Following the blinking lights helps take the measure of molecules

Lynn M. Savage

To gain a better understanding of the biochemical processes that occur at or near cell membranes, scientists need to get in and take measurements of the particles that are involved.

A number of metrological techniques are available, including those as simple as optical and fluorescence microscopy, but these either exhibit too low resolution or, as in the case of Förster resonance energy transfer, too high.

Now researchers at the University of North Carolina at Chapel Hill, led by Nancy L. Thompson, have exploited the propensity of quantum dots to blink on and off to devise a microscopy method that enables them to measure distances in the 10- to 100-nm range.

They reasoned that the position of individual particles within a group could be determined by acquiring images of the point spread function of each one. This is feasible only because the particles fluctuate between “off” and “on” states of fluorescence, blinking for the duration of their emission.

As reported in the October Biophysical Journal, the investigators tested this notion by placing particles made by Quantum Dot Corp. of Hayward, Calif. (now part of Invitrogen Corp.), on a slide and imaging them with an Olympus microscope, a 60x objective with a 1.4 NA, an additional 1.6x magnification element, a 100-W mercury arc lamp and an intensified CCD camera from Stanford Photonics Inc. of California. They limited differences in fluorescence intensity by constraining the imaged regions of interest to ~400 x 400 pixels.

They found that single quantum dots could be identified by monitoring their fluorescing and nonfluorescing states, rejecting particles that were close to one another, particles close to the boundary of the region of interest, nonspecifically bound clusters of particles and random clusters that remained from the manufacturing process. They determined the centroids of each particle’s point spread function, and, by overlapping all of the possible pairs of quantum dots in pixel-by-pixel layering of the regions of interest, they calculated the distances between the individual particles within a few tens of nanometers.

Researchers have measured nanoscale distances using blinking quantum dots. Representative images of a pair of quantum dots separated by a 122-base-pair stretch of DNA acquired in an instance where one (1), both (2) or none (3) of the quantum dots were in their fluorescent “on” state (top; scale bar = 1 mm). Integrated fluorescence intensity of the observed pair of quantum dots (line) and of the background (dots) as a function of frame number shows the selected instances (1, 2, 3) of the representative images (bottom). In this case, the two quantum dots were found to be separated by 47 ±16 nm. Courtesy of B. Christoffer Lagerholm.

To test the usefulness of the technique for biological applications, they measured the length of a 122-base-pair stretch of DNA. At an average length of 3.4 Å per base pair, the DNA fragment should have been ~42 nm. Using quantum dots attached to strands of the DNA, the scientists measured the length of each fragment to be 48 ±8.5 nm. The discrepancy possibly results from the technique adding in the size of the particles themselves.

The researchers noted that spontaneous aggregation of the particles can confound the measurement technique, but that, once this problem is solved, it will offer advantages over alternative methods and may be suitable for characterizing such structures as cell membrane lipid microdomains, or lipid “rafts.”