Exchangeable fluorophore labels in super-resolution fluorescence microscopy

We present fluorophore labels that transiently and repetitively bind to their targets as probes for various types of super-resolution fluorescence microscopy. Transient labels typically show a weak affinity to a target, and exchange constantly with the buffer that constitutes a reservoir with a large amount of intact probes, leading to repetitive binding events to the same target (we refer to these labels as “exchangeable labels”). This dynamic labeling approach is insensitive to common photobleaching and yields a constant fluorescence signal over time, which has been successfully exploited in SMLM, STED, single-particle tracking and super-resolution optical fluctuation imaging (SOFI). We discuss properties of suitable exchangeable labels and experimental parameters for optimal performance for the different super-resolution methods. In addition, we show how to combine different classes of exchangeable labels for high-quality multicolor super-resolution imaging.