Principles of Image Formation

When an electron beam strikes an object, several things can happen. If the electron does not strike an atom in the sample, it will continue to travel in a straight line until it hits the imaging screen. If the electron does come into contact with the sample, it can either bounce off elastically, that is, without any loss of energy, or inelastically, i.e., transferring some of that energy to the atom. In an inelastic bounce, the amount of energy transferred from electron to sample is variable and random. Therefore, when the electron eventually reaches the imaging plane, it has an unknown energy and angle of incidence. This particular electron will then generate noise in the image. However, if the electron bounced elastically, it's energy is constant, and the law of conservation of momentum will determine the angle at which it will bounce. This electron can be used to give high resolution information on the sample. Note that an electron can have an angle of reflection greater than 90. Electrons that follow such a trajectory are termed 'back reflected electrons' and are generally not used in electron microscopy.

Contrast arises when there is interference between electrons coming in from different angles. Electrons that interact with the sample are bent away from their original path, and will thus interfere (either constructively or destructively) with the main electron beam. If a small objective aperture is used, electrons that get deflected at a greater angle are blocked, and the contrast of the image is enhanced. However, electrons with a high deflection contain high resolution information and are therfore lost. A balance needs to be achieved between having good contrast and having a high resolution.

Projections of a solid ball and hollow sphere as seen in a transmission electron microscope
Figure 1. Differentiating between a solid ball (left) and a hollow sphere (right) in a transmission electron microscope.

Because the electrons of a TEM pass through the sample before hitting the imaging screen, they contain information on the inside structures of that sample. If a hollow sphere and a solid ball are held in front of a flashlight, both will cast an identical shadow: a solid black disk. In a TEM, however, the two projections will be different. The solid ball will generate a disk that is very dark in the center and progressively drops off in darkness towards the edges (Figure 1 left). The hollow sphere will also cast a circular image. However, the darkness gradient will be in the opposite direction, with the edge being the darkest, and the middle the lightest (Figure 1 right). This happens because the electron beam passes through the least amount of matter in the middle of a hollow sphere. The darkness of the image is proportional to the electron absorbency properties of the material used.

Electron microscopes can also be used to generate and view diffraction patterns of samples. These are usually from samples that contain a repeated motif, such as 2D crystals (sheets one layer thick) of proteins. In the back focal plane of the objective lens, a difraction image is formed. Depending then, on the positioning of the intermediate lens, the difraction pattern or the image will be magnified and displayed on the view screen.


Preparation Techniques: Grids