20.109(S16):TEM (Day4)

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20.109(S16): Laboratory Fundamentals of Biological Engineering

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TEM overview

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. TEM enables 0.14 nm resolution. At this resolution, one can distinguish individual planes of atoms – making TEM an excellent materials characterization instrument. The visualization of single atoms allows one to determine many parameters that describe the structure of the material, such as crystal plane spacing. Additionally, TEMs can be equipped to perform energy-dispersive X-ray spectroscopy analysis (EDX/EDS/XEDS) or electron energy-loss spectroscopy analysis (EELS). The former, EDX, enables identification of the atomic composition of heavier elements in the material. EDX has limited detection of elements below sodium (Na) on the periodic table and no detection of elements below carbon (C). The latter, EELS, is most sensitive to lighter elements, typically from carbon to the 3d transition metals. EELS can reveal atomic composition, electronic properties of the valence and conduction band of a material, surface properties, and chemical bonding.

Although we will be using TEM for materials characterization, it should also be noted that TEM is used for the analysis of biological materials as well. 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. Rather than imaging single atoms, biological samples display contrast of components via different densities. Areas of higher density show up as a darker color than areas of low density, creating a “shadow image." 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.

In TEM, 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 ångström-sized objects and of cellular details down to near atomic levels.

How does a TEM work?

The principles behind transmission electron microscopy are analogous to the principles behind an optical microscope. Instead of light and glass lenses, however, the TEM utilizes an electron beam focused via electromagnetic lenses. Since the TEM utilizes an electron beam rather than visible light, the microscope must also function under a vacuum. This condition allows the electrons to remain traveling in a straight line because they have no air particles with which to collide.

Specifically, at the top of the column of a TEM, a high voltage electron beam is emitted by a cathode and focused via a condenser lens. Then the beam is transmitted through the specimen. The resulting image is then magnified through a series of electromagnetic lenses until it is recorded by a fluorescent screen or camera.

Sample preparation for TEM

Copper TEM grid with carbon mesh, photographed at 60X magnification
We will be imaging our phage nanocomposites via TEM. To prepare for TEM, you previously deposited your samples on a very thin TEM grid. TEM grids can be made of many kinds of materials. Each design, however, includes lines of a conductive metal which disperses the electron beam and thereby prevents destruction of the sample from the harsh beam. Our TEM grids are composed of a copper mesh with carbon mesh is strung between the grids. Once a sample has been applied to our TEM grids, we will only visualize the portions of the sample contacting the carbon mesh, along with any imperfections in the carbon mesh itself.

Today will visualize the nanowires you generated on M3D1 (with instructors' input to precipitate the iron onto the phage) to confirm that they were biomineralized with FePO4.


Part 1: Examining TEM images

  1. Today you will image the TEM grids that you prepared on Day 2. Due to space limitations, only 2-3 groups can join Prof. Belcher and Jifa for the imaging at a time.
  2. Use the downtime to work on your Biomaterials engineering mini-report or your Research proposal presentation.

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