An automated tool for 3d tracking of single molecules in living cells
Authors: Gardini L., Capitanio M., Pavone FS.
Autors Affiliation: Univ Florence, LENS European Lab Nonlinear Spect, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, I-50019 Sesto Fiorentino, Italy; Ist Nazl Ottica, I-50125 Florence, Italy
Abstract: Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell’s environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.
KeyWords: Single-molecule fluorescence imaging; 3D tracking; living cells imaging; off-focus imaging; Fluorescent Beads; Quantum Dots