Bleed-Through in Fluorescence Imaging

The problem

Seeing things in the wrong filter set

Bleed-through occurs when you can see fluorescence from a neighboring channel in the channel of interest. This can occur in experiments that involve labeling with more than one fluorophore at a time.

Figure 1. Signals from two different fluorophores can appear in the same channel if the emission/excitation of the fluorophores are not both carefully matched to filters. An example of bleed-through is shown, where the signal from a fluorophore detected in the TRITC filter also shows in the FITC filter.

These HUVECs were stained with ReadyProbes ActinRed™ reagent, and the peroxisomes were labeled with a primary antibody for peroxisomes, and then detected with a fluorescently labeled secondary antibody (Alexa Fluor 488 secondary antibody). In this experiment the actin label was imaged using a TRITC filter, and the peroxisome labeling was imaged using a FITC filter. The image above was taken with the FITC filter. You can easily see the expected fluorescence signal from the peroxisome labeling, but you can also see bleed-through from the actin labeling. In this case the fluorescence signal from the actin stain bleeds through into the FITC channel—these two fluorophores should not have been used together in this experiment.