AAT Bioquest

What strategies can I use to reduce photobleaching in live-cell imaging?

Posted April 6, 2023


There are four strategies you can use to reduce photobleaching in live-cell imaging:

  1. Reduce the intensity of light - Reducing the intensity of light exposure is one of the most common strategies used to reduce photobleaching in live-cell imaging. This can be done by using a light source with fewer photons such as LEDs, mercury lamps and xenon-arc lamps. Reducing bright light lowers the frequency of excitation-emission cycles, which helps to extend the life of fluorescent molecules and slow down photobleaching. However, it can also reduce emission signals. It’s important to find a balance in order to obtain a high quality image. 
  2. Reduce sample exposure to light - The shorter time exposure to light reduces photobleaching by decreasing the frequency of excitation-emission cycles in live-cell imaging. Lower light exposure can be achieved by proper incubation of samples in the dark, storing fluorophores in the dark, and turning off your microscope’s light between images, 
  3. Use antifade mounting media - Using antifade mounting media slows down fading and photobleaching in samples that need to be mounted. Different fluorophores require different mounting media. You may need to conduct a range of optimization experiments in order to identify the most suitable medium.  
  4. Use newer fluorophores - Newer fluorophores have greater photostability and are more resistant to bleaching as compared to older fluorophores that are more susceptible to fading. While photobleaching is not entirely avoidable, you can reduce its occurrence using these four strategies.
Additional resources

Circumventing photodamage in live-cell microscopy

FluoroQuest™ PLUS Antifade Mounting Medium