Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 15, Pages 6829-6834Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0912894107
Keywords
single molecule; tracking; membrane dynamics; lipid rafts
Categories
Funding
- Leibniz-Prize
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We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (similar to 10-20 nm standard deviation or similar to 20-40 nm full-width-at-half-maximum) and temporal resolution (<1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on <20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of similar to 15 ms and <4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization.
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