Abstract
A confocal microscope set-up has been modified so that three different fluorescent markers can be studied. The three fluorophores (FITC, Texas Red and Cy5) are detected at the same time, i.e. simultaneous scanning is employed. We have shown that simultaneous scanning optimizes the precise overlay of the three colour components of an image (in contrast with sequential scanning where shifts between the colour components may occur). To suppress optical cross-talk between the three fluorophores, intensity-modulated multiple-beam scanning, IMS, has been employed. Cross-talk between the fluorescence signals has been measured. Results show that cross-talk is virtually eliminated by using the IMS technique. Measurements have been performed on a triple-stained immunofluorescence specimen in order to show the applicability of the technique in biological research. Results are presented that show that the technique presented here improves the accuracy of colocalization, position and intensity measurements for studies on stationary as well as on dynamic structures.
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