Scientific Method / Science & Exploration

Detecting the absorption of light from just a single molecule

Researchers show that they can detect the absorption from a single molecule. …

One of the not-so-secret desires of the optical imaging community is "single molecule" imaging. This is usually not quite what it seems though. Most of us don't expect to get a picture of where all the atomic nuclei or electrons are. Instead, what we usually think of is being able to say the optical signal from a particular location is due to a single molecule. So, in that respect, it is probably more accurate to call this single molecule location mapping.

In that vein, a paper in Nature Photonics shows how to detect the absorption from just a single molecule. Unfortunately, they call it "Single-molecule imaging by optical absorption," which is only true if the molecule were something like one hundred times bigger than it actually is and looks like a round blob. Despite the inaccurate title, the paper itself is still quite interesting.

There's a cool factor involved in saying "I can see single molecules." 

The way a molecule responds to light depends very strongly on its immediate environment. But, normally, an optical beam is so big in diameter that it is difficult to set up an experiment where only a single molecule is sitting in the beam at a time. Add to that the fact that molecules just don't absorb much light—one photon at a time every few nanoseconds means that the total absorbed power is something like 63pW, and that's only if you drive the molecule as hard as it can go—and you end up with a very tough experiment.

A molecule sitting in free space and not near anything that might disturb it will absorb a narrow range of colors. The win is this: once it feels the forces associated with other nearby molecules, the color of light it will absorb changes. So, picture a single molecule, say a dye molecule, surrounded by a water and proteins. If we can continuously monitor what color of light it absorbs, then we can figure out what is going on around the dye molecule. Going further, if we can do this for a whole area, then we can learn more than just where molecules are, but how they respond to their environment.

You've got the goal. How to get there? Well you will probably use some of the technology developed by Celebrano and colleagues. They have created a microscope that tracks absorption by focusng two different colors of laser light very tightly onto a sample that only contains a only few dye molecules. The dye molecules are the only species that strongly absorb one of the particular colors chosen. 

But lots of things weakly interact with the same light. The reality is that everything scatters light—changes its direction, meaning that it won't be collected by the microscope optics and counts as being absorbed. Absorption is also an issue. Resonant absorption, the type mentioned above, is the strongest form, but non-resonant absorption, which happens at every color in the spectrum, becomes significant when you are looking at single molecules.

That makes the name of the game "count only the missing light that is due to the dye molecule." That is where the two laser colors come in. One color is resonantly absorbed by the dye, while the other is not. All the scattering and non-resonant absorption effect both colors of light equally. So, if we pass both light beams through the sample and then subtract the non-resonant absorption and scattering from the resonant absorption, we can figure out when and where the laser light hits a dye molecule.

This is a very clever technique with high sensitivity and Celebrano and coworkers present very clean results. But... I can't help feeling that the whole thing is a little overblown. You will have noticed that I kept referring to dye molecules. That is because dye molecules have two properties that make this experiment much easier: they absorb strongly—really really strongly—and they can emit light at a different color after they absorb (we call this fluorescence). In other words, they had the strongest possible signals and could be tracked by their fluorescence, which is much easier than tracking their absorption.

It should be noted that they did not need the fluorescent, and they confirmed that by making measurements on dye molecules that were unable to emit light. They really are able to measure absorption from a single molecule... as long as it is a strong absorber.

The other thing missing in this bit of research is that they use two fixed colors of light. If your molecule doesn't absorb near either of those two, you are out of luck. With this system, you can only count on figuring out which portion of the molecular population is sitting in an environment where it is likely to absorb light that is the color of the laser you have on offer. Not hugely useful.

Finally—and this is the biggy—the researchers use an artificially dilute sample. That is there is only one dye molecule in the focus of the laser beam at any one time. In natural samples, this is very difficult to arrange, which makes the prospect of single molecule imaging—as opposed to location mapping of individual molecules in a dilute sample—much more daunting.

I told you it was interesting, but mainly for what is yet to be done.

Nature Photonics, 2011, DOI: 10.1038/nphoton.2010.290

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