The Transmission Electron Microscope (TEM) achieves its remarkable resolution by “illuminating” samples using an electron beam in a vacuum rather than using a conventional light source in air. Since the electron beam passes through the sample that is being examined, the sample must be sufficiently thin and sufficiently sturdy to be hit by electrons in a vacuum. It’s important to remember that many biological materials are damaged or destroyed by the incoming electrons and that the TEM can image only the species that survive this harsh treatment. The denser parts of the sample will absorb or scatter some of the electron beam, and it’s the scattered electrons or those that pass through the sample that are focused using an electromagnetic lens. This “electron shadow” then strikes a fluorescent screen, giving rise to an image that varies in darkness according to the sample's density. For samples that are amenable to TEM, this form of examination can allow observation of angstrom-sized objects and of cellular details down to near atomic levels.
Copper TEM grid with carbon mesh, photographed at 60X magnification
Samples are applied to a wafer-thin "grid" before being loaded into the TEM and placed under vacuum. The grid can be made of many kinds of materials. All have lines of a conductive metal, in our case copper, that disperse the electron beam and thereby help keep the sample from being blown to bits by the energy in the beam. A carbon mesh is strung between the metal lines. Once a sample has been applied to the grid, it's only the portions that come to rest on the carbon mesh can be visualized, along with any imperfections in the carbon mesh itself.
Today you'll wash the nanowires you synthesized last time and visualize a sample of them by TEM. The rest of the nanowires will be stored in a mortar until next time, when you'll grind them with some carbon and teflon for assembly into a battery.
map from John to TEM facilities
Before you begin today's protocols, be sure to make some notes in your lab notebook about the appearance of the solution of nanowires. The next steps are devised to collect and wash the CTAB off the nanowires. You'll end up with some concentrated nanowires to observe by TEM and also to dry for assembly into a battery next time.
Part 1: Collect nanowires
- Vortex the 50 ml falcon tubes and then sonicate the nanowires for 2 minutes. This step will break up large clumps of nanowires that may have formed.
- Centrifuge your 50 ml falcon tubes in the clinical centrifuge at 3K for 10 minutes.
- Pour off the supernatant and resuspend the pellet in 1 ml of sterile water. You may need to vortex to resuspend the nanowires in the water.
- Move 0.1 ml of your nanowires to an eppendorf tube and pool with team using same concentration of Ag+. Use this pool of nanowires for Part 2 of today's protocol.
- Add 9 ml of 100% EtOH to the remainder of the nanowires for Part 3 of today's protocol. Note that Part 2 and Part 3 of today's protocol can be done in parallel.
Part 2: TEM sample preparation
Each group should prepare a TEM grid. This will allow for some duplicates to be visualized in case the grid is damaged or different kinds of EM are being performed.
- Vortex the nanowire pool for 1 minute at least and immediately remove 10 ul of the nanowire suspension to place on the silver/black side (not the copper-looking side) of the TEM grid that you have balanced in the specialized tweezers. Treat the grid with care and use the tweezers only on the edge to minimize damaging the delicate mesh.
TEM grid balanced in tweezers, wrong side up!
- Allow the nanowires to settle onto the grid undisturbed for 20'.
- Remove the droplet from the grid by touching the edge with a Kimwipe thereby wicking the solution off the grid.
- Wash the grid by adding 10 uL of sterile H2O onto the grid.
- Allow the grid to sit with water for 3 minutes and then wick dry.
- Wash the grid by adding 10 ul of 100% EtOH onto the grid.
- Allow the grid to sit with EtOH for 3 minutes and then wick dry, if it hasn't air dried.
- Place the grid into the TEM grid holder to transport to the TEM facility (13-1012). A member of the teaching faculty will be in the facility to examine your samples with you.
Part 3: Harvesting the remainder of your nanowires
- Pool your sample with any group experimenting with the same ratio of gold and silver as you have tested.
- Centrifuge the sample of nanowires in EtOH using the clinical centrifuge (3K 10').
- Decant the supernatant and add 20 mL of sterile water and resuspend the centrifuged materials by vortexing if necessary.
- Harvest using the clinical centrifuge (3K 10').
- Decant the supernatant and add 20 ml of 100% EtOH to resuspend the nanowires.
- Harvest the nanowires in the clinical centrifuge one last time (3K 10').
- Decant the supernatant and wash the nanowires into a mortar with 100% EtOH.
- Allow the nanowires to settle and remove the clear solution above them with a 1 ml pipetman.
- Loosely cover the mortar with aluminum foil and set the covered mortar in the 30° incubator until next time. The majority of the EtOH will evaporate at 30°, though a vacuum oven will be used to fully dry the nanowires for next time.
For next time
You should now be considering the presentation materials themselves and if you can, start working on the materials you will use to describe your idea. Reconsult the specific directions for what you'll need as well as the more general guidelines for all oral presentations.