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Recent developments of fluorescent probes beyond approved indocyanine green (ICG) – itself increasingly spreading into new imaging applications like lymphatic mapping, arthritis imaging and tumor surgery – exploit various photophysical and biochemical mechanisms to monitor molecular events with higher specificity and accuracy. Emphasizing nanoparticulate formulations, targeted conjugates, activatable probes, probes with a sensor function and multimodality probes, this review discusses advantages and limitations of each type of probe, thereby critically assessing the desired translation into the clinic.