Over the last several years there has been increased interest in multicolor fluorescence experiments. The development of additional dyes, more fluorescent protein variants and novel fluorescent labels such as quantum dots gives us far more opportunities than ever before for fluorescence imaging in live cells and animals. This has heightened the need for more advanced methods to discriminate between different dyes and fluorescent markers simultaneously. In response to this need, multispectral imaging has become an important tool for many biologists.