Fluorescence microscopy is a powerful enabling tool for biological discovery, albeit its effective penetration depth and resolving capacity are limited due to intense light scattering in living tissues. The recently introduced short-wave infrared cameras and contrast agents featuring fluorescence emission in the second near-infrared (NIR-II) window have extended the achievable penetration to about 2 mm. However, the effective spatial resolution progressively deteriorates with depth due to photon diffusion. Here we introduce diffuse optical localization imaging (DOLI) to enable super-resolution deep-tissue fluorescence microscopy beyond the limits imposed by light diffusion. The method is based on localization of flowing microdroplets encapsulating lead sulfide (PbS)-based quantum dots in a sequence of epi-fluorescence images acquired in the NIR-II spectral window. Experiments performed in tissue mimicking phantoms indicate that high-resolution detection of fluorescent particles can be preserved over 4 mm depth range, while in vivo microangiography of murine cerebral vasculature can be accomplished through intact scalp and skull. The method further enables retrieving depth information from planar fluorescence image recordings by exploiting the localized spot size. DOLI operates in a resolution-depth regime previously inaccessible with optical methods, thus massively enhancing the applicability of fluorescence-based imaging techniques.

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用于 NIR-II 窗口中无创深部脑微血管造影的漫射光学定位成像

荧光显微镜是一种强大的生物发现工具，尽管由于活组织中强烈的光散射，它的有效穿透深度和分辨能力受到限制。最近推出的短波红外相机和在第二个近红外 (NIR-II) 窗口中具有荧光发射的造影剂已将可实现的穿透扩展到约 2 毫米。然而，由于光子扩散，有效空间分辨率随着深度而逐渐恶化。在这里，我们引入了漫反射光学定位成像 (DOLI)，以实现超越光漫射限制的超分辨率深组织荧光显微镜。该方法基于在 NIR-II 光谱窗口中获得的落射荧光图像序列中封装硫化铅 (PbS) 基量子点的流动微滴的定位。在组织模拟体模中进行的实验表明，可以在 4 毫米深度范围内保持对荧光粒子的高分辨率检测，而小鼠脑血管系统的体内微血管造影可以通过完整的头皮和头骨完成。该方法还能够通过利用局部光斑尺寸从平面荧光图像记录中检索深度信息。DOLI 在以前光学方法无法达到的分辨率深度范围内运行，从而大大提高了基于荧光的成像技术的适用性。