Photoactivation of silicon rhodamines via a light-induced protonation
Photoactivatable fluorophores are important tools to investigate dynamic processes in cells. With the advent of super-resolution microscopy techniques based on single-molecule localization, these fluorophores have found even more applications. However, to make these techniques available for routine live-cell imaging, brighter, cell-permeable fluorophores are required. In our research, we develop new photoactivatable synthetic fluorophores based on the silicon rhodamine scaffold. This class of fluorophores has ideal properties for live-cell imaging: excitation and emission maxima in the far-red, high extinction coefficient, high quantum yield, photostability and cell-permeability. Instead of using bulky photolabile groups, we made use of photochemical concepts that require smaller structural modifications and generated a far-red photoactivatable fluorophore. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy. We showed that this fluorophore can be used not only in fixed-cells, but also for following the fast dynamics of mitochondria by single-molecule localization microscopy in live-cells. Most excitingly, we could distinguish the unlabeled interior of the mitochondria from their labeled outer membrane in several cases, showcasing the power of this probe in combination with super-resolution microscopy.