Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information, Using a two-detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal-to-noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.