Biological Sample Preparation

Electron microscopy is a crucial technique used in the life sciences to visualize microstructures that would otherwise be too small to observe. Scanning electron microscopy (SEM) can reveal topographical information about biological samples such as the shapes of peripheral proteins on cell membranes. Transmission electron microscopy (TEM), though requiring significantly more preparatory work, provides information about structures within the interiors of biological samples, such as the shapes of organelles within a cell. As an advanced imagery technique, electron microscopy (EM) requires advanced sample preparation techniques in order to prevent damage to both the sample and the electron microscope, especially in the case of biological samples.

Dangers of Imaging Biological Samples

There are several obstacles in the way of having an unprepared biological sample, such as a recently dead insect, being imaged under an electron microscope. These obstacles result in 3 main issues: poor image quality, damage to the EM instrument, and damage to the sample.

Outgassing

Outgassing occurs when an object releases trapped gases when placed in a vacuum, resulting in damaging or contamination of the vacuum components. All unprepared biological samples will have some air in them, typically dissolved in some fluid which permeates a tissue. When a vacuum is pulled, these gases rapidly expand, potentially damaging equipment by evaporating or even sublimating contaminants, and potentially damaging the sample by increasing its temperature and ripping apart tissues due to the expansion of its gases. These contaminants will interfere with vacuum seals and can even get 'kicked up' (sucked upwards in a drift of gases) into machine components like apertures (thin strips of metal meant to regulate the electron beam). Meanwhile, the sample will be destroyed due to heat denaturing proteins and gas expansion ripping through microstructure.

Moisture

All unprepared biological samples will have some amount of moisture in them. Liquids such as water can evaporate and contribute to outgassing, but can also create a humid environment within the sample chamber resulting in contamination. Water vapor can then get in the path of electrons and deflect/eject them in unpredictable directions, resulting in lesser image quality and damage to the instrument.

Conductivity

Most unprepared biological samples will be nonconductive or poorly conductive. This results in poor image quality and charging of the sample, which can heat it up and degrade it.

Degradation

Biological samples are made of molecules that are