IMPROVED FLUOROPHORE SEPARATION WITH IMS CONFOCAL MICROSCOPY

SOME of the most extensively used fluorophores for multicolour fluores cence labelling of tissue, such as Lucifer yellow, fluorescein, Cyanin e-3.18. and rhodamine, all have the disadvantage of being difficult to separate spectrally due to their broad and overlapping emission spect ra. With the intensity-modulated multiple-beam scanning (IMS) techniqu e, cross-talk between labels can be reduced to a fraction of that seen using conventional excitation and detection technique. Thus, cross-ta lk to the 'red' channel decreased with about one order of magnitude fo r the 'green' fluorophores Lucifer yellow and FITC. For the tested 're d' fluorophores, the amount of cross-talk to the green channel was red uced by 60-90%