Cornell University scientists have developed the means to optically watch a single biological molecule at a time.
Until now, researchers were constrained from seeing individual molecules of an enzyme (a complex protein) interacting with other molecules under a microscope at relatively high physiological concentrations -- their natural environment -- by the wavelength of light, which limits the smallest volume of a sample that can be observed. This, in turn, limits the lowest number of molecules that can be observed in the microscope's focal spot to more than 1,000. Internal reflection microscopes have managed to reduce the number of molecules to about 100. But because this number is still far too high to detect individual molecules, significant dilution of samples is required.
The researchers have discovered a way around these limitations, and in the process reduced the sample being observed 10,000-fold to just 2,500 cubic nanometers (1 nanometer is the width of 10 hydrogen atoms, or 1 billionth of a meter), by creating a microchip that actually prevents light from passing through and illuminating the bulk of the sample. The microchip, engineered from aluminum and glass in the Cornell Nanoscale Science and Technology Facility, a NSF-funded national center, contains 2 million holes (each called a waveguide), some as tiny as 40 nanometers in diameter, or one-tenth of the wavelength of light.
Small droplets of a mixture containing enzymes and specially prepared molecules were pipetted into wells on the microchip and placed the chip in an optical microscope. Each of the chip's holes is so tiny that light from a laser beam is unable to pass through and instead is reflected by the microchip's aluminum surface, with some photons "leaking" a short distance into the hole, on the bottom of which an enzyme molecule is located.
These few leaking photons are enough to illuminate fluorescent molecules, called fluorophores, attached as "tags" to nucleotides (molecules that make up the long chains of DNA) in the sample. In this way, the researchers were able to observe, for the first time, the interaction between the ligand (the tagged nucleotide) and the enzyme in the observation volume (the region of the mixture that can be seen).
The problem until now has been seeing exactly how long an interaction between a biological molecule and an enzyme takes and how much time elapses between these interactions. This is complicated by the need to distinguish those molecules interacting with the protein and those just passing by. "A freely moving molecule will come in and out of the observation volume very quickly -- on the order of a microsecond. But if it interacts with the enzyme it will sit there for a millisecond," says Levene. "There are three orders of magnitude difference in the length of time that we see this burst of fluorescence. So now it's very easy to discriminate between random occurrences of one ligand and a ligand interacting with the enzyme."
Says Webb: "We see only one fluorescent ligand at a time, so we can now follow the kinetics [movement and behavior] in real time of individual reactions." He adds, "We can actually see the process of interaction."
This is pretty impressive. The problem with biology has always been that most important mechanisms are shaped and operate on such a small scale that it is hard to figure out how exactly biological systems function. Anything that makes it easier to watch smaller scale phenomena can be very beneficial in speeding up the rate at which biological systems can be taken apart and figured out.
A logical extension of this technique would probably be to use quantum dots in place of the flourophores. Quantum dots last longer and they can be tuned to emit light at many different frequencies. That way different molecules emitting at different frequencies can be watched at the same time.
|Share |||Randall Parker, 2003 February 12 05:10 PM Biotech Advance Rates|