The problem

Starting out bright but dimming with illumination

Photobleaching of your fluorescence signal during imaging can occur with a variety of fluorophores. The photochemical destruction of the fluorophore is observed as a fading of the fluorescence signal while you are doing your imaging experiment.

Fading can be a problem when you want to image dim or low-abundance targets or when you are attempting quantitative analysis. If you want to perform any type of image quantitation, it is important to take into account any loss of fluorescence due to photobleaching, because it can skew your quantitative data and give you false results.

Side-by-side stained cell images showing an intensely fluorescent image (right panel) that dims over time with exposure to illumination from the microscope (right panel).

Figure 1. HeLa cells were fixed and labeled with FITC-conjugated phalloidin. Coverslips were mounted in 50% glycerol (in PBS). Panel (A) shows the initial intensity of the fluorophore, while panel (B) shows the photobleaching that occurs after 36 seconds of constant illumination.

What you can do

Try a different dye

Some dyes have been formulated to be more resistant to photobleaching during illumination. If you experience a lot of photobleaching with the dye you have chosen, you may want to see if other available dyes could work better in your experimental setup.

Try using neutral-density filters

You can also try using neutral-density filters to reduce the number of photons illuminating your sample (but your signal will also be dimmer), or change the gain settings on your microscope to use less light. Just be sure you use the same settings for all the samples you image if you want to compare them.

Minimize exposure

The easiest way to minimize photobleaching is to minimize the sample’s exposure to excitation illumination. You could do this several different ways:

  • Find the area you want to image, and focus using transmitted light. Then take the image you need using fluorescence.
  • Find an area of your sample and focus using fluorescence. Then go to a neighboring area and take the image you need.
  • Find the area you want to image, but focus using a suboptimal exposure time. You can also incorporate binning to minimize the exposure to light.

Create a photobleach curve

Some fluorophores are quite stable and you don’t have to worry much about photobleaching. However, some fluorophores are not very stable at all. You also may be interested in seeing small changes in fluorescence intensity. In either of these cases, you may need to create a photobleach curve. You can use the photobleach curve to normalize for loss of fluorescence intensity due to photobleaching and not due to your experimental conditions (i.e., drug treatment).

Use mounting media with antifade protection

If you are imaging fixed cells and need to prevent photobleaching, there are commercially available mounting media that can provide antifade protection. How well they work will depend on what fluorophore you are using. You may need to try different formulations to see which works for you. See the mounting media section.