Towards a spectrum-based bar code for identification of weakly fluorescent microparticles

Spectrally resolved detection of fluorescent probes can be used to identify multiple labeled target molecules in an unknown mixture. We study how the spectral shape, the experimental noise, and the number of spectral detection channels affect the success of identification of weakly fluorescent beads on basis of their emission spectra. The proposed formalism allows to estimate the performance of the spectral identification procedure with a given set of spectral codes on the basis of the reference spectra only. We constructed a simple prism-based setup for spectral detection and demonstrate that seven distinct but overlapping spectral codes realized by combining up to three fluorescent dyes bound to a single bead in a barcode-based manner can be reliably identified. The procedure allows correct identification even in the presence of known autofluorescence background stronger than the actual signal.