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.

In vivo imaging of hormone receptors provides the opportunity to visualize target tissues under hormonal control in live animals. Detecting longer-wavelength photons in the second near-infrared window (NIR-II, 1000–1700 nm) region affords reduced photon scattering in tissues accompanied by lower autofluorescence, leading to higher spatial resolution at up to centimeter tissue penetration depths. Here, we report the conjugation of a small molecular NIR-II fluorophore CH1055 to a follicle stimulating hormone (FSH-CH) for imaging ovaries and testes in live mice. After exposure to FSH-CH, specific NIR-II signals were found in cultured ovarian granulosa cells containing FSH receptors. Injection of FSH-CH allowed live imaging of ovarian follicles and testicular seminiferous tubules in female and male adult mice, respectively. Using prepubertal mice, NIR-II signals were detected in ovaries containing only preantral follicles. Resolving earlier controversies regarding the expression of FSH receptors in cultured osteoclasts, we detected for the first time specific FSH receptor signals in bones in vivo. The present imaging of FSH receptors in live animals using a ligand-conjugated NIR-II fluorophore with low cell toxicity and rapid clearance allows the development of non-invasive molecular imaging of diverse hormonal target cells in vivo.