What Is The Magnification Of A Electron Microscope?

Electron microscopes use subatomic particles called electrons to magnify objects. The electrons are fired at the sample very fast. When electrons travel at speed they behave a bit like light, so we can use them to make an image. But because electrons have a smaller wavelength than visible light they can reveal very tiny details. This makes electron microscopes more powerful than light microscopes. A light microscope can magnify things up to 2000x, but an electron microscope can magnify between 1 and 50 million times depending on which type you use.

Electron Microscope vs. Light Microscope

Light Microscope

Uses visible light to magnify the image of the object. Light microscopes allow us to see things such as cells, parasites, and some bacteria. A light microscope can magnify things up to 2000x.

Electron Microscope

Uses subatomic particles called electrons to magnify objects. Because electrons have a smaller wavelength than visible light they can reveal very tiny details. An electron microscope can magnify between 1 and 50 million times depending on which type you use. To see much smaller things, including viruses and structures inside cells, such as DNA, we need a more powerful type of microscope.

Why Electrons Instead of Photons

Electrons are charged, which means their beams are focused with magnetic coils instead of with glass lenses (as used for photons, which don't have a charge), which allows for the smaller wavelengths. Electron beams can be focused using magnets. Light has to be focused using a lens, but at very small wavelengths (x-rays) it's impossible to make a lens at present. This means you can use electron beams at smaller wavelengths than light.

Wavelength and Resolution

The smaller the wavelength, the greater the resolution — the ability to distinguish between two adjacent points. The higher the resolution, the greater the magnification possible. The wavelengths being used in the instrument is more important than the size of the particle in terms of the magnification difference between optical and electron microscopy.

Scanning Electron Microscope (SEM) Magnification

Scanning electron microscopes (SEMs) can magnify 3-dimensional objects — perhaps a bed bug or a fruit seed. The outside surface of the object is scanned, which is how the scanning electron microscope gets its name.

• Electrons are fired towards the sample. The electrons move very fast, and when they hit the sample they bounce off its outside surface.
• The bounced-back electrons are detected by a screen, which then makes an image we can see on a computer.
• An SEM can magnify a sample by about one million times (1,000,000x) at the most.
• Because a sample can be used in its natural state, the SEM is the easiest electron microscope to use.
• The final image looks 3D and shows you the outside of your sample.

Transmission Electron Microscope (TEM) Magnification

Transmission electron microscopes (TEMs) are a bit different to SEMs. To look at something using a TEM the sample must be sliced into a very thin section and prepared specially. The electrons pass, or transmit, through the thin section of the sample, which is how the transmission electron microscope gets its name.

• Electrons are fired very fast towards the sample, just like in an SEM.
• Because the sample is so thin, when the electrons hit the sample, they pass through it.
• After passing through the sample, the electrons reach a screen where the image appears. The image can be seen on the screen itself, or on a computer screen.
• A TEM can magnify a sample up to 50 million times (50,000,000x). This is far more than the SEM.
• However, it takes a long time to prepare a sample for TEM, which makes TEMs harder to use.
• The final image you see from a TEM looks 2D — it shows a thin section through your sample.

TEM Magnification Ranges by Project Type

One of the first questions that a new user of any sort of microscope asks is usually "what magnifications should I use when collecting images?" The most simple answer is that one needs to use a magnification high enough to see what is interesting. Both the sample itself and the goal of the work are important in determining the proper magnification(s) to use.

1. Protein binding projects require a magnification high enough to see the binding protein itself (which will depend on that protein's size) but do not require the atomic resolution necessary to show nano-particle crystallinity. Most binding proteins will be several nanometers (nm) in diameter, and magnifications in the range of 20,000x to 40,000x are usually sufficient for such work.
2. If the goal is to discuss amino acid side-chains at the binding surface, the magnification will need to be high enough to see the atomic structure of the binding interface itself, likely to be on the order of 60,000x to 80,000x. High resolution modeling of atomic structures into a biological system can require magnifications anywhere from 20,000x to 80,000x.
3. A cell biologist tracing membrane systems through an entire organelle would require lower magnifications because of the large area/volume necessary to image. Bacteria and most cellular organelles are usually measured in terms of several micrometers (µm), while macromolecular complexes are measured in 10s or 100s of nm. Magnifications as low as 2500x to 10,000x may be sufficient for membrane tracing through large volumes of material.
4. On the JEOL JEM 3200FS, graphite spacing can be seen at as low a magnification as 80,000 (or perhaps even 60,000) and the gold spacing can be seen at 100,000 (and even 80,000 in some images).

Magnification and Resolution in SEM

At the heart of any microscopy technique lies two crucial concepts: magnification and resolution. While often used interchangeably, these terms represent distinct aspects of image formation and image interpretation.

Magnification

Magnification is the ratio between two measurements, which implies that two objects are needed for a correct evaluation of the value. The first object is the sample. The second is a picture of it. Although the sample will not change its size, the picture can be printed in an infinite number of different sizes, meaning magnification is a relative number. When storing a digital image of the sample, resizing the image causes the magnification number to become ostensibly wrong. Magnification is thus of no practical use in the scientific field.

Resolution

The theoretical resolution of a microscope is expressed as the minimum distance between two distinct points that can still be distinguished in an image (i.e., Rayleigh criterion). Smaller resolution values indicate a higher resolving power. Image formation is diffraction-limited, meaning the incident radiation (be it light or electrons) will blur the image of the object due to the bending of waves as they pass around the edge of an object. How much distortion, or blurring, depends upon the wavelength of the incident radiation.

Edge Resolution (Knife Edge Resolution)

Edge resolution is typically used to quantify the resolution of actual SEM systems. Knife edge resolution measures the distance between a "white" point and a "black" point from an intensity profile drawn perpendicular to an edge feature, where the definition of white and black are defined by arbitrary intensity values. Because edge resolution is not a standardized metric, it's always important to understand how it is being reported.

Field of View

What scientists use is a couple of parameters that describe the actual imaged area (field of view — the area that the microscope points at) and how sharp this image is (resolution). The field of view defines the size of the feature to be imaged. This value typically ranges between some millimeters (a bug) to few microns (the hair of a bug) and a couple of nanometers (the molecular macrostructure of the exoskeleton). With modern instruments, objects in the range of few hundred picometers can be imaged — and that is the average size of an atom.

Radiation Damage and Its Effect on Magnification Choice

Virtually all specimens examined in a TEM will eventually exhibit signs of radiation damage, though the electron dose needed to show a significant effect can differ enormously between biological and non-biological materials. The concept of radiation damage becomes extremely relevant to the choice of magnification when considering the electron dose that hits the specimen and the area of the recording device over which that dose is spread.

For example, if the electron dose is set to 10 e-/Ų, and the magnification changes from 20,000 to 40,000, the electron dose per unit area at the detector is reduced by a factor of 4. Fewer electrons per unit area equates to a lower signal (and lower signal-to-noise). If one needs or wants to severely limit the electron dose in terms of e-/Ų, there comes a point where the magnification is so high and the dose is so low that there is effectively no signal left at the detector. The lowered temperatures in cryo-EM are not really slowing the amount of damage, but rather the lowered temperatures are simply masking the effect of the damage for a brief while. It is also very important to note that while it is less obvious, radiation damage is still a major concern for most biological specimens embedded in negative stain.

Specimen Preparation and Resolution Limits

Just as a particular specimen can impose resolution limits on what can be observed, different specimen preparation techniques can also impose their own resolution limits. If the specimen is prepared in any sort of negative stain, recording images that could be used to attain resolutions significantly higher than 10 to 12 Å is a waste of time and resources.

The warning is simply that if it has been or can be shown that a specimen will yield results showing order only to resolution x, it is foolish to collect at least initially only images that contain significantly more information than that resolution: higher resolution images will necessitate higher magnifications, will have smaller fields of view (and fewer single particles, unit cells, etc.) and may require changes in the data collection strategy that make high resolution work much more difficult than if the goal is more modest resolution. The caveat is really intended to urge people starting any project to set modest goals and work towards higher resolution if and when a particular sample behaves well.