Owing to reduced light scattering and tissue autofluorescence, in vivo fluorescence imaging in the 1,000–3,000-nm near-infrared II (NIR-II) spectral range can afford non-invasive imaging at depths of millimetres within biological tissue. Infrared fluorescent probes labelled with antibodies or other targeting ligands also enable NIR-II molecular imaging at the single-cell level. Here we present recent developments in the design of fluorophores and probes emitting in the NIR-II window based on organic synthesis and nanoscience approaches. We also review advances in NIR-II wide-field and microscopy imaging modalities, with a focus on preclinical imaging and promising clinical translation case studies. Finally, we outline current issues and challenges for the wider adoption of NIR-II imaging in biomedical research and clinical imaging.