Fluorescence imaging is one of the most powerful techniques for visualization of the temporal and spatial biological events in living cells, and is employed in many fields of research. Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited photoinduced electron transfer (a-PeT), donor-excited photoinduced electron transfer (d-PeT), Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT) and spirocyclization. Especially, the a-PeT and d-PeT mechanisms, which have been established by our group, are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.