title: 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.
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy.|Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Gardini
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy.|Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Capitanio
authors.affiliation: LENS - European Laboratory for Non-linear Spectroscopy, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy.|Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto Fiorentino, Italy.|National Institute of Optics-National Research Council, Largo Fermi 6, 50125 Florence, Italy.|International Center of Computational Neurophotonics, Via Nello Carrara 1, 50019, Sesto Fiorentino (FI), Italy.
authors.forename: Francesco S
authors.initials: FS
authors.lastname: Pavone
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000737:chemistry / Q000378:metabolism*; D045663:Quantum Dots / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D005732:Gangliosides