Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP⁺ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1⁺ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

中文翻译：

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体内脂滴和肝囊巨噬细胞的多色双光子显微成像


脂滴 (LD) 储存能量并提供脂肪酸和胆固醇。 LD 是慢性非酒精性脂肪肝 (NAFLD) 的一个标志。最近，研究重点关注肝巨噬细胞在 NAFLD 中的作用。绿色荧光蛋白（GFP）用于标记生物成像分析中的特征目标。 Cx3cr1-GFP小鼠广泛用于研究肝脏巨噬细胞，例如NAFLD模型。在这里，我们开发了一种双光子显微观察工具来研究体内 LD2 标记的 LD 与 GFP 标记的肝被膜巨噬细胞之间的相互作用。 LD2是一种用于LD的小分子双光子激发荧光探针，在880 nm激发下呈现深红色（700 nm）荧光，具有较高的细胞染色能力和光稳定性以及较低的细胞毒性。该探针可以通过双光子显微镜（TPM）清晰地观察LD，并能够在Cx3cr1-GFP小鼠肝被膜中同时对GFP ⁺ 肝被膜巨噬细胞（LCM）进行体内成像。在NAFLD小鼠模型中，Cx3cr1 ⁺ LCM和LD随着脂肪肝疾病的进展而增加，通过活体3D TPM图像观察LCM的时空变化。 LD2 将有助于研究肝巨噬细胞和 LD 的相互作用和免疫作用，以更好地了解 NAFLD。