Li Wei, Li Xiaoyu, Zhang Chenshuang, Wang He, Zhu Yinru, Wang Yunyun, Yan Wei, Liu Liwei, Qu Junle
College of Physics and Optoelectronic Engineering, Shenzhen Key Laboratory of Photonics and Biophotonics, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen, Guangdong 518060, China.
Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Anal Chem. 2024 Jul 16. doi: 10.1021/acs.analchem.4c01303.
Two-photon fluorescence lifetime microscopy (TP-FLIM) is a powerful quantitative imaging technique that characterizes and analyzes the structure and function of biological samples through a combination of intensity and lifetime imaging. Because TP-FLIM is independent of the fluorescence signal intensity and the fluorophore concentration, it is widely used in high-throughput, high-content drug screening and clinical diagnostics. Second harmonic generation (SHG) imaging technology has the advantages of high spatial resolution and imaging depth inherent to nonlinear optical imaging. Second harmonics often appear in noncentrosymmetric structures. Collagen tissue in biological organisms is a good example of these structures, showing strong harmonic effects. Therefore, SHG has been widely used for imaging of specific tissue structure imaging. TP-FLIM technology is highly sensitive for quantitatively detecting changes in microenvironments. The objective of this study is to examine pathological pulmonary fibrosis slices using a combined approach of TP-FLIM and SHG technology. The fluorescence lifetime data of pulmonary collagen fibers are analyzed by using phasor plot analysis methods, and normal collagen fibers and fibrotic collagen fibers are distinguished by calculating the aspect ratio from the SHG images formed by the collagen fibers. Our study provides a new method for a deeper understanding of the pathological mechanisms and clinical diagnosis of pulmonary fibrosis and other collagen fiber-related disorders.
双光子荧光寿命显微镜（TP - FLIM）是一种强大的定量成像技术，它通过强度成像和寿命成像相结合的方式来表征和分析生物样品的结构与功能。由于TP - FLIM与荧光信号强度和荧光团浓度无关，因此它被广泛应用于高通量、高内涵药物筛选以及临床诊断。二次谐波产生（SHG）成像技术具有非线性光学成像固有的高空间分辨率和成像深度的优点。二次谐波通常出现在非中心对称结构中。生物体内的胶原组织就是这些结构的一个很好的例子，表现出强烈的谐波效应。因此，SHG已被广泛用于特定组织结构成像。TP - FLIM技术在定量检测微环境变化方面具有高度敏感性。本研究的目的是使用TP - FLIM和SHG技术的联合方法检查病理性肺纤维化切片。通过使用相量图分析方法分析肺胶原纤维的荧光寿命数据，并通过计算由胶原纤维形成的SHG图像的纵横比来区分正常胶原纤维和纤维化胶原纤维。我们的研究为更深入了解肺纤维化及其他胶原纤维相关疾病的病理机制和临床诊断提供了一种新方法。
