Multi-color single filter photobleaching analysis for measuring gene expression

Fluorescence microscopy is widely used in biomedical applications to quantitatively measure specific molecular signatures. It is often valuable to study several biosignatures simultaneously, such as in early stages of cancer diagnostics. Currently, multiple analyte can be observed based on the excitation and emission spectra of fluorescent dyes. However, the number of concurrent dyes is limited by the optical spectral bandwidth. We have developed a novel identification technique based on the photostability of different fluorophores. Fluorophore identification was demonstrated in mixtures of rhodamine 6B and rhodamine 6G. In addition, the concentration of each dye was successfully determined. Since the analysis is independent of the individual dye spectra, this technology can be easily integrated with existing spectra-based systems for expanding the ability of multi-analyte detection.