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The use of Lucifer yellow, bodipy, FITC, TRITC, RITC and Texas red for dual immunofluorescence visualized with a confocal scanning laser microscope

A new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo‐inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo‐inactivation. Bodipy fl and FITC give lower levels of cross‐talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross‐talk into images of RITC‐stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross‐talk into the green band‐pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488‐nm line of an argon ion laser is used as the excitation source.