High‐resolution fluorescence imaging, combined with the latest labeling techniques available today have yet been able to provide the necessary spatiotemporal resolution to record all cellular processes in live cells. Substituting the bulky fluorescent protein tags (such as GFP) currently used in live‐cell applications with much smaller fluorescent dyes that possess superior photophysical characteristics will markedly improve these advanced imaging techniques. Genetic code expansion and bioorthogonal labeling offer, for the first time, a non‐invasive way to specifically and directly attach such fluorescent dyes to proteins in live cells. Here we employ this strategy to directly tag α‐tubulin in live mammalian cells. By screening different conditions we have optimized the system for quantitative high‐resolution recording of microtubules in live cells. We will present data demonstrating the feasibility and efficiency of the approach and will discuss the advantages and limitations of using genetic code expansion for quantitative high‐resolution microscopy.
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