Quantitative comparison of methodologies for translation site imaging in living cells

Single-molecule imaging of translation sites in living cells has enabled the dynamics of protein synthesis to be investigated with high spatial and temporal resolution. These methodologies utilize the interaction between a multimerized epitope tag and its cognate fluorescent single-chain variable fragment or nanobody to detect nascent polypeptides as they emerge from the ribosome. Here, we present a systematic comparison of current methodologies and determine that the ALFA-tag reduces perturbations of mRNA expression and increases the fluorescent signal of translation sites.