To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered appearance of mitochondria.

Advances in TEM Sample Preparation

Cryofixation

In this technique, small cells and organelles can be frozen by plunge freezing, while larger cells and tissues can be frozen using high pressure. Plunge freezing is achieved by placing a small amount of sample on a cold EM grid and then rapidly plunging into liquid ethane filled in a reservoir and surrounded by liquid nitrogen. The rapid freezing ensures that water does not crystallize. In high-pressure freezing, liquid nitrogen is used to freeze the sample rapidly, and it is subjected to a ~2000 bar pressure to decelerate ice crystal formation. Cryofixed samples are sectioned in a cryo-ultramicrotome that consists of a frozen diamond knife.

Shadow casting

The rotating platform in a vacuum chamber holds the sample, which is then coated with heavy metal. The rotation of the sample ensures a metal coating on the front side and a shadow effect on the backside. The technique is particularly useful when studying ultrastructural details of bacteria or viruses, DNA, RNA, or isolated proteins.

Negative staining

Small samples, such as ribosomes, enzyme molecules, viruses, etc., can be imaged directly without sectioning. Samples are mixed with ammonium molybdate, uranyl acetate, or phosphotungstic acid, which are electron opaque. A metal layer is formed on the grid, except where the sample lies, creating a negative image of the sample. Contrast is achieved as a result of the pattern of metal distribution.

Preparing Desiccated and Non-desiccated Samples for SEM

SEM can image three types of samples, namely, (i) desiccated and conductive, (ii) desiccated and non-conductive, and (c) non-desiccated (wet). Naturally conductive samples that are desiccated can be easily mounted and imaged. However, desiccated and non-conductive samples must be coated with a conductive layer to protect the sample from overheating and improve the image quality. These samples are made conductive by coating the sample with a thin gold layer using a sputter coater. It uses argon ions to knock gold atoms off a gold plate and coat the sample surface with gold, making the surface conductive to electrons.

Wet samples can outgas in the vacuum environment of the microscope.

Wet SEM, also called Environmental SEM or ESEM, must control the water sublimation in the specimen during imaging. It is accomplished by first coating the sample with a thin layer of a membranous polymer that is transparent to electron beams and separates the wet specimen from the vacuum. Then the temperature is decreased, which decreases the saturated vapor pressure. These conditions allow instrument operation at a higher vacuum that preserves ultrastructural features of the specimen and reduces noise. A Deben Coolstage may also be used to image wet samples by freezing them to −25°C, which preserves the sample structure and prevents outgassing while electrons are bombarding it.