To prepare a specimen for scanning electron microscopy (SEM), it must typically be dried and treated with fixatives. Fixatives are used to reduce artifacts, such as the shriveling that can be caused by the drying process. After fixation and drying, the specimen is sputter-coated with a thin layer of metal, such as gold, before observation.

SEM Specimen Preparation

In a scanning electron microscope (SEM), a backscatter electron detector is a specific type of detector that identifies electrons traveling directly back from the sample after being struck by the electron beam.

Backscatter Electron Detector

In a scanning electron microscope (SEM), secondary electron detectors are utilized to capture electrons that travel to the sides after the sample is hit by the electron beam.

Secondary Electron Detector

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) serve different observational purposes in microbiology. TEM requires extremely thin sections of a specimen and is utilized to visualize internal structures, such as organelles and the interior of membranes. In contrast, SEM is used to view the detailed, three-dimensional surfaces of specimens, ranging from very small samples to larger objects like pollen grains.

TEM vs. SEM

When samples are prepared for viewing using a scanning electron microscope (SEM), they must be dehydrated using an ethanol series and be even drier than necessary for a transmission electron microscope (TEM). Critical point drying achieves this by using inert liquid carbon dioxide under pressure to displace the water from the specimen.

SEM Critical Point Drying

A scanning electron microscope (SEM) is an instrument that forms highly detailed images of the surfaces of specimens. It operates by directing a beam of electrons at the specimen, which knocks off electrons from its surface. These emitted electrons are detected and displayed on a monitor, producing images with a distinct three-dimensional appearance. Scanning electron microscopy is utilized to view the external surfaces of both very small samples and larger objects, such as pollen grains. Some electron microscopes can achieve magnifications up to $${}2,000,000	imes$$.

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Electron microscopy utilizes short-wavelength electron beams, focused with magnets, to significantly increase magnification and resolution compared to visible light. While light microscopes are limited by visible light wavelengths, electrons behave as waves with much smaller wavelengths, allowing an electron microscope to produce sharp images with magnifications ranging from $${}20	imes$$ to $${}100,000	imes$$ or more. This resolving power enables the visualization of subcellular and molecular structures. However, electron microscopy cannot be used on living material due to the harsh methods required to prepare the specimens prior to viewing.

Electron Microscopy

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The transmission electron microscope (TEM) is a type of electron microscope that functions analogously to a brightfield microscope. It utilizes an electron gun to release an electron beam that is directed from above the specimen and focused using a magnetic lens. The electrons pass through the specimen and are then projected onto a detector to capture the image. TEM is particularly useful for observing small, thin specimens, such as tissue sections and subcellular structures.

Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

While electron microscopy can be utilized to view the structures of biofilms, it has significant drawbacks. The preparation process requires dehydrating the specimen, which can distort the biofilm and introduce undesirable artifacts, making it less ideal than techniques like confocal microscopy for observing living microbial communities.

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