Fluorescent chemical dyes are relatively small, can have good photophysical properties and span the color spectrum, making them attractive alternatives to fluorescent proteins for labeling proteins. Researchers are actively developing tools for labeling proteins with dyes in living cells.

Improving tools for protein labeling. Credit: Marina Corral Spence/Nature Publishing Group

To be useful for most applications, methods for incorporating dyes into proteins must allow specific labeling of proteins. On this front, several tools already exist, such as SNAP and Halo tags, FlAsH and ReAsH, and hexahistidine tags. These tools involve genetic tagging of target proteins with a small protein or peptide that can specifically bind appropriate dyes. An alternative method involves the incorporation of non-natural amino acids into proteins during translation; here the non-natural amino acids either are fluorescent or can be made fluorescent via click chemistry.

Although these methods are gaining popularity, they suffer from issues such as limited utility in multiplexed imaging, low labeling efficiency and the number and quality of dyes that can cross the membrane of a living cell. Such dyes are an active area of research. Future work will undoubtedly improve labeling efficiency, which will enable enhanced quantitative imaging; improve available dyes, which could increase multiplexing and decrease the light dosages necessary for imaging; and reveal entirely new methods for protein tagging.

Specific protein labeling will also enhance super-resolution imaging in fixed and living cells. As resolving power approaches tens of nanometers, labeling issues come to the fore. For example, labeling with an antibody adds ∼10 nm to the structure of interest, and using a secondary antibody doubles this added size. As an alternative, researchers are developing nanobodies, which are smaller, single-chain antibodies for use in labeling, as well as using the alternative strategies described above.