Subject: Re: microscope magnifications

Date: Fri Jan 16 15:43:57 1998
Posted by Ricky J. Sethi
Position: PhD

Hi James,

Thanks for your question. Your question is very interesting but I think it's more instructive to rephrase it a bit. You asked what the highest magnifications for microscopes are. Magnification is the measure of how much a microscope can enlarge the specimen under study. Although it is always possible to enlarge an image as much as you want (e.g., by projecting it on a screen), you lose the fineness of detail that is the main purpose of the study (i.e., you get a bigger, blurrier image).

For this reason, a better measure for the effectiveness of a microscope is it's resolving power, or it's limit of resolution. The limit of resolution is the separation at which 2 objects can still be seen as distinct (i.e., the ability to distinguish 2 points close together as being separate, rather than one blurry point). You can see this for yourself when you're out driving at night on a long stretch of highway; when a car first approaches you from far away in the opposite direction, all you see is a blurry point of light. It could be a motorcycle or a car. As it comes closer, you slowly begin to resolve the image (point of light), into 2 distinct points (the 2 headlights). The resolving power is how far apart 2 objects can be and still be seen as 2 distinct objects; the resolving power of the naked eye is about 0.1mm (i.e., it can distinguish 2 objects as being distinct if they're separated by as little as 0.1mm).

Thus, the magnification of light microscopes is limited to their lower resolving power but the electron microscopes can go to much higher magnifications before reaching their limit of resolution. Typical magnifications for light microscopes are about 1,000 to 1,500 times the actual size whereas electron microscopes offer magnifications of 100,000 times or more!

Now, you might be wondering why microscopes can't resolve to arbitrary magnifications. This is due to the interference and diffraction of light because of the wave nature of light. Light is a quantum particle which means that it exhibits both particle and wave aspects, depending on how you look at it. It is somehow neither a particle or a wave and yet both a particle and a wave (quantum weirdness rearing it's ugly head).

The main thing to remember is that light can be treated as a wave. And every wave has an associated wavelength. The average wavelength of visible light is about 550 nm (nanometers). Objects can be distinguished, or resolved, as 2 distinct objects as long as the distance between them is more than 1/2 the wavelength of the light being used. Thus, for ordinary (compound) light microscopes, objects can be distinguished only if they are separated by more than about 275 nm. Objects that are closer than that can't be resolved and appear as one, blurry object no matter how much you enlarge them (increased magnification without increased resolution=bigger, blurrier image). So why are electron microscopes able to achieve a higher resolution? Because electrons, like light, are also quantum particles; i.e., they also exhibit both particle AND wave aspects.

But the wavelengths associated with electrons are much smaller, on the order of 0.004 nm (for a moving electron with a certain energy; see, p.148 of Alberts, below). However, due to aberrations of electron lenses (just like there are aberrations in glass lenses there are also aberrations in the magnetic lenses used in electron microscopy), the resolving power of most modern elecron microscopes is about 0.1nm. Still, if you use electrons (in an electron microscope), you can achieve a much higher resolution than with light (in a light microscope). Biologically speaking, structures less than 275nm in width become apparent at these resolutions; cool things like the plasma membrane (approx. 7.5-10nm), ribosomes (15 to 25nm), microtubules (20 to 30nm), etc. suddenly become "visible" to us! On a side note, these same problems with magnifications and resolutions plague telescopes and modern astronomy.

Of course, there are many technical aspects to microscopy (lens aberrations, as already mentioned; chromatic aberrations for light microscopes; preparation and fixation of biological specimes since cells are colorless and translucent; contrast; radiation damage; etc.).

One final point: electron microscopes can be broken down into transmission electron microscopes (TEMs), scanning electron microscopes (SEMs), and scanning tunneling microscopes (STM). TEMs work in a similar way to light microscopes (they bounce electron radiation off specimens analagous to how light microscopes bounce light radiation off specimens) but while TEMs use the electrons that pass through the specimen to make the image (ignoring the electrons scattered from the electron dense, stained material), the SEM uses the electrons that are scattered from a thin metal layer that the sample is coated with. The sample is scanned with a very narrow beam of electrons and as these electrons are scattered off the coating, they are measured and used to control the intensity of a secondary beam of electrons which moves with the first, primary beam and forms the image on the computer.

The SEM gives a great 3-d picture with highlights and shadows but it can only examine sufrace features and the resolution is not that high (about 10nm with an effective magnification of 20,000 times, see Alberts below). Although SEMs are mainly used to look at the cell surface, they are smaller, simpler, and cheaper than their more powerful cousins. STMs, on the other hand, work by moving a "needle" up and down to get a 3 dimensional image of a specimen (this is dependent on another quantum effect, that of tunnelling; all you need to remember is that the needle has an electron "at the tip" and the microscope (actually, the computer) sees how much the needle goes up and down as it moves over a specimen, like the needle in a record player; thus, the STM "feels" the surface and allows us to make a detailed computer image of the surface of the specimen). Here are the relative resolving powers of light and electron microscopes (please refer to Alberts, p. 140, and Halliday and Resnick, p. 1061): Light Microscopes up to about 200 nm (about the size of bacteria) TEMs to about 0.1 nm (about the diameter of an atom!) and STMs can resolve features as small as 1/100 of an atomic diameter!!!

To put all this in perspective: with your eye, you can see objects as small as 0.1mm (e.g., a really thin hair), but with a light microscope, you can actually see objects as small as bacteria. And with an electron microscope, you can "see" down to the molecular, and even atomic, levels! I hope that helps; if any part is unclear or you'd like more details, please feel free to drop me an email.

Regards,

Rick.

P.S., if you'd like to look further into this, please refer to the following books at your library:

* Molecular Biology of The Cell, by Alberts, Bray, Lewis, et. al.
* Physics, by Halliday, Resnick, Krane.
* Biology, The Easy Way, by Edwards (a Barron's Guide).

The books are in order of "difficulty" (i.e., the first is the most "advanced" and the last is the easiest to browse through). You should also be able to find books specifically on Microscopy but if you just want a quick summary of concepts, you can check out the entries in an encyclopaedia, under microscopes and electron microscopes.