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Pushing microscopes to their limits (and beyond)

November 21, 2013
By Jeremie Fish (adkscienceguy@ , Adirondack Daily Enterprise

A long time ago, it seemed like light microscopes (the kind of microscope you use to look at things with visible light) had reached their limit.

As the science community decided to probe more deeply into nature, it began using microscopes that did not require the use of visible light and thus took the human eye out of the equation.

Scientists had to move away from the light telescope to probe more deeply because of an important limiting factor, the diffraction limit. To understand the diffraction limit, you need only to think of a newspaper example. The cartoons that are run in newspapers occasionally are made up of a bunch of little dots. From far away, your eyes see the individual dots as blended together to make a seemingly continuous picture. Yet when you get close enough, you can resolve the dots with your eyes and you can tell that there are individual dots.

A similar thing happens when you reach the diffraction limit. At that point, you can no longer resolve two different objects, and they appear as one blob. This happens because visible light, as well as the rest of the electromagnetic spectrum (such as infrared and microwaves), naturally tends to spread out as it propagates from place to place. This spreading out is related to the energy of the electromagnetic wave. The more energetic the wave is, the less diffraction occurs and the better the resolution (thus the smaller the object that can be "seen").

This is actually the reason that they switched from using red laser light to using blue laser light (blu-ray) because blue light can resolve dots of information that are closer together than red light can, so more information can be stored on the same size disk. (I am predicting then that the next big thing will be UV-ray disks).

This, of course, meant that you needed more energetic waves (thus going outside the range of visible light) to see smaller and smaller objects. Or you had to circumvent electromagnetic waves by other means such as using special properties of electrons to collect data leading to a variety of different electron microscopes that take "pictures" which are really just computer enhanced images.

When trying to study individual molecules however these other microscopes just don't do the job (at least not inside a living cell). The problem is that visible light has already reached its diffraction limit at this size, so what can we do? Well scientists have developed ingenious ways to skirt the diffraction limit using regular visible light. One of those ingenious ways is called Total Internal Reflection Fluorescence Microscopy (TIRF, or TIRFM). The beauty of TIRF is that utilizing a special feature of light (quantum tunneling), you can peer into the cell more deeply than regular light microscopy. There are other techniques as well (such as FRAP and FRET) that have allowed scientists to work their way around the diffraction limit. What makes these techniques exciting is that they use low enough energies to view what is happening in a live cell in real time, without killing the cell.

Of course, this has allowed for new processes to be discovered and has allowed us to march ever farther along the road of progress when it comes to an understanding of how to keep an individual healthy. There is still plenty of room for advances but we can be sure that human ingenuity never ceases to amaze, especially at doing things that at one time may have seemed impossible.

Jeremie is a Wilmington resident and Clarkson University graduate student.



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