Many promising attributes of ZnO nanoparticles (nZnO) have led to their utilization in numerous electronic devices and biomedical technologies. nZnO fabrication methods can create a variety of intrinsic defects that modulate the properties of nZnO, which can be exploited for various purposes. Here we developed a new synthesis procedure that controls certain defects in pure nZnO that are theorized to contribute to the n-type conductivity of the material. Interestingly, this procedure created defects that reduced the nanoparticle band gap to ∼3.1 eV and generated strong emissions in the violet to blue region while minimizing the defects responsible for the more commonly observed broad green emissions. Several characterization techniques including thermogravimetric analysis, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, Raman, photoluminescence, and inductively coupled plasma mass spectrometry were employed to verify the sample purity, assess how modifications in the synthesis procedure affect the various defects states, and understand how these alterations impact the physical properties. Since the band gap significantly decreased and a relatively narrow visible emissions band was created by these defects, we investigated utilizing these new nZnO for bioimaging applications using traditional fluorescent microscopy techniques. Although most nZnO generally require UV excitation sources to produce emissions, we demonstrate that reducing the band gap allows for a 405 nm laser to sufficiently excite the nanoparticles to detect their emissions during live-cell imaging experiments using a confocal microscope. This work lays the foundation for the use of these new nZnO in various bioimaging applications and enables researchers to investigate the interactions of pure nZnO with cells through fluorescence-based imaging techniques.