ID-3-P-2327 Towards the mechanism of photounbinding 

Recent studies have shown that fluorescently labeled antibodies can be reversibly dissociated from their antigen after illumination with laser light. This unexpected phenomenon is called photounbinding, and can be both boon and bane in fluorescence imaging approaches. While photounbinding should be avoided in typical quantitative fluorescence imaging, it has also the potential to become a valuable low-invasive tool of controlling binding assays. For both avoiding and utilizing photounbinding is vital to understand its mechanism. Unfortunately, pinpointing photounbinding is not straight forward as it often remains masked in an experiment as it first glance appearing is similar to photobleaching where the fluorophore becomes dark (non-fluorescent) at some point after laser illumination. However, photounbinding is substantially different, as it additionally creates an unbound (vacant) site.

To visualize photounbinding in a confocal microscope we immobilize GFP on a glass surface, recognize it by an antibody, laser scan a region of interest to induce photounbinding and subsequently rebound with a differently labeled antibody against GFP (see figure). We hypothesize that photobleaching is most likely driven by the production of Radical Oxygen Species (ROS). To test this hypothesis, we introduce a redox sensitive GFP (roGFP2) to determine the changes in the redox potential after illumination by laser light. Our results show that redox potential increase in the roGFP2 vicinity with increasing scanning iterations or/and laser power. As ROS production is considered to be local, the distance of the local ROS production site from the antibody antigen complex should play a key role for photounbinding. By varying the effective distance of the binding partner (antigen) to its fluorescent marker, we designed a protocol that allows for comparison of distance dependent rebinding patterns. First, we modified monoclonal antibodies by adding a single heterobifunctional amino spacer with either a short or a long linker length. Second, the fluorescence dye AlexaFluor555 was bound to the functional group at one side of the linker while the antibody was bound to the other site by an active sulfhydryl group. In a Fluorescence Resonance Energy Transfer experiment we finally verified the different distances between the acceptor (AlexaFluor555) and the donor (Green Fluorescent Protein GFP) molecules by Fluorescence Lifetime Imaging Microscopy and showed that photounbinding is strongly dependent on the linker length. Thus, we can pinpoint ROS production as one of the main mechanisms of photounbinding as rebinding can hardly be observed if the local ROS production site is kept away from the antibody by the long linker.