Time-gated (TG) fluorescence imaging (TGFI) has attracted increasing attention within the biological imaging community, especially during the past decade. With rapid development of light sources, image devices, and a variety of approaches for TG implementation, TGFI has demonstrated numerous biological applications ranging from molecules to tissues. The paper presents inclusive TG implementation mainly based on optical choppers and electronic units for synchronization of fluorescence excitation and emission, which also serves as guidelines for researchers to build suited TGFI systems for selected applications. Note that a special focus will be put on TG implementation based on optical choppers for TGFI of long-lived probes (lifetime range from microseconds to milliseconds). Biological applications by TG imaging of recently developed luminescent probes are described.