Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Abstract

Recent developments in fluorescence microscopy call for novel small molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy, is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present a rational design and synthetic route for a novel type of small molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal “click” chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe overall improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples. The fluorescent probe is tailor‐made for advanced live‐cell microscopy technologies with molecular features that go far beyond the usual requirements of spectral and photophysical properties for small‐molecule labels.