Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Spectral Imaging

Additive Properties of Emission Spectra


The process of linear unmixing of data obtained from spectral imaging is a mechanism for matching the spectral variations in the lambda stack with the known spectral variations for the fluorophores present in the specimen. For each pixel in the image, the fluorescence intensity of colocalized fluorophores having overlapping emission spectra will be the sum of the intensity for each individual fluorophore. This article explores how multiple spectra can be added to produce a composite emission spectrum similar to those encountered in spectral imaging of specimens labeled with multiple fluorophores.

In order to determine the spectral content of each pixel in an image, the simplest approach would be to match the summed spectrum from that pixel with all possible sum combinations from a library, similar to what investigators undertake when they are matching a fingerprint with a database. For example, if the measured summed spectrum was a very close match to the black curve, it could be concluded that the two fluorophores were evenly mixed in that pixel. Likewise, if the summed spectrum matched the black curve, it would indicate that the pixel contained 75 percent of one fluorophore and 25 percent of the other. In review, linear unmixing is a straightforward technique that compares a matrix representing the summed spectra measured in an image against a library of predicted spectra according to the best-fit parameters mandated by the software. After the contribution of each spectral component is determined, the lambda stack can then be segregated into individual images for each fluorophore. Thus, the intensity of an individual pixel in the unmixed spectral image represents the total measured pixel intensity multiplied by the proportion of each fluorophore spectra in that pixel. The result of linear unmixing is conversion of a lambda stack into individual images representing the signal profile for each fluorophore.

Contributing Authors

Adam M. Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.