Professor Watt Webb's group put 40-nanometer holes in the aluminum top layer of a 25 millimeter square chip. "Conventional wisdom would tell you that this is not a single- or multimode waveguide, since its size is ten times smaller than the light going through it. Rather, we call it a zero-mode waveguide," said postdoctoral fellow Michael Levene.

Single-mode waveguides are usually the same size as the light that passes through them, hence enabling only one wavelength of light to pass, while multimode waveguides are larger and allow several wavelengths to pass. But a waveguide that is ten times smaller than the light impinging on it should pass nothing — hence the term "zero-mode." However, the Cornell researcher found that edge effects enabled the 488-nm light to penetrate the 40-nm waveguides to a depth of about 10 nm.

"The holes are so small that we are able to isolate individual molecules inside them, and even though the light doesn't pass through, it penetrates far enough to see one fluorescent ligand at a time interacting with the molecule inside the hole," Levene said.

The microchip, fabricated with aluminum on a glass substrate, used only 90,000 of the waveguides in the latest experiment. However, future researchers will be able to perform massively parallel experiments by utilizing all two million holes. The chip was created at the Cornell Nanoscale Science and Technology Facility with funds from the National Science Foundation, the U.S. Department of Energy, and the National Institutes of Health.

(a) A laser beam is first expanded by a telescope (L1 and L2), then focused by a high-NA objective lens (OBJ) on a fluorescent sample (S). The epi-fluorescence is collected by the same objective, reflected by a dichroic mirror (DM), focused by a tube lens (TL), filtered (F), and passed through a confocal aperture (P) onto the detector (DET).

(b) Magnified focal volume (green) within which the sample particles (black circles) are illuminated. The focal volume is the distribution of laser illumination at the focus of the objective. On the other hand, the observation volume, contained within the focal volume, is the region in space where fluorescent molecules are both excited and detected.

(c) A typical fluorescence signal, as a function of time, measured for rhodamine green (RG) with excitation wavelength lx=488 nm.

(d) Portion of same signal in (c), binned, with expanded time axis and average fluorescence Fbar. The signal F(t) at time t is correlated with itself at a later time (t+t) to produce the autocorrelation G(t).

(e) Measured G(t) describing the fluorescence fluctuation of RG molecules due to diffusion only as observed by FCS. Assuming a Gaussian observation volume, G(t) can be least-squares fitted using various analytic functions to extract information about molecular concentration, brightness, diffusion, and chemical kinetics, for one or more diffusing fluorescent species.