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

1. Cleaning of microchips and coating with poly-L-lysine (PLL) and fibronectin-like protein (FLP)

1. Place two microchips with a SiN membrane (2.0 x 2.6 mm) in 50 mL of acetone. Handle the microchips carefully with the flat side facing up. Avoid breaking the edges by using flat-beak tweezers. Avoid touching the top surface of the microchips when handling with tweezers to prevent breakage of the SiN window.
   NOTE: One may also use polytetrafluoroethylene coated or carbon tip tweezers to prevent damage of the microchips.
2. Wash the chips for 2 min by carefully shaking the beaker, watching the microchips do not flip over.
3. Transfer the microchips to 50 mL of ethanol and wash for 2 min by carefully shaking the beaker. Ensure that the transfer is quick so that the excess acetone does not dry in.
4. Wash the microchips with 50 mL of water for 10 min.
5. Dip the microchips in a freshly prepared beaker of 20 mL of ethanol.
6. Place microchips onto a cleanroom tissue for drying.
7. Plasma clean the microchips with 11.5 sccm O₂ and 35 sccm Ar at 70 mTorr and radio frequency (RF)-target of 50 W for 5 min.
8. Place the microchips in a laminar flow hood for sterile cell work.
9. Prepare a solution of 0.01% PLL in water. Prepare a solution of 15 µg/mL FLP in phosphate-buffered saline (PBS).
10. Prepare a 24 well plate under the laminar flow hood and fill 5 wells individually with 1 mL of the solutions in the following order: well 1 – PLL; well 2 – water; well 3 – water; well 4 – FLP; well 5 – PBS and well 6 – PBS.
    NOTE: Conduct washing steps by dipping a microchip into the indicated 24 or 96 well for a few seconds using tweezers. Perform incubation steps by incubating the microchips in the 24 or 96 well in the indicated solution for indicated time and temperature. Transfer the microchips to another well within a few seconds.
11. Incubate the microchips in PLL solution for 5 min. Then wash the microchips in water twice.
12. Incubate the microchips in FLP for 5 min. Then wash the microchips in PBS twice.
13. Transfer both microchips to wells (one for each microchip) of a new 96 well plate prefilled with 50 µL of serum-free medium for cell seeding.
14. Incubate the microchips at 37 °C and 5% CO₂ until the cell suspension is prepared.

2. Seeding cells on SiN membrane microchips

1. Set up all supplies and equipment in a laminar flow hood to ensure sterile working.
2. Wash the breast cancer cell line, SKBR3, in a cell culture flask with growth medium once. Use Dulbecco’s modified eagle medium (DMEM) containing 10% fetal calf serum (FCS), and 1% non-essential amino acids (NEAAs) as growth medium.
3. Incubate the cells with 1 mL of the cell detachment solution until the cells detached from the flask.
4. Add 5 mL of growth medium to the detached cells in the flask. Transfer this suspension to a centrifuge tube.
5. Pipette 20 µL of the cell suspension into a hemocytometer to obtain the cell concentration. Use the following formula.
   .
6. Prepare a dispersed cell suspension of 2.5 x 10⁵ cells/mL. Calculate the needed amount of the prepared cell suspension by:
   
   and fill up with growth medium to the desired volume.
7. Add 100 µL of the cell suspension to the two wells of a 96 well plate containing the PLL and FLP coated microchips with the SiN membrane facing up and 50 µL of serum-free medium so that each well contains 25,000 cells.
8. Incubate the plate at 37 °C and 5% CO₂ for 5 min to wait for the cells to attach to the microchip.
   NOTE: At this point, cells can detach from the microchip because they have not adhered yet.
9. Check the density of the cells on the microchip with an inverted microscope. Make sure cells cover the window with sufficient space to flatten out and adhere (see Figure 1A).
   NOTE: More cells can be added at this point if necessary.
10. Transfer the microchips into new wells containing 200 µL of growth medium and incubate at 37 °C and 5% CO₂ overnight.
11. In the afternoon of the next day, transfer both microchips to serum-free medium (serum starvation medium) if cells have flattened out and adhered to a visually inspected confluency (i.e., the fraction of the window area covered with cells) of about 2/3 (see Figure 1B). Change to serum-free medium as needed to bring the cells in a defined starting condition as needed for comparison between different experiments²⁵.
12. Incubate at 37 °C and 5% CO₂ overnight.
    NOTE: Be aware that cell amounts may differ according to growth rates and cell morphology for other cell lines.

3. HER2 labeling and fixation

1. Prepare the solutions as described in Supplementary Table 1.
   1. Work under the laminar flow hood to ensure sterile working. Use a 96 well plate referred to as labeling plate I, and fill 6 wells per microchip with 200 µL of the labeling plate I solutions: PBS/BSA, PBS/BSA/GS, antibody mimetic, PBS/BSA, PBS/BSA, PBS/BSA. Use one row (one letter per row, e.g., A1 to A6) of the well plate per microchip. Warm the labeling plate to 37 °C.
   2. Under a fume hood, prepare a 24 well plate referred to as fixation plate, and fill 8 wells per microchip with 500 µL of the fixation plate solutions: CB, FA, CB, PBS, PBS, PBS, PBS/glycine, PBS/BSA.
      CAUTION: CB is acutely toxic by inhalation or oral ingestion and is hazardous to waters. Work under the fume hood with appropriate protection and dispose CB according to the safety data sheet (SDS). FA is corrosive and harmful for skin and health. Work under the fume hood and refer to the SDS for information about handling and disposal.
   3. Prepare a 96 well plate referred to as labeling plate II and fill 4 wells per microchip with 200 µL of the labeling plate II solutions: QDs, PBS/BSA, PBS/BSA, PBS/BSA.
      NOTE: Here, a 24 well plate is used to better see the microchips in the wells. To use less antibody mimetic (step 3.2) and QDs (step 3.4), use 96 well plates for these steps.
2. Label HER2 in the cells.
   1. Once these plates are ready, start labeling by placing the microchips into the wells of the first row of labeling plate 1.
   2. Wash the microchips with PBS/BSA in the 96 well plate marked as labeling plate I.
   3. Block unspecific sites to prevent unspecific binding of the antibody mimetic by incubating with PBS/BSA/GS for 5 min at 37 °C and 5% CO₂.
   4. Incubate with 200 nM antibody mimetic for 10 min at 37 °C and 5% CO₂.
   5. Wash microchip three times in PBS/BSA.
3. Fix the cells.
   1. Transfer the microchips to the 24 well fixation plate in the fume hood.
      NOTE: No sterile work is needed from here.
   2. Wash once with CB for a few seconds.
   3. Fix cells with 3% FA for 10 min.
   4. Wash once with CB and three times with PBS.
   5. Block free aldehyde groups of FA by incubating with PBS-glycine for 2 min.
   6. Wash microchips with PBS-BSA once.
4. Attach the QDs.
   1. Move the microchips to the 96 well labeling plate II.
   2. Incubate with 20 nM QDs for 12 min.
   3. Wash the microchips with PBS/BSA twice.
   4. Store the microchips in a well containing PBS/BSA.

4. Light microscopy of the fixed cells

1. Prepare a 3.5 cm diameter glass bottom dish with 2 mL of PBS/BSA solution.
2. Take the first microchips, place it upside down (cells facing down) into the glass bottom dish and place the dish in the fluorescence microscope. Lower the microchip slowly into the liquid to prevent damage on the cells.
3. Acquire differential interference contrast (DIC) and fluorescence images of every microchip with a 40x objective and the appropriate fluorescence channel.
   NOTE: Here, an excitation wavelength of 540-580 nm and an emitting wavelength of 607-683 nm is used to detect QD655.
4. Repeat the procedure for the second microchip.

5. Post-fixation

1. Perform all steps under the fume hood.
2. Fill 6 wells per microchip of a 96 well plate with 200 µL of the post-fixation solutions:
   CB, GA, CB, PBS, PBS, PBS.
   CAUTION: GA is hazardous to waters, harmful for skin, respiratory system, and eyes. Work under the fume hood and refer to the SDS for information about handling and disposal.
3. Place both microchips in their respective wells with the cells facing up.
4. Wash once with CB.
5. Fix cells with 2% GA for 10 min.
6. Wash once with CB.
7. Wash three times with PBS/BSA.
   NOTE: The first fixation step with FA already fixes the biological structure but a reduced level of membrane protein diffusion may still occur, possibly leading to label induced clustering due to the presence of multiple streptavidins per QD. Keep the time of the experimental steps from FA fixation to GA, therefore, as short as possible to minimize the diffusion of the proteins in the membrane.
8. Store in PBS/BSA to prevent osmotic shocks and 0.02% sodium azide (NaN₃) to prevent bacterial growth at 4 °C until graphene coating in a new well plate. Seal well plate with paraffin film to prevent drying. The microchips and cells are stable up to 2 weeks when stored at 4 ˚C.
   CAUTION: NaN₃ is hazardous to waters and acutely toxic by oral ingestion, for skin and the respiratory system. Work under the fume hood and refer to the SDS for information about handling and disposal.

6. Cleaning and transferring graphene onto salt crystals

1. Remove poly(methyl methacrylate) (PMMA)-graphene from PMMA-graphene-on-polymer (Figure 2A).
   1. Pipette a few droplets of water on the polymer around the PMMA-graphene.
      NOTE: Ensure that the PMMA-graphene easily comes off the supporting polymer as it floats on the water surface.
   2. Immerse the PMMA-graphene-on-polymer into water with an angle of 30-45° to release the PMMA-graphene.
      NOTE: The polymer should only be slightly wetted. Pipetting too much water on the polymer will lift up and possibly fold the PMMA-graphene.
2. Etch copper-based contaminants using a sodium persulfate solution (Figure 2B).
   NOTE: Commercial graphene grown on copper foil often contains sub-micrometer copper residues, which can be removed with a copper etchant solution²².
   1. Prepare a 50 mL solution of 0.42 M sodium persulfate in water.
   2. Transfer PMMA-graphene into the sodium persulfate solution with the graphene side down using a standard glass slide. PMMA-graphene will float on top of the solution.
   3. Leave the PMMA-graphene in the sodium persulfate solution overnight.
   4. Remove the PMMA-graphene from the sodium persulfate solution and place it on top of clean water using a glass slide. Let it float on the water for half an hour.
   5. Repeat the previous step a total of three times to remove all sodium persulfate residues from the PMMA-graphene.
3. Transfer the PMMA-graphene onto a sodium chloride (NaCl) crystal.
   1. Prepare a saturated solution of NaCl in water in a Petri dish.
   2. Transfer the PMMA-graphene on top of the NaCl solution with the graphene side down using a glass slide.
   3. Hold a NaCl crystal with tweezers and pick up the floating PMMA-graphene.
      NOTE: The size of the NaCl crystal should be slightly larger than the size of the PMMA-graphene to avoid folding the protruding graphene at the edge of the salt or contacting the graphene with tweezers. In these experiments NaCl crystal of 12 mm x 12 mm x 0.5 mm is used for picking up a 10 mm x 10 mm graphene sheet and supporting it afterwards.
   4. Hold the PMMA-graphene-on-salt vertically for 2 min to let excess water flow out.
   5. Let the PMMA-graphene-on-salt dry at room temperature for 30 min and bake it in an oven at 100 °C for 20 min to completely remove water.
4. Remove PMMA using an acetone wash (Figure 2C).
   1. Preheat acetone in a glass Petri dish to ~50 °C on a hotplate in the fume hood. Watch the temperature carefully to avoid fire.
   2. Immerse the PMMA-graphene-on-salt into the Petri dish filled with acetone and leave it to dissolve the PMMA for 30 min.
   3. Repeat the previous step a total of three times with new, clean acetone.
   4. Let the graphene-on-salt air-dry thoroughly before using it for sample preparation.

7. Graphene Coating

NOTE: The graphene coating procedure is schematically shown in Figure 3A.

1. Wash one microchip prepared with fixed cells and labeled HER2 in pure water to remove any residues of salt from the buffer. Place the microchip on a filter paper. The cells are visible as dark spots (Figure 3B).
2. Cut the multilayer graphene on NaCl crystal into a piece that fits the SiN window of a microchip using a razor blade.
3. Prepare a beaker of pure water and remove the graphene from the NaCl crystal by tilting the crystal about 45° angle with respect to the water surface and touching the water. The graphene will float onto the water surface (Figure 3C).
4. Catch the graphene with a metal loop from the surface of the water. The graphene will float within the droplet below the loop (Figure 3D).
5. Touch the upper surface of the microchip with the lower loop surface. The microchip will stick to the metal loop. The graphene can be seen on top of the microchip (Figure 3E).
6. Under a stereomicroscope, use filter paper to remove the remaining water from the microchip, so that the graphene covers all cells on the SiN window.
   NOTE: The graphene will move when blotted. Make sure the graphene stays on the top of the window by touching the microchip just with the edge of the filter paper.
7. Use tweezers to remove the microchip from the metal loop and place it onto a filter paper. The graphene is visible as a purple shimmer on the microchip (Figure 3F).
8. Transfer the microchip from the paper to a compartment Petri dish.
9. Pipette a droplet of water into one of the free compartments and close the lid to provide a water-saturated atmosphere.
10. Seal the compartment dish with paraffin film and store in the fridge at 4 °C if required for further measurements.

8. STEM

1. Adjust the STEM at 200 kV beam energy using an alignment/test sample for a probe size of at least 0.2 nm by adjusting the condenser lenses, a probe current I = 180 pA (information about the probe current for different microscope settings is provided by the manufacturer within 5% accuracy) and a beam convergence semi-angle of 13.2 mrad by inserting an aperture. Set the ADF STEM detector opening semi-angle range (inner and outer) to 68-280 mrad by adjusting the projector lens settings. Set the STEM image size to 2048 x 2048 pixels, and the pixel dwell time t = 6 µs.
2. Load the microchip with graphene-coated cells in a standard specimen holder for TEM in such a way that the cells are facing up.
3. Load the holder into the electron microscope.
4. Acquire an overview picture at magnification (M) = 800x (Figure 4) using the settings in step 8.1.
5. Identify a region of interest on a cell.
6. Image the QDs with an ADF detector at M = 80,000x at a pixel size d = 1.3 nm (Figure 4) using the settings in step 8.1.
7. Acquire an image of the area after exposure with a lower magnification (here, M = 50,000x) to show the exposed area (Figure 4).
8. Calculate the electron dose
   
   where e is the elementary charge. With the settings given in the above,
   
   and an error of 5%.
9. Acquire 20 images for a dose series with the same settings, but with t = 60 µs resulting in an accumulated dose of
   .
10. To confirm the presence of graphene, switch the microscope to TEM at M = 1,200x, select a region near a cell, and switch to diffraction mode. Record a diffraction pattern at an exposure time of 0.5 s, 2048 x 2048 x 3 pixels, and a selected area aperture of 50 µm (Figure 4).
11. At the end of the session, remove the sample from the microscope, place the microchip back into the compartment dish, seal the dish with paraffin film, and store it in the fridge at 4 °C if required for further measurements.
12. Select the second microchip prepared in the same way as the first microchip but without graphene coating.
13. Repeat steps 8.2-8.11 but now for this sample. Record a diffraction pattern with the same settings as in the above, as comparison (Figure 4).

9. Analysis

NOTE: For automated detection of QD positions in a STEM image, the analysis uses a plugin of local design for ImageJ (NIH), as described elsewhere²⁰. The plugin is available upon request.

1. The software automatically applies the following steps to detect particles in a STEM image:
   1. Apply a Gaussian filter to reduce pixel noise.
   2. Apply a Fast Fourier Transform (FFT) bandpass filter to obtain nanoparticles only.
   3. Set a threshold to binarize the image.
   4. Use a particle diameter of 10 nm with a tolerance factor of 2 for particle detection.
   5. Detect particle positions.
2. Measure the center-to-center distance between ten pairs of QDs per series. Here, 10 distances with varying size were measured per series.
3. Calculate the relative change in particle distance by comparing each particle distance to the distance in the first image using:
   .