Three-Dimensional Tracking of Quantum Dot-Conjugated Molecules in Living Cells

Year: 2018

Authors: Gardini L., Calamai M., Hatakeyama H., Kanzaki M., Capitanio M., Pavone FS.

Autors Affiliation: [Gardini, Lucia; Calamai, Martino; Capitanio, Marco; Pavone, Francesco Saverio] LENS European Lab Nonlinear Spect, Sesto Fiorentino, Italy.
[Gardini, Lucia; Calamai, Martino; Pavone, Francesco Saverio] INO Natl Inst Opt, Sesto Fiorentino, Italy.
[Hatakeyama, Hiroyasu] Tohoku Univ, Frontier Res Inst Interdisciplinary Sci, Sendai, Miyagi, Japan.
[Kanzaki, Makoto] Tohoku Univ, Grad Sch Biomed Engn, Sendai, Miyagi, Japan.
[Capitanio, Marco; Pavone, Francesco Saverio] Univ Florence, Dept Phys & Astron, Sesto Fiorentino, Italy.

Abstract: Here, we describe protocols for three-dimensional tracking of single quantum dot-conjugated molecules with nanometer accuracy in living cells using conventional fluorescence microscopy. The technique exploits out-of-focus images of single emitters combined with an automated pattern-recognition open-source software that fits the images with proper model functions to extract the emitter coordinates. We describe protocols for targeting quantum dots to both membrane components and cytosolic proteins.


KeyWords: Cytosolic proteins; Lipids; Living cells; Out-of-focus imaging; Plasma membrane proteins; Quantum dots; Single-molecule localization; Three-dimensional tracking; Ganglioside GM1; Cell labeling; Human; Membrane component