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.

Cell image showing fluorescence from two different color dyes appearing in a single filter set, rendering them indistinguishable in the experiment.   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.

What you can do to minimize bleed-through

  • Choose your fluorophores and filters wisely. Aim to use the most suitable filter for each fluorophore you want to image.
  • Using fluorophores with narrow emission spectra can also help minimize bleed-through.
  • When designing experiments with multiple fluorophores, choose fluorophores that do not have spectral overlap. For example, if you want to label with three colors, try to use dyes that fit with the DAPI, FITC, and Texas Red® filter sets.