Significance Fluorescence-based optical imaging is an important tool allowing researchers and clinicians to molecularly probe wide-ranging biological structures and processes. To break through the traditional molecular imaging window spanning from the visible to the near-infrared (NIR)-I (400–900 nm) region for imaging multiplicity, newly designed and ultrapurified fluorescent probe-antibody conjugates with fluorescence emissions in the NIR-II region (1,000–1,700 nm) have been developed. These NIR-II probes can reduce background autofluorescence for deep-tissue molecular imaging in a 3D imaging mode. These probes open up more and deeper nonoverlapping molecular imaging channels for complex biological systems.