SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
ANALYSIS METHOD FOR MEASURING SUBMICROSCOPIC
DISTANCES WITH BLINKING QUANTUM DOTS
B Christoffer Lagerholm 1, Laurel Averett 1, Gabriel E Weinreb 1, Ken Jacobson 1 and Nancy L Thompson 1*
1 University of North Carolina
* To whom correspondence should be addressed. E-mail: nlt{at}unc.edu.
Submitted on December 6, 2005
Revised on January 28, 2006
Accepted on 14 June 2006
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Abstract |
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A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function (PSF) of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the PSFs determined. Images of the blinking QDs were then overlapped in software, pixel-by-pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122 base pair double stranded DNA fragment.
Key Words:
Fluorescence Microscopy, Fluorescent Nanoparticles, Lipid Rafts, Membrane Microdomains, Photophysics, Receptor Clustering
