Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy.

Multicolor fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles, or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using fluorescence resonance energy transfer (FRET). These techniques have become invaluable to all biologists from cytogeneticists to zoologists. Resolving multiple, co-localized chromosome probes and individual fluorescent species in corals share the common problem of spectral overlap. Even in simple systems where two or three fluorochromes are used, spectral overlap or cross-talk can be difficult to eliminate, limiting the ability to distinguish one signal from another with any confidence. However, this problem is not unique to the biologist; geologists who use remote sensing techniques have been struggling with the problem for a long time. In fact, their problems are actually worse because many geological spectra are very similar and the number of possible spectral classes is much larger than the number of fluorescent probes in a biology experiment. Nonetheless, these issues have been tackled successfully, and biologists have recently begun to employ these methods to decipher multiple spectral signatures within cells and organisms, a process known as emission fingerprinting. Here, we review some approaches that have been used to enhance spectral resolution in fluorescence microscopy.