Which probes can be used for SPT?
There are several elements to consider when choosing a probe for SPT. First, the probe needs to be photoactivatable, photoconvertible or photoswitchable to enable tracking of single molecules. Second, the probe and staining protocol must be compatible with living cells.
Traditionally, fusion proteins that are photoactivatable (PA) or photoconvertible (PC) are chosen for SPT. Because they are genetically encoded in the strain used, they require no staining with an external cell-permeable probe, thereby greatly simplifying the procedure and reducing non-specific signal to almost nothing. With recent genome editing technique, it is even possible to tag endogenous proteins, limiting artefacts related to protein overexpression. mEOS, PAmCHERRY, and DENDRA2 are examples of PA and PC fusion proteins that have been commonly used in SPT.
While it can be possible to use photoswitchable STORM dyes, it is not as common. First, the standard STORM staining protocol including photoswitchable probes (such as AF647, CF680 etc.) is immunofluorescence, which is incompatible with living cells. Second, most of these probes require the use of a photoswitching buffer and of high laser power, both of which are toxic for cells. However, a few photoswitchable probes and staining techniques happen to be compatible with living cells and, therefore, SPT. For example, the SNAP tag and Halo tag systems allow staining of target proteins in living cells, and several SNAP- and Halo- compatible fluorophores are photoswitchable, such as TMR, or JF dyes designed by the Lavis Lab in Janelia Farm. These staining procedures are usually quite straightforward and the imaging that follows fairly simple; however, the downside is that there can be a lot of non-specific staining, adding noise to the final data.