Figure 1.

Comparison of permanent (high-affinity or covalent) and exchangeable (transient) labeling. (A) Permanent labels are constantly exposed to light irradiation and inevitably photobleached. Therefore, the fluorescent signal deteriorates, making prolonged imaging more complicated. In contrast, the continuous exchange of low-affinity labels with undamaged ones from cytosol or medium increases the apparent photostability of labeling. Red stars denote fluorescent labels, pink stars denote photobleached labels. (B) High-affinity labels already bound to target structures, due to their large size, can sterically interfere with the binding of other label molecules. Alternatively, frame-by-frame accumulation of low-affinity labels’ positions followed by frames merging increases effective labeling density. (C) Bulky labels, continuously bound to target structures, may affect the dynamics and functioning of the latter. In addition, some labels, such as fluorescently labeled taxol or fluorescent proteins could drastically disturb cell activity. However, in the case of low-affinity labeling, target molecules remain untagged most of the time and therefore their functioning is less hindered.