Design of a four channel spectral analyzer to resolve linear combinations of two fluorescent spectra

Advances in molecular probes and fluorescence labeling require sophisticated spectral analysis. For the analysis of spectral data one frequently uses an RGB system (3D color system), dedicated filters for each n basic spectra (nD), or a spectral analyzer (mD, m>n). We present a method for designing spectral filters that optimally resolve all basic spectra as well as all linear combinations of two basic spectra. A three-channel color system may distinguish a large number of fluorophors, but cannot uniquely identify the basic spectra that constitute a linear combination. We present a four-channel color system that optimally resolves all basic spectra as well as all linear combinations of two basic spectra. The design strategy yields a set of spectral filters with transmissions between 0 and 1, which can be manufactured. “High-dimensional” spectra will be projected onto a four-dimensional sub-space in which the task can be accomplished. The design strategy incorporates a noise model that provides robustness over a range of signal-to-noise ratios.