Sensorless adaptive optics in the second near-infrared window for deep vascular imaging in vivo

We have experimentally validated the use of sensorless adaptive optics (AO) to enhance laser scanning confocal microscopy in the second near-infrared (NIR II) spectral range, termed as AO-NIR II confocal microscopy. This approach harnesses a NIR II fluorophore, excited by an 808 nm wavelength and emitting beyond 1000 nm, to visualize intricate structures in deep brain tissues with the intact skull. By leveraging the reduced scattering and aberrations in the NIR II spectrum, we successfully captured a three-dimensional (3D) vascular structure map extending 310 µm beneath the skull. AO typically boosts the fluorescence signal by approximately 2–3 times, leading to a superior contrast and diminished smearing effects. Consequently, small blood vessels at various depths can be clearly visualized, which might otherwise remain undetectable without AO corrections.