In standard light microscopy, there must be at least 1000 molecules present in the microscope's focal spot because of restrictions on sample volume caused by the wavelength of light. Even internal reflection microscopes still require sample volumes containing about 100 molecules. Therefore scientists must significantly dilute the sample to examine individual molecules.
The Cornell technique, in contrast, could look at sample volumes that are 10,000 times smaller - it required just 2500 cubic nm. That's because the nanofabricated device contained holes so small (about one-tenth of the wavelength of light) that they stopped light travelling very far into the sample.
"To get to these [relatively high, naturally occurring] concentrations and still observe one event at a time you need a very small volume," said Michael Levene of Cornell. "Otherwise you would have all those molecules milling around and we couldn't tell which were interacting with our enzyme."
The device, which is 25 mm wide, consisted of an 89 nm thick film of aluminium on a glass coverslip. The aluminium layer contained 25 wells, each with 90,000 holes inside: the smallest of these holes were about 40 nm in diameter. Light falling on the device was mainly reflected from the aluminium surface, although some photons were able to "leak" a short distance into the holes.
These photons then illuminated fluorescent molecules (known as fluorophores) attached as tags to nucleotides in the sample. In this way, the researchers were able to examine individual interactions between the tagged nucleotides and the enzyme molecules in their sample.
"[The technique] provides a very powerful way of looking at fluctuations and variability in behaviour of individual enzyme molecules," added Watt Webb of Cornell. "We are seeing those variations and they are huge. Observing them with such detail was hardly accessible until this experiment."
The technique could have applications in drug discovery or in creating a new method for DNA sequencing that can read the genetic code from a single DNA molecule.
The researchers reported their work in Science.