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.