Imaging is a critical component of clinical care, with applications that include screening, diagnosis, therapy-planning, and monitoring of treatment. The limits of detection of most common imaging modalities (e.g., X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), and computer tomography (CT)) prevent the visualization of deep-tissue structures at the cellular level with sufficient sensitivity and resolution. Fluorescence microscopy techniques enable high-resolution imaging, but they are not suited for diagnostics at the macroscopic scale. Optical imaging, which uses visible, ultraviolet, or infrared light, is a promising technique that is relatively safe and low-cost. It can be used with near infrared (NIR) probes, which penetrate through deep tissue. Imaging in the second near-infrared (NIR-II) window provides a 100-fold improvement in the signal-to-noise ratio, further improving the method. Combining NIR-II-emitting probes with hyperspectral imaging allows for in vivo imaging at depths of up to ~9 cm in biological tissues with single-cell resolution.