Electron microscopy is used when the greatest resolution is required, and when the living state can be ignored. The images produced in an electron microscope reveal the so-called ultrastructure of cells. There are two different types of electron microscope – the transmission electron microscope (TEM) and the scanning electron microscope (SEM). In the TEM, electrons that pass through the specimen are imaged. In contrast, in the SEM, electrons that are reflected back from the specimen (secondary electrons) are collected, and the surfaces of specimens are imaged.
The equivalent of the light source in an electron microscope is the electron gun. When a high voltage of between 40 000 and 100 000 volts (the accelerating voltage) is clamped between the cathode and the anode, a tungsten fi lament emits electrons (Figure 1). The negatively charged electrons pass through a hole in the anode, forming an electron beam. The beam of electrons passes through a stack of electromagnetic lenses (referred to as the column). Focussing of the electron beam is achieved by changing the voltage across the electromagnetic lenses. When the electron beam passes through the specimen, some of them are scattered, while others are focussed by the projector lens onto the detector. The detector can be a phosphorescent screen, photographic film or a digital camera. Since electrons have a limited penetration power, specimens for TEM must be thin (50–100 nm) to allow them to pass through.
Fig1. Light and electron microscopy. Schematic that compares the path of light through a compound light microscope (LM) with the path of electrons through a transmission electron microscope (TEM). Light from a lamp (LM) or a beam of electrons from an electron gun (TEM) is focussed at the specimen by glass (LM) or electromagnetic condenser lenses (TEM). For the LM, the specimen is mounted on a glass slide with a coverslip placed on top, and for the TEM the specimen is placed on a copper electron microscope grid. The image is magnified with an objective lens (glass in the LM and electromagnetic lens in the TEM), and projected onto a detector with the eyepiece lens in the LM or the projector lens in the TEM. The detector can be the eye or a digital camera in the LM or a phosphorescent viewing screen or digital camera in the TEM.
Thicker specimens can be viewed by using a higher accelerating voltage, for example in the intermediate-voltage electron microscope (IVEM) or the high- voltage electron microscope (HVEM), which use acceleration voltages of 400 000 V and 1 MV (= 106 V), respectively. Stereo images can be collected by acquiring two images at 8–10° tilt angles. Such images are useful in assessing the 3D relationships of organelles within cells when viewed in a stereoscope or with a digital stereo- projection system.
