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Bioengineering

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Published: December 20, 2021
doi:
10.3791/63411
, , ,
1Department of Electronic and Computer Engineering,The Hong Kong University of Science and Technology, 2State Key Laboratory of Molecular Neuroscience,The Hong Kong University of Science and Technology, 3Center of Systems Biology and Human Health,The Hong Kong University of Science and Technology, 4Molecular Neuroscience Center,The Hong Kong University of Science and Technology, 5Department of Biology, School of Life Sciences,Southern University of Science and Technology

Summary

受激拉曼散射（SRS）显微镜允许根据特定化学键的固有振动对生物分子进行无标记成像。在该协议中，描述了集成SRS和双光子荧光显微镜的仪器设置，以可视化活小鼠脊髓中的细胞结构。

Abstract

受激拉曼散射（SRS）显微镜能够基于内在分子振动对自然微环境中的生物组织进行无标记成像，从而为亚细胞分辨率下生物过程的 体内 研究提供了完美的工具。通过将双光子激发荧光（TPEF）成像集成到SRS显微镜中，组织的双模态 体内 成像可以从多个角度获取关键的生化和生物物理信息，这有助于了解细胞代谢，免疫反应和组织重塑等所涉及的动态过程。在该视频协议中，介绍了TPEF-SRS显微镜系统的设置以及动物脊髓的 体内 成像方法。脊髓作为中枢神经系统的一部分，在大脑和周围神经系统之间的通信中起着至关重要的作用。髓鞘富含磷脂，包围并隔离轴突，以允许作用电位的盐传导。脊髓髓中髓鞘的 体内 成像对于研究神经退行性疾病和脊髓损伤的进展非常重要。该协议还描述了动物制备和 体内 TPEF-SRS成像方法，以获取高分辨率的生物图像。

Introduction

拉曼显微镜1，2正在成为一种强大的无标记方法，可以根据生物分子中各种化学键的特征频率对生物组织进行成像。由于其非侵入性和适应性强的成像能力，拉曼显微镜已被广泛用于成像生物组织中富含脂质的成分，如髓鞘3，4，5，脂肪细胞6，7和脂滴8，9，10.受激拉曼散射（SRS）信号作为受激拉曼增益（SRG）或受激拉曼损耗（SRL）获得的受激拉曼散射（SRS）信号是无背景的，显示出与自发拉曼散射的完美光谱相似性11，12。此外，SRL和SRG线性依赖于分析物浓度，允许对生化组分进行定量分析9，11，13。双光子激发荧光显微镜（TPEF）由于其固有的光学切片能力，深度渗透深度和低光毒性，已被广泛用于体内生物成像14，15，16。然而，TPEF成像的性能取决于荧光标签的特性，并且由于宽带荧光光谱8，17，18，19，可分辨颜色的数量受到限制。无标记SRS成像和基于荧光的TPEF成像是两种互补的成像方式，它们的组合可以提供丰富的组织生物物理和生化信息。这两种成像模式都基于非线性光学（NLO）过程，允许简单地集成到一个显微镜系统中。SRS和TPEF成像的结合，即所谓的双模态成像，可以对细胞和组织进行高维成像和分析，从而促进对复杂生物系统的全面理解。具体而言，与飞秒（fs）SRS技术相比，皮秒（ps）SRS显微镜可以实现具有高光谱分辨率的化学键成像11，从而可以区分生物组织中的多种生化成分，特别是在拥挤的指纹区域20，21。此外，与另一种集成了相干反斯托克斯散射（CARS）显微镜的常用双模态NLO显微镜系统相比，SRS在光谱和图像解释以及检测灵敏度方面表现出优于CARS的性能11。SRS-TPEF显微镜已被用作研究各种生物系统的有力工具，例如秀丽隐杆线虫9，22，非洲爪蟾蝌蚪脑5，小鼠脑23，24，脊髓25，26，周围神经27和脂肪组织7等。

脊髓与大脑一起构成了中枢神经系统（CNS）。在生理和病理条件下，可视化CNS体内的细胞活动对于理解CNS疾病的机制28，29，30和开发相应的疗法至关重要31，32，33。髓鞘包裹和绝缘轴突以实现高速动作电位传导，在CNS的发展中起着重要作用。脱髓鞘被认为是白质疾病的标志，如多发性硬化症34。此外，脊髓损伤后35，髓鞘碎片可以调节巨噬细胞活化，导致慢性炎症和继发性损伤36。因此，活体小鼠模型中髓鞘与神经元和神经胶质细胞的体内成像对了解CNS疾病的动态过程有很大帮助。

在该协议中，描述了自制TPEF-SRS显微镜的基本设置程序，并介绍了小鼠脊髓的双模态 体内 成像方法。

Protocol

本工作所进行的所有动物程序均按照香港科技大学（科大）实验动物设施的指引进行，并已获科大动物伦理委员会批准。设置和操作TPEF-SRS显微镜需要激光处理的安全培训。处理激光时，始终佩戴波长范围合适的激光安全护目镜。 1. TPEF-SRS显微镜的设置（设置原理图见 图1） 使用与锁模镱光纤激光器连接的集成光学参数?…

Representative Results

使用Thy1-YFPH转基因小鼠进行脊髓轴突和髓鞘的体内双模态成像，其在背根神经节传入神经元中表达EYFP（图3）。这些标记的传入神经元将感觉信息从周围神经传递到脊髓，中央分支位于脊髓背柱中。使用TPEF-SRS显微镜，可以使用无标记SRS成像清晰地观察密集分布的髓鞘，并且可以使用TPEF成像观察稀疏标记的YFP轴突。通过双模型成像显示，轴突被一层厚厚的髓鞘紧密包?…

Discussion

在该协议中，详细描述了TPEF-SRS显微镜的基本设置。对于SRS成像，泵浦和斯托克斯光束在OPO内部在时间和空间上重叠。然而，这种重叠在通过显微镜系统后可能会被破坏。因此，泵和斯托克斯光束共定位的空间和时间优化对于实现最佳SRS成像是必要的，也是至关重要的。泵浦和斯托克斯光束之间的时间延迟与两束光束的光路差有关，这取决于光学元件在显微镜系统中的色散38。当…

Disclosures

The authors have nothing to disclose.

Acknowledgements

本署透过拨款16103215、16148816、16102518、16102920、T13-607/12R、T13-706/11-1、T13-605/18W、C6002-17GF、C6001-19E、N_HKUST603/19、创新及科技署（ITCPD/17-9）、大学教育资助委员会卓越领域计划（AoE/M-604/16， AOE/M-09/12）及香港科技大学（科大）资助RPC10EG33，支持这项工作。

Materials

#2 Forceps Dumont 11223-20 For laminectomy
10X objective Nikon CFI Plan Apo Lambda 10X
25X objective Olympus XLPLN25XSVMP2
Burn cream Betadine
Camera Sony α6300
Current amplifier Stanford research SR570
Current photomultiplier modules Hamamatsu H11461-01
D2 665 nm long-pass dichroic mirror Semrock FF665-Di02-25×36 For directing epi-fluorescence signal to the detection module
D3 700 nm short-pass dichroic mirror Edmund 69-206 For separating SRS from TPEF detection path
Depilating cream Veet
FS1 975 nm short-pass filter Edmund 86-108 For blocking stokes beam
FS1 Bandpass filter Semrock FF01-850/310 For blocking stokes beam
Fs2 Bandpass filter Semrock FF01-525/50 For selecting YFP signal
Fs2 Shortpass filter Semrock FF01-715/SP-25 For blocking fs excitation laser beam
Half-wave plate Thorlabs SAHWP05M-1700
High-speed photodetector MenloSystems FPD 310-F For checking Stokes beam modulation
Iodine Betadine
IR Scope FJW FIND-R-SCOPE Infrared Viewer 2X Kit Model 84499C2X
Iris Thorlabs CPA1
L1 Thorlabs AC254-060-B-ML
L10 Thorlabs LA4052-A
L2 Thorlabs LA1422-B
L3 Thorlabs AC254-050-B
L4 Thorlabs AC254-060-B-ML
L7 f=100 mm, AB coating
L8 Thorlabs LA4874-A
L9 Thorlabs AC254-035-B-ML
Lock-in amplifier APE
Mirror Thorlabs PF10-03-P01
Motorized flipper Thorlabs MFF101/M
multifunctional acquisition card National Instrument PCIe-6363
Oscilloscope Tektronix TDS2012C
Photodiode APE For detecting SRS signal
Picosecond laser source APE picoEmerald
Polarizing beam splitter Thorlabs CCM1-PBS252/M
Power meter Newport 843-R
Saline Braun
Scan lens L5 Thorlabs SL50-CLS2
Scanning mirror Cambridge Technology 6215H
Silicone gel World Precision Inc. KWIK-SIL
Ti:sapphire fs laser Coherent Chameleon Ultra II
Tube lens L6 Thorlabs TTL200-S8
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Tags

In Vivo ImagingTwo-photon FluorescenceStimulated Raman ScatteringMicroscopySubcellular ResolutionChemical SpecificityCellular MetabolismImmune ResponseNeurodegenerative DiseasesSpinal Cord InjuryTitanium Sapphire LaserOPOPhotodetectorOscilloscopeEOMPump LaserWavelengthOptical Alignment

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Wu, W., Li, X., Qu, J. Y., He, S. In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy. J. Vis. Exp. (178), e63411, doi:10.3791/63411 (2021).
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