3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition.

Abstract:

:Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Gardini L,Capitanio M,Pavone FS

doi

10.1038/srep16088

subject

Has Abstract

pub_date

2015-11-03 00:00:00

pages

16088

issn

2045-2322

pii

srep16088

journal_volume

5

pub_type

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