When scientists make observations of matter, they in most cases observe large numbers of molecules and base their conclusions about a “typical” molecule on the results. Inherent in this fundamental approach is an averaging process. This can be illustrated by the following “thought experiment.” Imagine a can containing millions of dice. Each die in the container, after it is thrown, can send out its own signal to a detector, which receives the signals from all the dice simultaneously and averages them. Imagine, now, that all the dice are thrown simultaneously. If the most probable result occurs–that one-sixth of the dice land as “ones,” one-sixth as “twos,” etc.–the detector reports the sum of all signals, which is then divided by the total number of dice to give the result: 3.5. The observer thus learns that the typical die, when thrown, comes up 3.5! The point is that there is no such thing as a typical die. Furthermore, if we had to rely solely on the average result to describe the throw of dice, we couldn’t play Monopoly. Individual throws of individual dice (or pairs of dice) are much more interesting and useful.

Scientists realize that the averaging process inherent in scientific observation conceals the detailed behavior of individual molecules, and thus interest has been growing in the observation of individual molecules. (A single molecule corresponds to 1/Avogadro’s number, or 1.66 X 10^-24 moles, or 1.66 ymol [yoctomol]. One chemist suggested in jest recently that the single-molecule unit should receive the name guacamole.) Viewing a single molecule is a tall order for several reasons. One reason is that a single molecule must be isolated in a way that permits its observation. Another reason is that the signal that is received from that molecule must be strong enough, and the detector sensitive enough, to create an observable signal.

Chemists have been able to isolate and immobilize single molecules and atoms by using them as doping agents in films, crystals, or gels. One of the most common techniques for observation is fluorescence, a process in which a molecule, after absorbing light, re-emits it at a longer wavelength. Because fluorescence occurs over a background of zero–that is, there is no other light of the same wavelength present from which the signal has to be extracted–fluorescence is a very sensitive detection method.

The behavior of single molecules can be illustrated by the work of a group of chemical physicists at the University of California, San Diego, led by W. E. Moerner, who entrapped a jellyfish protein called green fluorescent protein in a polyacylamide gel. (1) Irradiation of a sample containing a large number of such molecules produces a continuous fluorescence. However, when the individual target molecules are sufficiently dispersed, in a very small sample, and are examined by a very sensitive technique (e.g., fluorescence), the number of molecules observed in a given time period is small enough that the signal from each can be observed. We are still observing a group of molecules, but the group of molecules is so small that the contributions of individual molecules can be distinguished. When such a single-molecule system is observed during continuous irradiation, it “flickers” on and off in an irregular manner. This flickering behavior is seen in the fluorescence of green fluorescent protein dispersed in a gel. (The picture a left shows the backbone of this protein.) You may view an animation of the flickering emission from green fluorescent protein. Such flickering behavior is presumably the result of fluctuations in the protein’s structure, its environment, or both. Analysis of the flickering can in principle lead to insight about the underlying processes.

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