Department of Chemistry, Stanford University, Stanford, CA 94305.
Bio-X, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2021 Feb 9;118(6). doi: 10.1073/pnas.2023888118.
Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.
具有深层组织穿透深度和高时空分辨率的非侵入性光学成像是在活体哺乳动物中纵向研究单细胞水平生物学的重要手段，但由于光散射而具有挑战性。在这里，我们开发了近红外二区（NIR-II）（1000 至 1700nm）结构照明光片显微镜（NIR-II SIM），具有超长的激发和发射波长，分别高达约 1540nm 和 1700nm，抑制了光散射，从而能够对具有深层轴向穿透深度的组织进行大体积三维（3D）成像。将结构照明集成到 NIR-II 光片显微镜中，通过大约两倍的降低背景和提高空间分辨率。在体内，我们进行了非侵入性的斜角 NIR-II SIM，对小鼠 CT26 肿瘤微环境进行了 3D 容积多重分子成像，纵向地以单细胞水平测绘出对免疫疗法的反应，包括细胞因子磷酸鸟嘌呤（CpG）、Toll 样受体 9（TLR-9）激动剂与 OX40 抗体联合治疗的 CD4、CD8 和 OX40。NIR-II SIM 为活体哺乳动物中免疫细胞的非侵入性容积分子成像提供了另一种工具。
