Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.

7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin-phalloidin, fluorescent in the blue region of the spectrum. Coumarin-phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit triple immunofluorescence microscopy of the cytoskeleton with fluorescein and rhodamine conjugates together with coumarin-phalloidin.