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,
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.”
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