In vivo label-free confocal imaging of the deep mouse brain with long-wavelength illumination

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1-micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.