Sometimes the bleed-through of the blue nuclear label into the green channel seems higher than usual. There is a real phenomenon behind this increase; it's called photoconversion.

It is known for the blue nuclear labels DAPI, Hoechst and Vybrant DyeCyle Violet, but can happen to other labels too. Red-shifts of up to 80 nm have been observed in real samples, e.g. cy5-Beta-catenin on polyvinylidene (PVDF) membrane.

Note that the in-solution values stated in reference may differ from spectra in other conditions.

DAPI, Hoechst 33258 and Vybrant DyeCyle Violet Exhibit Photoconversion

Spectral Characteristics of the Red-shifted Label

Too High Label Concentrations Can Diminish Signal

DAPI Spectral Shift

DAPI Spectral Characteristics

DAPI Red-shift Conditions

Hoechst 33258 Red-shift

Hoechst Spectral Characteristics

Vybrant DyeCyle Violet

 Vybrant DyeCyle Violet also exhibits the photoconversion red-shift of it's spectra.

Mitigating the Red-shift

Sources