Fluorogenic probes for live-cell imaging

The development of novel fluorogenic probes and their use in live-cell imaging lead to a plethora of discoveries in biological research. Herein I report on novel red fluorogenic probes for live-cell super-resolution microscopy of various cellular organelles. First, I describe a new silicon rhodamine fluorophore termed SiR595. As most other SiR derivatives, SiR595 exists in an equilibrium between two chemical forms, a fluorescent zwitterion and a non-fluorescent spirolactone. Compared to regular SiR, the equilibrium of SiR595 is shifted towards the non-fluorescent, uncharged spirolactone. When coupled to appropriate targeting ligands, the resulting SiR595-based probes show high cell permeability. Furthermore, SiR595-based probes are fluorogenic as binding to their targets shifts the equilibrium towards the fluorescent zwitterion. I took advantage of these properties to develop novel red fluorogenic probes for live-cell imaging of Halo-tagged proteins, microtubules, F-actin and DNA. I was furthermore able to show that all of these probes are compatible with live-cell STED nanoscopy. Furthermore, I developed fluorogenic probes for live cell imaging of centrosomes. The centrosome is the principal microtubule-organizing center in animal cells. We targeted polo-like kinase 4 (Plk4), a serine/threonine-protein kinase, which localizes to centrioles throughout the cell cycle. To label Plk4 in live cells, we used Centrinone, a potent inhibitor of Plk4. Conjugating Centrinone to SiR595, I obtained and characterized several fluorogenic SiR595-Centrinone probes. SiR595-Centrinone probe labels overexpressed GFP-Plk4(K41M) during live-cell imaging and could be used to characterize the spatial distribution of GFP-Plk4(K41M) using live-cell STED nanoscopy. While the here introduced probes for Plk4 are not bright and fluorogenic enough to detect endogenous Plk4, the work provides the foundation for the development of such probes.