Biomaterials for in vivo fluorescence imaging are required to be biocompatible, nontoxic, photostable and highly fluorescent. Fluorescence must be in the near infrared (NIR) region of the electromagnetic spectrum to avoid absorption and autofluorescence of endogenous tissues. NIR fluorescent polystyrene nanoparticles may be considered ideal biomaterials for in vivo imaging applications. These NIR nanoparticles were prepared by a swelling process of polystyrene template nanoparticles with a hydrophobic NIR dye dissolved in a water-miscible swelling solvent, a method developed for preparation of nonbiodegradable nanoparticles, for NIR fluorescent bioimaging applications. This method overcomes common problems that occur with dye entrapment during nanoparticle formation such as loss of fluorescence and size polydispersity. Fluorescence intensity of the nanoparticles was found to be size dependent, and was optimized for differently sized nanoparticles. The resulting NIR nanoparticles were also found to be more fluorescent and highly photostable compared to the free dye in solution, showing their potential as biomaterials for in vivo fluorescence imaging. In vivo fluorescence imaging requires the use of near IR fluorescent imaging agents, to avoid the absorbance and autofluorescence of endogenous tissues that occur at visible wavelengths. Near IR dye was encapsulated into polystyrene nanoparticles via a swelling process. The dye encapsulation process was performed for a series of differently sized nanoparticles, without affecting the relatively uniform size distribution of each size. The method used was found to be appropriate for nanoparticles averaging approximately 20-100 nm in size. The resulting nanoparticles were highly fluorescent and highly photostable, and with appropriate surface modification, have potential for biological and in vivo imaging applications.