Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.

Polo-like kinase (Plk4) is a serine/threonineprotein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy. Such direct labeling of Plk4 results in an increased resolution in STED imaging compared with using anti-Plk4 antibodies, underlining the importance of direct labeling strategies for super-resolution microscopy. We anticipate that Cen-TCO will become an important tool for investigating the biology of Plk4 and of centrioles.

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