Three-dimensional tracking of quantum dot-conjugated molecules in living cells

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-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.