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Recording of dual-labeled specimens using frequency-multiplexed confocal imaging and intensity-modulated two-wavelength excitation

We demonstrate the possibility to use intensity-modulated excitation and frequency multiplexing in combination with lock-in detection to make multi-parameter measurements with a confocal scanning laser microscope. This approach can be used, for example, when studying dual-labelled fluorescent specimens. Frequency-multiplexed confocal imaging has the potential to reduce a main problem in connection with detection of multiple-labelled specimens, namely cross-talk between the recorded signals from the two fluorophores. In addition, it has the advantage compared with the traditional method that the fluorescent spectra are utilized much more efficiently, thereby greatly improving signal quality.