Polo-like kinase (Plk4) is a serine/threonine-proteinkinase thatis essential for biogenesis of the centriole organelle and is enrichedat centrioles. Herein, we introduce Cen-TCO, a chemical probe basedon the Plk4 inhibitor centrinone, to image Plk4 and centrioles inlive or fixed cultured human cells. Specifically, we established abio-orthogonal two-step labeling system that enables the Cen-TCO-mediatedimagingof Plk4 by STED super-resolution microscopy. Such direct labelingof Plk4 results in an increased resolution in STED imaging comparedwith using anti-Plk4 antibodies, underlining the importance of directlabeling strategies for super-resolution microscopy. We anticipatethat Cen-TCO will become an important tool for investigating the biologyof Plk4 and of centrioles.