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.

Near-Infrared II Semiconducting Polymer Dots: Chain Packing Modulation and High-Contrast Vascular Imaging in Deep Tissues.

Fluorescence imaging in the second near-infrared (NIR-II) window has attracted considerable interest in investigations of vascular structure and angiogenesis, providing valuable information for the precise diagnosis of early stage diseases. However, it remains challenging to image small blood vessels in deep tissues because of the strong photon scattering and low fluorescence brightness of the fluorophores. Here, we describe our combined efforts in both fluorescent probe design and image algorithm development for high-contrast vascular imaging in deep turbid tissues such as mouse and rat brains with intact skull. First, we use a polymer blending strategy to modulate the chain packing behavior of the large, rigid, NIR-II semiconducting polymers to produce compact and bright polymer dots (Pdots), a prerequisite for in vivo fluorescence imaging of small blood vessels. We further developed a robust Hessian matrix method to enhance the image contrast of vascular structures, particularly the small and weakly fluorescent vessels. The enhanced vascular images obtained in whole-body mouse imaging exhibit more than an order of magnitude improvement in the signal-to-background ratio (SBR) as compared to the original images. Taking advantage of the bright Pdots and Hessian matrix method, we finally performed through-skull NIR-II fluorescence imaging and obtained a high-contrast cerebral vasculature in both mouse and rat models bearing brain tumors. This study in Pdot probe development and imaging algorithm enhancement provides a promising approach for NIR-II fluorescence vascular imaging of deep turbid tissues.