We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 × 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells.