Fluorescence microscopy has become an indispensable tool to investigate the dynamics of macromolecules directly in living cells on length scales ranging from the nanoscale via super-resolution microscopy (SRM) to the macroscale by light sheet technology. Advances in these microscopy techniques and the desire to perform experiments with high spatial and temporal resolution in living cells increase the requirements imposed on the fluorophores used. Tailor-made synthetic small-molecule fluorophores in combination with innovative labeling strategies help to overcome these challenges and continue to push the boundaries in live-cell microscopy. This Review discusses important advances in improving the performance of synthetic fluorophores for live-cell applications and how synergistic effects can be produced by using clever labeling strategies. We detail how synthetic fluorophores advance different microscopy modalities including live-cell SRM and showcase how they can be implemented into bi

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