Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique

Abstract A new technique for simultaneous recording of multiple fluorophores has been implemented in a confocal scanning laser microscope. Dual excitation wavelengths, intensity-modulated at different frequencies and two lock-in amplifiers tuned to the corresponding frequencies are used. In this way two fluorophores can be independently, albeit simultaneously, excited. This technique, Intensity-modulated Multiple-beam Scanning (IMS) microfluorometry, has the potential to virtually eliminate the cross-talk between fluorophores that often occurs when recording multiple-labelled specimens. Furthermore, it will offer simultaneous information about both the excitation and emission spectra of the fluorophores used. Also, variations in decay time over the image area can be studied independently and simultaneously for two fluorophores.