细胞器靶向的细胞微环境双光子荧光探针的设计合成及其在生物成像中的应用研究

Real-time monitoring of intracellular micro-environments in organelles has an important significance to study the physiological and pathological processes in cells. Fluorescent probe has become an effective approach to monitor intracellular micro-environments in organelles. However, most of fluorescent probes for intracellular micro-environments in organelles are based on one-photon fluorophores, which are excited by the excitation source in a rather short excitation wavelength that limits their biological applications because of the shallow penetration depth, strong photobleaching and high autofluorescence in vivo. In this project, due to the advantages of two-photon fluorescent probes such as deeper penetration depth, lower autofluorescence and reduced photodamage and photobleaching in vivo, we attempt to combine it with organelle-targetable fluorescent probes and develop a series of novel organelle-targetable (Golgi apparatus, endoplasmic reticulum, mitochondria and lysosomes) two-photon fluorescent probes for bio-related intracellular micro-environments (pH and viscosity), in order to expand the target organelles for the study on intracellular micro-environments and achieve real-time monitoring of intracellular micro-environments in cells, tissues and animals, which will provide a valuable theoretical basis and technical support for the study on intracellular micro-environments-related physiological and pathological processes and the early diagnosis of intracellular micro-environments-related diseases.

实时监测细胞器细胞微环境，对研究细胞的生理与病理进程具有重要意义。荧光探针已经成为细胞器细胞微环境监测的有效方法。然而，目前细胞器靶向的细胞微环境荧光探针大多数是基于单光子荧光基团设计的，通常具有激发光波长较短导致的生物样品穿透深度小、光漂白严重、易受到生物体内背景荧光干扰等缺点，在生物成像方面存在一定的局限性。基于双光子荧光探针的生物样品穿透深度大、生物成像背景荧光低、光损伤与光漂白作用小等优点，本项目将双光子荧光探针与细胞器靶向荧光探针相结合，选取生物相关的细胞微环境（pH、粘度）作为研究对象，开发一系列新型的细胞器（高尔基体、内质网、线粒体、溶酶体）靶向的细胞微环境双光子荧光探针，拓展细胞器细胞微环境研究的靶向范围，实现细胞、组织、活体中细胞微环境的动态监测，为细胞器细胞微环境相关的细胞生理与病理进程的研究以及疾病的早期诊断提供有价值的理论依据和技术支持。

荧光探针已经成为细胞器内生命活动研究或生物信息检测的有效方法，对更准确的研究细胞的生理与病理进程具有重要意义。本项目针对目前细胞器靶向的荧光探针通常具有的激发光波长或者发射光波长较短导致的生物样品穿透深度小、光漂白严重、生物体内背景荧光干扰严重等缺点，利用双光子荧光探针以及近红外荧光探针的生物样品穿透深度大、生物成像背景荧光低、光损伤与光漂白作用小等优点，构建了一系列新型的溶酶体靶向的次氯酸与pH值近红外荧光探针、线粒体靶向的次氯酸双光子荧光探针。项目组研究了这一系列探针在体外、细胞、组织或小鼠活体中对次氯酸或 pH 的检测性能与成像效果，并将其中某些探针用于活体中炎症疾病的实时监测，取得了良好的效果。上述探针具有优异细胞器靶向性能、良好的光稳定性、近红外长波长发射或双光子激发等特点，可获得较大的成像深度以及较小的背景荧光干扰，在组织、活体生物成像中具有一定的优势，为溶酶体与线粒体内相关的细胞生理与病理进程的研究以及疾病的早期诊断提供有价值的理论依据和技术支持。