Microglia are an essential population of resident immune cells in the central nervous system (CNS) and retina. These microscopic cells possess sub-cellular processes that make them challenging to image due to limited resolution and contrast. The baseline behavior of microglial processes in the living retina has been poorly characterized, and yet are essential to understanding how these cells respond under conditions of health, development, stress and disease. Here we use in vivo adaptive optics scanning light ophthalmoscopy combined with time-lapse imaging and quantification of process motility, to reveal the detailed behavior of microglial cells in a population of healthy mice. We find microglial processes to be dynamic at all branch-levels, from primary to end-protrusions. Cell-processes remodel at average speeds of 0.6 ± 0.4 µm/min with growth and deletion bursts of 0–7.6 µm/min. Longitudinal imaging in the same mice showed cell-somas to remain stable over seconds to minutes, but show migration over days to months. In addition to characterizing in vivo process motility and Sholl analysis using a microglial reporter mouse, we also demonstrate that microglia can be imaged without fluorescent labels at all. Phase-contrast imaging using safe levels of near-infrared light successfully imaged microglia soma and process remodeling with micron-level detail noninvasively, confirmed by simultaneous imaging of fluorescent microglial cells in transgenic mice. This label-free approach provides a new opportunity to investigate CNS immune system noninvasively without requiring transgenic or antibody labeling which could have off-target effects of changing normal microglial behavior. Additionally, CNS microglia study can now be conducted without the need for cranial window surgery which have the potential to change their behavior due to local or systemic inflammation.

中文翻译：

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活体视网膜中小胶质细胞个体过程的动态成像

小胶质细胞是中枢神经系统 (CNS) 和视网膜中必不可少的常驻免疫细胞群。这些微观细胞具有亚细胞过程，由于分辨率和对比度有限，因此难以成像。活体视网膜中小胶质细胞过程的基线行为没有得到很好的表征，但对于了解这些细胞在健康、发育、压力和疾病条件下如何反应至关重要。这里我们使用vivo自适应光学扫描光学检眼镜结合延时成像和过程运动的量化，揭示健康小鼠群体中小胶质细胞的详细行为。我们发现小胶质细胞过程在所有分支级别都是动态的，从初级到末端突起。细胞过程以 0.6 ± 0.4 µm/min 的平均速度重塑，生长和缺失的爆发速度为 0–7.6 µm/min。相同小鼠的纵向成像显示细胞体在几秒钟到几分钟内保持稳定，但在几天到几个月内显示出迁移。除了在体内表征使用小胶质细胞报告鼠标处理运动性和 Sholl 分析，我们还证明了小胶质细胞可以在没有荧光标记的情况下成像。使用安全水平的近红外光的相衬成像成功地对小胶质细胞体进行了成像，并以非侵入性的方式利用微米级细节进行了过程重塑，这通过转基因小鼠中荧光小胶质细胞的同时成像得到证实。这种无标记方法为无创研究 CNS 免疫系统提供了新的机会，无需转基因或抗体标记，这可能具有改变正常小胶质细胞行为的脱靶效应。此外，CNS 小胶质细胞研究现在可以在不需要颅窗手术的情况下进行，颅窗手术有可能因局部或全身炎症而改变其行为。