多光子成像技术的生物医学应用新进展

李少强; 耿俊娴; 李艳萍; 刘雄波; 彭晓; 屈军乐; 刘丽炜; 胡睿

doi:10.7498/aps.69.20201039

摘要

多光子成像技术由于具有低侵入性、强穿透力、高空间分辨率等优点, 自问世以来便成为生物医学研究的有力工具, 在癌症病理、神经疾病及脑功能成像等方面取得了一系列较好的研究成果. 目前, 应用较为广泛的多光子成像技术是双光子激发荧光显微成像技术, 其在生物医学应用中具有较大的发展潜力. 本文详细阐述了多光子成像技术在多色成像、功能成像及成像深度等方面的生物医学应用新进展, 包括多色双光子激发荧光显微成像、双光子激发荧光寿命显微成像、双光子光纤内窥成像和三光子显微成像技术, 并简要介绍这几种多光子成像技术的原理与特性, 最后展望其未来发展前景.

关键词:

Abstract

In contrast to single photon excitation fluorescence imaging, laser scanning confocal imaging, and wide-field imaging, the multi-photon imaging has advantages of minimal invasion and deeper penetration by using near-infrared (NIR) laser source. Moreover, it can carry out three-dimensional high-spatial-resolution imaging of biological tissues due to its natural optical tomography capability. Since its advent, multi-photon imaging has become a powerful tool in biomedicine and achieved a series of significant discoveries in cancer pathology, neurological diseases and brain functional imaging. In the past decade, as a major form of multi-photon imaging techonoogy, two-photon excited fluorescence microscopy imaging has a great potential in biomedical applications. In order to satisfy the practical biomedical applications, multi-photon imaging technologies have made significant breakthroughs in improving the deficiencies of traditional 2PEF in multi-color imaging, functional imaging, live imaging and imaging depth, such as multicolor two-photon excitation fluorescence microscopy, two-photon fluorescence lifetime imaging microscopy, two-photon fiber endoscopic imaging, and three-photon microscopy imaging technology. For example, multicolor two-photon excitation fluorescence microscopy is demonstrated to achieve simultaneous imaging of multiple fluorophores with multiple wavelenth excitation lasers or continuous spectrum. In addition, the two-photon fluorescence lifetime microscopic imaging provides a method to achieve high-resolution three-dimensional imaging of biological tissue with multi-dimensional information including fluorescence intensity and lifetime. In addition, two-photon optical fiber endoscopic imaging with small system size and mimal invasion is developed and used to image the tissue inside the deep organ. Finally, two-photon excitation fluorescence microscopy technique still has relatively strong scattering for brain functional imaging in vivo. Therefore, the imaging depth is limited by the signal-to-background ratio. Three-photon microscopic imaging technique can achieve higher imaging depth and a desired signal-to-noise ratio by extending the wavelength from 1600 nm to 1820 nm because the attenuation of the excitation light in this wavelenth range is much smaller. In this article, we briefly introduce the principles and applications of these multi-photon imaging technologies, and finally provide our view for their future development.

Keywords:

作者及机构信息

深圳大学物理与光电工程学院, 教育部/广东省光电子器件与系统重点实验室, 深圳　518060

通信作者: 胡睿, rhu@szu.edu.cn

基金项目: 国家科技部重点研发项目(批准号: 2017YFA0700402)、国家自然科学基金(批准号: 61525503, 61722508, 61620106016, 61835009, 61935012, 61961136005)和深圳市自由探索项目(批准号: JCYJ20180305124902165)资助的课题

Authors and contacts

College of Physics and Optoelectronic Engineering, Shenzhen University, Ministry of Education/ Guangdong Key Laboratory of Optoelectronic Devices and Systems, Shenzhen 518060, China

Corresponding author: Hu Rui, rhu@szu.edu.cn

Funds: Project supported by the National Key Research and Development Program of China (Grant No. 2017YFA0700402), the National Natural Science Foundation of China (Grant Nos. 61525503, 61722508, 61620106016, 61835009, 61935012, 61961136005), and the Shenzhen Free Exploration Project, China (Grant No. JCYJ20180305124902165)

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施引文献

图 1 2PEF、3PEF过程能级图. 2PEF和3PEF都是非直接激发辐射过程, 存在非辐射能量转移. 图中$ {\rm{\nu }}_{\rm{p}} $为吸收光子频率, $ {\rm{\nu }}_{\rm{f}} $为发射荧光频率, $ {\rm{\nu }}_{\rm{NR}} $为非辐射能量转移.

Fig. 1. Energy level diagram of 2PEF and 3PEF process. Both 2PEF and 3PEF are indirect excitation radiation processes, and there is non-radiative energy transfer. In the figure, $ {\rm{\nu }}_{\rm{p}} $ is the frequency of absorbed photons, $ {\rm{\nu }}_{\rm{f}} $ is the frequency of emitted fluorescence, and $ {\rm{\nu }}_{\rm{NR}} $ is the non-radiative energy transfer.

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图 2 激光扫描多色双光子激发荧光显微镜系统示意图. 图中各部分为: Femtosecond Laser, 飞秒激光器; Optical isolator, 光隔离器; Mirror, 反射镜; HWP (half-wave plate), 半波片; Lens, 透镜; PCF (photonic crystal fiber), 光子晶体光纤; FL (fiber launcher), 光纤耦合器; Filter, 滤光片; Scanners, 扫描振镜; DM (dichroic mirror), 二向色镜; PMT (photomultiplier tube), 光电倍增管探测器; Monochromator, 单色仪; Obj (objective), 物镜

Fig. 2. Schematic diagram of laser scanning multicolor two-photon fluorescence microscope system. The abbreviations in the figure are as follows: HWP, half-wave plate; PCF, photonic crystal fiber; FL, fiber launcher; DM, dichroic mirror; PMT, photomultiplier tube; Obj, objective.

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图 3 NLS-LSS-mKate1标记细胞核(红色)和GalT-ECFP标记高尔基(蓝色)的肿瘤细胞的多色双光子激发荧光显微成像^[16]. 比例尺: 10 µm

Fig. 3. Multicolor two-photon excited fluorescence microimaging of tumor cells with NLS-LSS-mKate1 labeled nucleus (red) and GalT-ECFP labeled Golgi (blue)^[16]. Scale bar: 10 µm.

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图 4 小鼠皮层组织的连续三维多色成像^[23]

Fig. 4. Continuous three-dimensional multicolor imaging of mouse cortical tissue^[23].

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图 5 常见的荧光寿命测量方法及双光子TCSPC-FLIM成像系统示意图　(a) 频域法; (b) TCSPC法; (c) 基于TCSPC的双光子FLIM成像系统示意图. 图中各部分为: Femtosecond Laser, 飞秒激光器; BS (beam splitter), 分光镜; Scan Lens, 扫描镜; Tube Lens, 镜筒透镜; DM (dichroic mirror), 二向色镜; Obj (objective), 物镜; Filter, 滤光片; PMT (photomultiplier tube), 光电倍增管探测器; Reference Beam, 参考光; PD (photodiode), 光电二极管; TCSPC, 时间相关单光子计数法

Fig. 5. Schematic diagram of common fluorescence lifetime measurement methods and imaging systems: (a) Frequency domain method; (b) TCSPC method; (c) schematic diagram of a two-photon FLIM imaging system based on TCSPC. The abbreviations in the figure are as follows: BS, beam splitter; DM, dichroic mirror; Obj, objective; PMT, photomultiplier tube; PD, photodiode; TCSPC, time-correlated single photon counting.

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图 6 利用双光子FLIM技术揭示肝脏切片上的癌症转移^[33]

Fig. 6. Using two-photon FLIM technology to reveal cancer metastasis on liver slices^[33].

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图 7 小鼠视网膜毛细血管成像^[43]　(a) 双光子荧光强度图像; (b) 图7(a)中的局部血管; (c) 图7(b)对应的荧光寿命图像. 比例尺: 25 µm

Fig. 7. Imaging of mouse retinal capillaries^[43]: (a) Two-photon fluorescence intensity image; (b) local blood vessel in Fig.7 (a); (c) fluorescence lifetime image corresponding to Fig.7 (b). Scale bar: 25 µm.

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图 8 双光子光纤内窥系统示意图. 图中各部分为: Femtosecond Laser, 飞秒激光器; FL (fiber launcher), 光纤耦合器; PBF (photonic band-gap fiber), 光子带隙光纤; Mirror, 反射镜; DM (dichroic mirror), 二向色镜; Lens, 透镜; Filter, 滤光片; PMT (Photomultiplier tube), 光电倍增管探测器; DAQ (data acquisition), 数据采集; DCF (double-clad fiber), 双包层光纤; Endomicroscope Probe, 内窥镜探头

Fig. 8. Schematic diagram of a two-photon fiber endoscopic system. The abbreviations in the figure are as follows: FL, fiber launcher; PBF, photonic band gap light; DM, dichroic mirror; PMT, Photomultiplier tube; DAQ, data acquisition; DCF, double-clad fiber.

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图 9 用于双光子内窥镜的空气-二氧化硅DC-PCF设计及系统成像图^[55]　(a) 光纤纤芯示意图, 二氧化硅部分为灰色, 空气部分为黑色; (b) 双包层光纤纤芯截面示意图; (c) DC-PCF具有灵活性; (d)−(k) 组织样本的无标记双光子光纤内窥成像, 红色为TPEF信号, 绿色为SHG信号. (d), (e) 大鼠尾肌腱; (f) 鼠耳. D: 真皮; E: 表皮; IC: 内部软骨; (g)健康人类肺部样品(肺泡区域); (h) 小鼠动脉; (i)−(k)健康人类肺部样品里3个位置的细胞外基质. 比例尺: 50 µm

Fig. 9. Design and system imaging diagram of an Air-silica DC-PCF for a two-photon endoscope^[55]: (a) Schematic diagram of the optical fiber core, the silica part is gray, and the air part is black; (b) the cross-sectional schematic view of the double-clad fiber core; (c) DC-PCF is flexible; (d)−(k) Unlabeled two-photon fiber endoscopy imaging of tissue samples, red is TPEF signal, green is SHG signal; (d), (e) rat tail tendon; (f) mouse ear. D: dermis; E: epidermis; IC: internal cartilage; (g) healthy human lung samples (alveolar regions); (h) mouse arteries; (i)−(k) extracellular matrix at 3 locations in healthy human lung samples. Scale bar: 50 µm.

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图 10 激光扫描三光子显微镜示意图. 图中各部分为: Fiber Laser, 光纤激光器; HWP (half-wave plate), 半波片; PBS (polarization beam splitter), 偏振分束器; Mirror, 反射镜; Lens, 透镜; PCF (photonic crystal fiber), 光子晶体光纤; Scan Mirror, 扫描镜; Scan Lens, 扫描透镜; Tube Lens, 镜筒透镜; DM (dichroic mirror), 二向色镜; Filter, 滤光片; Obj (objective), 物镜; PMT (photomultiplier tube), 光电倍增管探测器

Fig. 10. Schematic diagram of a laser scanning three-photon microscope. The abbreviations in the figure are as follows: HWP, half-wave plate; PBS, polarization beam splitter; PCF, photonic crystal fiber; DM, dichroic mirror; Obj, objective; PMT, photomultiplier tube.

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图 11 小鼠组织模型衰减谱及活体成像^[60]　(a) 基于米氏散射和吸水率的组织模型的衰减谱; (b) FVB/N小鼠脑血管三光子图像的三维重构; (c) B6.Cg-Tg(Thy1-Brainbow1.0)HLich/J小鼠脑内神经元三光子图像的三维重构

Fig. 11. Attenuation spectrum and in vivo imaging of mouse tissue model^[60]: (a) Attenuation spectrum of tissue model based on Mie scattering and water absorption; (b) three-dimensional reconstruction of three-photon image of FVB/N mouse cerebrovascular; (c) B6.Cg-Tg (Thy1-Brainbow1.0) three-dimensional reconstruction of three-photon images of neurons in the brain of HLich/J mice.

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图 12 活体小鼠脑血管的三光子FLIM成像^[75]

Fig. 12. Three-photon FLIM imaging of cerebral blood vessels in living mice^[75].

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图 13 活体小鼠三光子脑血管成像图, 2100 µm的成像深度为目前最深^[79]

Fig. 13. Three-photon cerebrovascular imaging of living mice. The imaging depth of 2100 µm is currently the deepest^[79].

下载: 全尺寸图片幻灯片

表 1 双光子FLIM监测NADH和FAD的工作原理^[28]

Table 1. Working principle of NADH and FAD monitoring by two-photon FLIM^[28].

名称	本质	主要分布	工作原理	与FLIM联系	生理功能
NADH	烟酰胺腺嘌呤二核苷酸(NAD)的还原态, 一种还原型辅酶	线粒体和细胞质	在氧化还原反应中, NADH作为氢和电子的供体, NAD作为氢和电子的受体	NAD与脱氢酶结合时, 激发和发射都有蓝移, 同时荧光量子产率增加. NADH跟蛋白质结合后显示较长的寿命成分. FLIM 还可以计算这些物质的相对含量	改善能量水平、保护细胞、促进神经递质的产生等
FAD	黄素腺嘌呤二核苷酸, 某些氧化还原酶的辅基	线粒体	FAD参与体内各种氧化还原反应, 在生物氧化系统中起传递氢的作用	游离的FAD分子显示较长的寿命成分, 和蛋白质结合的FAD 分子显示较短的寿命成分	可做成活性型维生素B2, 用于神经性耳鸣、脑动脉硬化等

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