Biological imaging has revealed many new biological findings, among them GFP and other fluorescent proteins and small molecule based fluorescent probes have been widely used, especially in the past two decades. In this tutorial review the design concept and application of chemical probes are described—these are FRET based probes, Zn²⁺ probes and MRI probes. Fluorescence resonance energy transfer (FRET) has been used extensively for the design principle for fluorescent probes. One of the most significant advantages of probes with FRET modulation is that these can enable ratiometric measurement in living cells, which reduces the artefacts from microscopic imaging systems. The design strategy for the development of a small molecular FRET probe is described, in terms of avoiding close contact between donor fluorophore and acceptor fluorophore in aqueous solution. Furthermore, a strategy to design a FRET probe with modulating overlap integral of donor and acceptor is introduced. Numerous tools for Zn²⁺ sensing in living cells have become available in the last decade. Among them, fluorescence imaging using fluorescent probes has been the most popular approach. Some of these probes can be used to visualize Zn²⁺ in living cells. Some of the biological functions of Zn²⁺ were clarified using these probes, especially in neuronal cells, which contain a high concentration of free Zn²⁺. Real-time imaging of enzyme activities in vivo offers valuable information in understanding living systems and in developing medicine for various types of diseases. Magnetic resonance imaging (MRI) is expected to be one of the most promising in vivo imaging techniques in the scientific and medical fields. One of the most promising nuclide for MRI is ¹⁹F. ¹⁹F atoms are concentrated in the form of solid salts mostly in bones and teeth in our bodies. The MRI signal of the intrinsic ¹⁹F is hardly detectable. A novel design strategy for ¹⁹F MRI probes in detecting protease activity is described.