Fluorescence microscopy techniques are considered an essential bioimaging tool for their remarkable sensitivity, high spatial resolution, and specificity in providing direct visualization of structures and processes within biological specimens. The basic principle of fluorescence microscopy lies in the emission of light from fluorescence materials, simply called fluorophores, which are attached to specific structures under observation. Understanding the working principle and the types of fluorophores used in different fluorescence microscopy techniques is crucial for bioimaging research. This chapter is designed to cover a comprehensive overview of a series of fluorophores and optical microscopy techniques useful for modern bioimaging applications. The discussion starts with a familiarization of different kinds of fluorescence materials, including quantum dots, nanoparticles, organic dyes, and fluorescent proteins, along with their characteristics, advantages, and suitability for biological imaging. Several surface modification and bioconjugation techniques are described to improve and modify these fluorescence materials to target biological structures precisely. The chapter further explores various fluorescence microscopy techniques, from conventional microscopy, including widefield, confocal, two-photon microscopy, and total internal reflection microscopy, fluorescence lifetime imaging microscopy (FLIM) to modern super-resolution microscopy techniques such as structural illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and stochastic optical reconstruction microscopy (STORM). The principle of operation, technical requirements, ideal applications, advantages and disadvantages of each microscopy technique are presented to provide better insights into their appropriate applications and limitations. This chapter intends to provide an introductory reference for bioimaging researchers in advancing optical microscopy instrumentation and designing new fluorophores for applications in neuroscience, cell biology, cancer research, and regenerative medicine.