The control of subcellular localization of proteins and their interaction with other partners in vivo are important parameters that provide clues to their function and regulation. The ability to simultaneously track multiple protein species with high resolution should provide a valuable assay system to study and characterize various types of posttranslational control pathways. In this work, we established the system and a method involving "spectral profiling" for the resolution of four different fluorescent protein tags in the same viewing field using digital imaging technology. With these techniques, we have (a) developed new derivatives of mGFP5, which is commonly used in the plant field, that are about three times brighter; (b) demonstrated that four spectrally distinct fluorescent proteins (cyan, green, yellow, and red) that are fused to a transcription factor could be stably expressed in nuclei and distinguished in tobacco (Nicotiana tabacum) mesophyll cells; and (c) shown that interaction between partners of a dimeric transcription factor can be detected by measuring fluorescence resonance energy transfer. These technologies should help one to study protein-protein interactions efficiently, especially for nuclear proteins under in vivo conditions.