Non- negative matrix factorization:a novel method to determine reference spectra of fluorescent dyesion a cellular environment

All spectrally resolved fl uorescence-based methods depend critically on the availability of accurate reference spectra. Very often spectra in a cellular environment do not agree with those measured in a cuvette. Also, for a given measurement instrument-specifi c corrections have to be applied. We recently developed a method to extract reference spetra from standard fl uorescence image stacks, such as those provided by a Zeiss Meta microscope. This method also allows one to separate spectral components of several simultaneously present dyes on a pixel by pixel basis. It should be ideal to complement lux-FRET measurements, providing reference spectra in the presence of autofl uorescence.