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 the design and synthesis of a 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 improves quality and conditions for regular and single-molecule localization microscopy on live-cell samples.
Keywords: bioorthogonal chemistry; click chemistry; fluorescent probes; super-resolution imaging; tetrazines.
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.