TEM images with metadata of dose rate and time stamps embedded in the image file.

The TEM video of the nanocrystal etching can be broken down into each of the individual frames. For each frame, the nanocrystal can be outlined using image analysis software. From the outline, the major and minor axis of the nanorod can be determined.

The two-dimensional outline can be cut along the major axis. Each of these new outlines of half of the nanorod can be used to reconstruct the three-dimensional shape by rotating the outline around the major axis. After watching this video, you should have a good understanding of how to fabricate graphene liquid cells for imaging nanomaterials using electron microscopy.

Though this method can be applied to nanomaterials, it can also be used to study soft materials or biological materials in their native liquid environment.