Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids.
We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (approximately 10-20 nm standard deviation or approximately 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 approximately 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.
Sahl
Steffen J
SJ
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Leutenegger
Marcel
M
Hilbert
Michael
M
Hell
Stefan W
SW
Eggeling
Christian
C
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D007668:Kidney / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D048328:Potoroidae; D011336:Probability; D015203:Reproducibility of Results; D012815:Signal Processing, Computer-Assisted; D013997:Time Factors
D008563:Membrane Lipids