Beyond photobleaching, laser illumination unbinds fluorescent proteins

Area of Science:

• Cell biology
• Biophysics
• Microscopy

Background:

• Confocal and two-photon fluorescence microscopy are vital tools in cell biology.
• Common issues include photobleaching and phototoxicity.
• Photounbinding, the light-induced dissociation of molecules, is an underrecognized problem.

Purpose of the Study:

• To investigate the phenomenon of photounbinding in fluorescence microscopy.
• To determine if laser excitation can cause antibody-ligand dissociation.
• To assess the impact of photounbinding on quantitative binding studies.

Main Methods:

• Utilized confocal and two-photon fluorescence microscopy.
• Employed fluorescently labeled anti-Green Fluorescent Protein (GFP) antibodies and immobilized GFP in vitro.
• Measured dissociation using laser intensities typical for FRAP, photolysis, and FCS.
• Visualized and quantified photounbinding by rebinding a second fluorescent antibody.

Main Results:

• Both one- and two-photon excitation induced dissociation of fluorescent anti-GFP antibodies from GFP.
• For two-photon excitation, photounbinding and photobleaching thresholds were identical.
• Single-photon excitation allowed separation of photounbinding from photobleaching.
• Photounbinding occurred only when at least one binding partner was fluorescently labeled.
• GFP remained functional for reassociation after photoinduced dissociation.

Conclusions:

• Photounbinding is a significant artifact in fluorescence microscopy, particularly for quantitative binding studies.
• This effect can be masked or misinterpreted as photobleaching.
• Researchers must consider photounbinding when designing and interpreting fluorescence-based binding experiments, especially those involving labeled antibodies.