Multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) have been developed for cancer targeting and imaging in living animals. Compared with small molecule dyes, the intense fluorescence emission of QDs makes it easier to track single protein molecules, they are remarkably resistant to photobleaching, their narrow emission spectrum facilitates imaging of many proteins simultaneously, and their large two-photon cross sections allow in vivo imaging at greater depths. Since QDs have become commercially available, their use to study protein trafficking has grown rapidly. As an example, in vivo targeting studies of human prostate cancer growing in nude mouse show that the QD probes can be delivered to tumor sites by both enhanced permeation and retention (passive targeting) and by antibody binding to cancer-specific cell surface biomarkers such as prostate-specific membrane antigen (active targeting). Using both subcutaneous injection of QD-tagged cancer cells and the systemic injection of multifunctional QD probes, multicolor fluorescence imaging of as few as 100-1000 cancer cells can be achieved under in vivo conditions. These results suggest that QD probes and spectral imaging can be combined for multiplexed imaging and detection of genes, proteins, and small-molecule drugs in single living cells, and that this imaging modality can be adopted for real-time visualization of cancer cell metastasis in live animals.