This paper describes the preparation of semiconducting polymer dots (Pdots) and their application for
single-molecule imaging and biosensing. The Pdots possessed high fluorescence brightness, with a 16 nm Pdot being ~9
times brighter than 13 nm Quantum Dots (Qdots). The surface of Pdots was successfully conjugated with streptavidin,
which made Pdots suitable for specific subcellular labeling and targeting. The interior composition of Pdots was also
successfully modified, through which the Pdots obtained additional functionalities. We demonstrated the utility of gold nanoparticle embedded Pdots in dual-modality imaging. We also demonstrated that Rhodamine B embedded Pdots were able to function as ratiometric temperature sensor in live-cell imaging mode.