Imaging and Reconstruction of Three-Dimensional Quantum Dot Trafficking in Live Cells by Spinning Disk Confocal Microscopy.

Protocols are provided for time-lapse tracking of three-dimensional quantum dot (QD) movement in live cells using spinning disk confocal microscopy. The workflow employs a combination of a commercially available spinning disk confocal system and open-source ImageJ plugins, as well as the popular Imaris 3D/4D image analysis software. Implementation of the protocol streamlines all phases of the experiment: volumetric live-cell imaging, subsequent QD localization in 3D with nanometer accuracy, and continuous trajectory reconstruction. Detailed instructions are described for preparing QD control samples, labeling live cells with QD conjugates, and defining the appropriate parameters for image acquisition and QD trajectory reconstruction.