Huang Lin, Zhou Xin, Liu Qihao, MacAulay Calum E, Tang Shuo
Department of Electrical and Computer Engineering, University of British Columbia, Vancouver, V6 T 1Z4, Canada.
Department of Integrative Oncology, BC Cancer Research Center, Vancouver, V5Z 1L3, Canada.
Biomed Opt Express. 2020 Jan 6;11(2):624-635. doi: 10.1364/BOE.381473. eCollection 2020 Feb 1.
A multimodal multiphoton microscopy (MPM) is developed to acquire both two-photon microscopy (2PM) and three-photon microscopy (3PM) signals. A dual-wavelength Er-doped fiber laser is used as the light source, which provides the fundamental pulse at 1580 nm to excite third harmonic generation (THG) and the frequency-doubled pulse at 790 nm to excite intrinsic two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Due to their different contrast mechanisms, the TPEF, SHG, and THG images can acquire complementary information about tissues, including cells, collagen fibers, lipids, and interfaces, all label-free. The compact MPM imaging probe is developed using miniature objective lens and a micro-electro-mechanical scanner. Furthermore, the femtosecond laser pulses are delivered by a single mode fiber and the signals are collected by a multimode fiber, which makes the miniaturized MPM directly fiber-coupled, compact, and portable. Design considerations on using the dual excitation wavelengths are discussed. Multimodal and label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples. The miniaturized multimodal MPM is shown to have great potential for label-free imaging of thick and live tissues.
一种多模态多光子显微镜（MPM）被开发用于获取双光子显微镜（2PM）和三光子显微镜（3PM）信号。使用双波长掺铒光纤激光器作为光源，它提供1580 nm的基频脉冲以激发三次谐波产生（THG），以及790 nm的倍频脉冲以激发固有双光子激发荧光（TPEF）和二次谐波产生（SHG）。由于它们不同的对比度机制，TPEF、SHG和THG图像可以获取关于组织的互补信息，包括细胞、胶原纤维、脂质和界面，所有这些都是无标记的。紧凑的MPM成像探头是使用微型物镜和微机电扫描仪开发的。此外，飞秒激光脉冲由单模光纤传输，信号由多模光纤收集，这使得小型化的MPM直接光纤耦合、紧凑且便携。讨论了使用双激发波长的设计考虑因素。在生物样品上展示了通过TPEF、SHG和THG进行的多模态无标记成像。小型化多模态MPM显示出对厚组织和活组织进行无标记成像的巨大潜力。
