Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

This method keeps biological samples in a native environment and reduces electron beam induced artifact. The main advantage is that this technique is applicable to whole cells. So cells don't need to be dehydrated, stained, or sectioned before electron microscopy.

The ability to image receptors on cancer cells in the context of the whole intact plasma membrane is a major aspect that can lead to new therapy approaches and the development of new anticancer drugs. I will demonstrate the work with cells, graphene-coating, and microscopy, and Sercan Keskin will show how to prepare the graphene. To begin, add 100 microliters of cell suspension to two wells of a 96 well plate containing PLL and FLP coated microchips with the silicon nitride membrane facing up and 50 microliters of serum-free medium.

Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for five minutes to allow cells to attach to the microchip. Check the density of the cells on the microchip with an inverted microscope and make sure that the cells cover the window with sufficient space to flatten out and adhere. To label HER2 in the cells, place the microchips into the wells of the first row of the labeling plate.

Then, wash the microchips with PBS BSA. To prevent unspecific binding of the antibody mimetic, incubate the microchips with PBS BSA GS for five minutes. Then with 200 nanomolar antibody mimetic for 10 minutes at 37 degrees Celsius and 5%carbon dioxide.

To remove the PMMA graphene from the polymer, pipette a few droplets of water on the polymer around the PMMA graphene. Then, immerse it in water with an angle of 30 to 45 degrees to release the PMMA graphene. To etch copper-based contaminants, prepair a 50 milliliter solution of 0.42 molar sodium persulfate in water.

Use a standard glass slide to transfer PMMA graphene into the sodium persulfate solution with the graphene side down. The PMMA graphene will float to the top of the solution. Leave it there overnight.

On the next day, transfer the PMMA graphene from the sodium persulfate solution into clean water. Let it float in the water for half an hour. Repeat the wash a total of three times to remove all sodium persulfate residues from the PMMA graphene.

Then, transfer the PMMA graphene onto a sodium chloride crystal. Prepare a saturated solution of sodium chloride in water in a Petri dish and transfer the PMMA graphene on top of the solution with the graphene side down. Hold the sodium chloride crystal with tweezers and pick up the floating PMMA graphene.

Then, hold it vertically for two minutes to let excess water flow out. Let it dry to room temperature for 30 minutes and bake it in an oven at 100 degrees Celsius for 20 minutes. Completely remove the water.

Preheat acetone in a glass Petri dish to approximately 50 degrees Celsius on a hot plate in the fume hood. Watch the temperature carefully to avoid fire. Immerse the PMMA graphene on salt into the Petri dish filled with acetone and leave it to dissolve the PMMA for 30 minutes.

Let the graphene on salt air-dry thoroughly before using it for sample preparation. Wash one prepared and labeled microchip in pure water to remove any residues of salt from the buffer and place it on filter paper. The cells should be visible as dark spots.

Use a razor blade to cut the multilayer graphene on the sodium chloride crystal into a piece that fits the silicon nitride window of a microchip. Remove the graphene from the sodium chloride crystal by dipping it in a beaker of water, tilting the crystal at a 45 degree angle with respect to the surface. Catch the graphene with a metal loop from the surface of the water, then touch the upper surface of the microchip with the lower loop surface, causing the microchip to stick to the metal loop.

The graphene can be seen on top of the microchip. Under a stereomicroscope, use filter paper to remove the remaining water from the microchip so that the graphene covers all cells on the silicon nitride window. Place the microchip onto a filter paper with tweezers.

The graphene is visible as a purple shimmer on the microchip. Transfer the microchip from the paper to a compartment Petri dish. Pipette a droplet of water into one of the free compartments and close the lid to provide a water saturated atmosphere.

Then, seal the compartment dish with paraffin film and store it in the fridge at four degrees Celsius. Set the STEM to 200 kilovolt beam energy using an alignment sample for a probe size of at least 0.2 nanometers by adjusting the condenser lenses. Set a probe current of 180 Picoampere and a beam convergence semi-angle of 13.2 milliradians by inserting an aperture.

Set the ADF STEM detector opening semi-angle range to 68 to 280 milliradians by adjusting the projector lens settings. Then, set the STEM image size to 2048 by 2048 pixels and the pixel dwell time to six microseconds. Load the microchip with graphene-coated cells in a standard specimen holder for tem in such a way that the cells are facing up and load the holder into the electron microscope.

Acquire an overview picture at 800 X magnification. Then, identify a region of interest and image of the quantum dot nanoparticles at 80, 000 X with a pixel size of 1.3 nanometers. Optimally seeded cells are shown here.

The window is covered, but there is sufficient space to allow them to flatten out and adhere. When too many cells were seeded on a microchip, there was insufficient space for all cells to adhere. After 24 hours, more than half of the cells didn't flatten.

If too few cells are seeded, the silicon nitride window will end up with a large empty space after 24 hours. DIC and fluorescence images of optimally seeded cells are shown here, demonstrating successful labeling of HER2. STEM was performed on graphene-coated and non-coated SKBR3 cells.

The insets in the 800 X magnification images were imaged at 80, 000 X.The quantum dot nanoparticles are visible as bright spots at 50, 000 X magnification. To investigate the effect of electron beam illumination on the sample, STEM images were acquired in an image series with an accumulating electron dose. Representative results for non-coated and graphene-coated samples are shown here.

The exposure of non-coated samples led to bright structures appearing on the cell surfaces, which did not occur with any of the graphene-coated samples. The average relative distance change of particle pairs stayed below 1.3%for non-coated samples and below 0.8%for the coated samples. Following this procedure, different kinds of membrane proteins can be imaged on whole cells using other microscopy methods.

This technique makes it possible to image proteins in the intact plasma membrane to gain more information about the organization of the plasma membrane and the interaction between proteins.