新型多光子成像技术研究进展

石玉洁; 张广杰; 陆政元; 应亚宸; 贾荟琳; 席鹏

doi:10.3788/CO.20181103.0296

新型多光子成像技术研究进展

doi: 10.3788/CO.20181103.0296

1.
北京大学元培学院, 北京 100871
2.
北京大学工学院生物医学工程系, 北京 100871

基金项目:

科技部重大仪器专项 No.2013YQ03065102

国家自然科学基金 No.61475010

国家自然科学基金 No.61729501

科技部重点研发专项 No.2013YQ03065102

详细信息

作者简介:
石玉洁(1996-), 女, 内蒙古乌兰察布人, 北京大学本科生, 2014年至今就读于北京大学元培学院整合科学专业, 主要从事化学生物学方面的研究。E-mail:shiyujie21@pku.edu.cn

张广杰(1994—)，女，山东济南人，博士研究生，2017年于北京林业大学获得学士学位，现为北京大学工学院生物医学工程系博士研究生，主要从事光学成像、光声成像方面的研究。E-mail:jiezgm@126.com

席鹏(1978—)，男，山西朔州人，博士，北京大学特聘研究员，博士生导师，2003年在中国科学院上海光机所获博士学位，主要从事超分辨显微成像方面的研究。E-mail：xipeng@pku.edu.cn

中图分类号: O439
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出版历程
- 收稿日期: 2018-02-26
- 修回日期: 2018-03-28
- 刊出日期: 2018-06-01

Advances in multiphoton microscopy technologies

1.
Yuanpeng College, Peking University, Beijing 100871, China
2.
Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China

Funds:

National Instrument Development Special Program No.2013YQ03065102

National Natural Science Foundation of China No.61475010

National Natural Science Foundation of China No.61729501

Ministry of Science and Technology of China, Key Research and Development Projects No.2013YQ03065102

More Information

Corresponding author: ZHANG Guang-jie, E-mail:jiezgm@126.com

摘要

摘要: 相比于传统的光学成像技术，近年来获得快速发展的新型多光子成像技术具有穿透深度大，组织光损伤小，信噪比高，且可方便进行光学层析成像的特点，故而被广泛应用于包括脑、肿瘤、胚胎在内的多种活体组织成像中。本综述回顾了新型多光子成像技术的诞生与发展历程，包括微型化双光子成像技术、双光子内窥技术和三光子成像技术，概括分析了其基本原理与成像特点，讨论了这一领域具有代表性的最新研究成果，重点总结了其在生物学基础研究领域和临床医学诊断中的主要应用，并展望了其未来的应用与发展前景。可以预见，随着激光器和光探测技术的不断进步，多光子成像技术将会得到更大的发展与更加广泛的应用。
- 多光子成像 /
- 微型化双光子成像技术 /
- 双光子内窥技术 /
- 三光子成像技术
Abstract: Compared with traditional optical imaging techniques, the fast-developing multiphoton microscopy technologies possess multiple advantages, such as deep penetration, low tissue photo-damaging, high signal-to-noise ratio, and excellent optical sectioning ability. Therefore, they have been widely applied in tissue-level microscopy in vivo for brains, tumors and embryos. This article reviews the recent development of new multiphoton microscopy technologies, including miniaturized two-photon microscopy, two-photon endoscopy, and three-photon microscopy. The review briefly illustrates their principles and characteristics, introduces the latest progresses in these areas, summarizes their main applications in basic research and clinical diagnosis, and discusses their potential application and development in the future. With the advances in laser devices and optical detectors, multiphoton microscopy will become an important tool for biomedical research with broad applications.
- multiphoton microscopy /
- miniature two-photon microscopy /
- two-photon endoscopy /
- three-photon microscopy

HTML全文

图 1 二维MEMS扫描镜的显微照片。(a)3.2 mm×3.0 mm硬模中的750 μm× 750 μm扫描镜；(b)内轴；(c)外轴^[12]

Figure 1. Micrograph of a two-dimensional MEMS scanner (a)750 μm ×750 μm scanning mirror in a 3.2 mm×3.0 mm die; (b)inner axis; (c)outer axis^[12]

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图 2 携带式双光子显微镜。(a)总览；(b)扫描镜整体焊线封装到印刷电路板上；(c)显微镜模型图(剖面观)；(d)激光照明(红)和荧光收集(绿)光路^[9]

Figure 2. Portable two-photon microscope. (a)overview; (b)the scanner die is wirebonded onto electrodes on the printed circuit board (PCB); (c)computer-aided-design model of the microscope, in a cut-away view; (d)laser illumination(red arrows) and fluorescence collection(green arrows) pathways^[9]

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图 3 FHIRM-TPM结构及外观。(a)微型化显微镜的光学部件和机械组装；(b) FHIRM-TPM实物照片(指尖和佩戴在小鼠上)^[13]

Figure 3. Structure and appearance of FHIRM-TPM. (a)Optical components and mechanical assembly of the miniature microscope; (b)photographs of a FHIRM-TPM on a fingertip and mounted to the head of a mouse^[13]

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图 4 光纤横截面示意图。(a)传统单模光纤；(b)高反射率光子晶体光纤；(c)双包层光纤^{[11, 20]}

Figure 4. Schematic of fiber cross-section. (a)A standard fibre consisting of two bulk materials; (b)a high-index guiding PCF with a solid silica core; (c)double-clad fiber^{[11, 20]}

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图 5 扫描方式^[21]

Figure 5. Scanning mechanisms^[21]

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图 6 微型物镜与纵向聚焦位移。(a)定制物镜示意图；(b)样品侧纵向聚焦位移^[25]

Figure 6. Miniature objective and longitudinal focal shift. (a)schematic of the customized miniature objective; (b)sample-side longitudinal focal shift^[25]

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图 7 鼠肾脏缺血-再灌注体内模型双光子荧光内窥成像^[25]

Figure 7. Endomicroscopy two photon fluorescence redox imaging of a mouse kidney ischemia-reperfusion model in vivo^[25]

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图 8 SSFS光源及三光子激光扫描显微镜示意图^[37]

Figure 8. Schematic of SSFS and three-photon microscope setup^[37]

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图 9 三光子成像结果图(a)脑部血管；(b)神经元^[37]

Figure 9. Three-photon imaging results. (a)Brain vasculature; (b)neurons^[37]

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表 1 近年报道的微型化双光子显微镜性能对比

Table 1. Comparison of miniaturized two-photon microscope′s characteristics reported recently

产品	头部佩戴重量	分辨率	扫描速度(频率、帧频)	扫描机制
Helmchen et al.(2001)^[10]	25 g	亚细胞尺度	300~800 Hz(线扫描)	光纤探针扫描
Piyawattanametha et al.(2006)^[12]	-	~1 μm	~3.5 kHz(线扫描)	MEMS扫描镜
Piyawattanametha et al.(2009)^[9]	2.9 g	1.29±0.05 μm(横向)10.3±0.3 μm(轴向)	15 fps(光栅扫描)~1 kHz(线扫描)	MEMS扫描镜
Zong et al. FHIRM-TPM(2017)^[13]	2.15 g	0.64±0.02 μm(横向)3.35±0.37 μm(轴向)	40 fps(光栅扫描)10 kHz(线扫描)	MEMS扫描镜

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表 2 双光子与三光子成像部分特性的比较

Table 2. Comparison of selected features of two-photon and three-photon microscopy

特性	双光子成像	三光子成像^[37]
成像深度	脑-400 μm 血管-600 μm^[40]	脑-1 120 μm 血管-1 300 μm
激光脉冲	100 fs/pulse, 100 MHz^[40]	65 fs/pulse, 1 MHz
可用激发光波长	700~1 000 nm	1 000~1 700 nm
轴向分辨率(FWHM)	~1.6 μm^[41]	~4 μm(激发光波长1 700 nm)

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