JoVE Journal > Chemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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화학

基于线扫描振动和频生成显微镜的多模态非线性高光谱化学成像

Published: December 01, 2023
doi:
10.3791/65388
, , ,
1Department of Chemistry and Biochemistry,UC San Diego, 2State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics,Chinese Academy of Sciences, 3Materials Science and Engineering Program,UC San Diego, 4Department of Electrical and Computer Engineering,UC San Diego

Summary

开发了一种多模态快速高光谱成像框架，用于获取宽带振动和频生成（VSFG）图像以及明场二次谐波生成（SHG）成像模式。由于红外频率与分子振动共振，揭示了对称性允许样品的微观结构和介观形态学知识。

Abstract

振动和频生成 （VSFG） 是一种二阶非线性光信号，传统上作为一种空间分辨率为 ~100 μm 的光谱技术用于研究界面处的分子。然而，光谱学对样品的异质性不敏感。为了研究介观异质样品，我们与其他人一起将VSFG光谱的分辨率极限降低到~1μm水平，并构建了VSFG显微镜。这种成像技术不仅可以通过成像解析样品形态，还可以在图像的每个像素点上记录宽带VSFG光谱。作为一种二阶非线性光学技术，其选择规则能够可视化生物学、材料科学和生物工程等中常见的非中心对称或手性自组装结构。在本文中，将引导观众了解一种倒置透射设计，该设计允许对未固定的样本进行成像。这项工作还表明，VSFG显微镜可以通过将其与神经网络函数求解器相结合来解析单个自组装板材的化学特定几何信息。最后，对不同样品在明场、SHG和VSFG构型下获得的图像进行了简要讨论，讨论了VSFG成像所揭示的独特信息。

Introduction

振动和频生成 （VSFG） 是一种二阶非线性光学技术1,2，已被广泛用作光谱学工具，以化学分析允许对称性的样品 3,4,5,6,7,8,9,10,11,12,13， 14,15,16,17,18,19,20,21,22。传统上，VSFG已应用于界面体系8,9,10,11（即气-液、液-液、气-固、固-液），这些体系缺乏反转对称性 – 这是VSFG活性的要求。VSFG的这一应用提供了埋藏界面12,13，界面14,15,16,17,18处水分子的构型以及界面19,20,21,22处化学物质的丰富分子细节。

尽管VSFG在确定界面处的分子种类和构型方面具有很强的实力，但其在测量缺乏反转中心的材料的分子结构方面的潜力尚未得到发挥。这在一定程度上是因为材料的化学环境、成分和几何排列可能具有不同质性，而传统的 VSFG 光谱仪具有大约 100 μm2 的大照明区域。因此，传统的VSFG光谱法报告了样品在典型的100 μm2照明区域内的集合平均信息。这种集成平均可能导致具有相反方向的有序域之间的信号抵消和对局部异质性的错误表征 15,20,23,24。

随着高数值孔径 （NA） 和基于反射的显微镜物镜（Schwarzschild 和 Cassegrain 几何形状）的进步，它们几乎没有色差，VSFG 实验中两个光束的焦点尺寸可以从 100 μm 2 减小到 1-2 μm2，在某些情况下可以减小亚微米25。包括这项技术进步在内，我们小组和其他公司已将VSFG开发成显微镜平台20,23,26,27,28,29,30,31,32,33,34,35,36。最近，我们实现了一种倒置光学布局和宽带检测方案37，该方案可以无缝收集多模态图像（VSFG、二次谐波产生（SHG）和明场光学）。多模态成像允许使用光学成像快速检查样品，将各种类型的图像关联在一起，并在样品图像上定位信号位置。通过消色差照明光学器件和脉冲激光照明源的选择，该光学平台允许未来无缝集成其他技术，例如荧光显微镜38 和拉曼显微镜等。

在这种新的安排中，已经研究了分层组织和一类分子自组装（MSA）等样本。这些材料包括胶原蛋白和仿生学，其中化学成分和几何组织对材料的最终功能都很重要。由于VSFG是二阶非线性光信号，因此它对分子间排列39,40特别敏感，例如分子间距离或扭曲角，使其成为揭示化学成分和分子排列的理想工具。这项工作描述了核心仪器的 VSFG、SHG 和明场模式，该仪器由泵浦光学参量放大器 （OPA） 的掺镱腔固体激光器、自制的多模态倒置显微镜和耦合到二维电荷耦合器件 （CCD） 探测器的单色器频率分析仪27 组成。提供了分步的构造和对齐程序，以及完整的设置部件列表。本文还以MSA为例，对MSA进行了深入分析，MSA的基本分子亚基由一个分子十二烷基硫酸钠（SDS）（一种常见的表面活性剂）和两个分子的β-环糊精（β-CD）（本文称为SDS@2 β-CD）组成，以说明VSFG如何揭示有组织物质的分子特异性几何细节。还已经证明，MSA的化学特定几何细节可以使用神经网络函数求解器方法确定。

Protocol

1. 高光谱线扫描VSFG显微镜 激光系统使用以 1025 nm ± 5 nm 为中心的脉冲激光系统（参见 材料表）。激光器设置为 40 W、200 kHz（200 μJ/脉冲），脉冲宽度为 ~290 fs。注意：确切的重复频率可能会有所不同，高重复频率激光通常更适合这种VSFG显微镜。 将种子激光器的输出引导到商用光学参量放大器 （OPA） 中，以产生中红外 （MIR） 光束（参见 材料表?…

Representative Results

图5：SDS@β-CD的分子结构、形貌和电位取向 。 （A） SDS@β-CD的俯视图和（B）侧视图化学结构。（C）样品平面上介尺度片片的代表性异质样品分布。分子亚基在底物上可能具有不同的取向和排列，这是未知的。该数字已根据 Wagner …

Discussion

最关键的步骤是从 1.42 到 1.44。为了获得光学空间分辨率，必须很好地对准物镜。收集发射信号、中继并将扫描光束投射为入口狭缝处的一条线也很重要。正确的对齐将保证最佳的分辨率和信噪比。对于典型的样品，如 SDS@2 β-CD 100 μm x 100 μm 片材，具有高信噪比的高分辨率图像（~1 μm 分辨率）需要 20 分钟。这已经比以前版本的仪器24,26 更快。通过更高…

Disclosures

The authors have nothing to disclose.

Acknowledgements

仪器开发得到了 Grant NSF CHE-1828666 的支持。ZW、JCW 和 WX 由美国国立卫生研究院、美国国立普通医学科学研究所资助，资助 1R35GM138092-01。BY由中国科学院青年创新促进会（CAS，2021183）支持。

Materials

1x Camera Por Thorlabs WFA4100 connect a camera to a microscope or optical system
25.0 mm Right-Angle Prism Mirror, Protected Gold Thorlabs MRA25-M01 reflect light and produce retroreflection, redirecting light back along its original path
3” Universal Post Holder-5 Pack Thorlabs UPH3-P5 hold and support posts of various sizes and configurations
30 mm to 60 mm Cage Plate, 4 mm Thick Thorlabs LCP4S convert between a 30 mm cage system and a 60 mm cage system
500 mm Tall Cerna Body with Epi Arm Thorlabs CEA1500 provide the function of enabling top illumination techniques in microscopy
60 mm Cage Mounted Ø50.0 mm Iris Thorlabs LCP50S control the amount of light passing through an optical system
60 mm Cage Mounting Bracket Thorlabs LCP01B mount and position a 60 mm cage system in optical setups
Air spaced Etalon SLS Optics Ltd. Customized generate narrow-band 1030 nm light 
Cage Plate Mounting Bracket Thorlabs KCB2 hold and adjust mirrors at a precise angle
CCD Andor Technologies Newton  2D CCD for frequency and spatial resolution
Collinear Optical Parametric Amplifier Light Conversion Orpheus-One-HP Tunable MID light generator
Copper Chloride Thermo Fischer Scientific A16064.30 Self-assembly component
Customized Dichroic Mirror Newport Customized selectively reflects or transmits light based on its wavelength or polarization
Ext to M32 Int Adapter Thorlabs SM1A34 provide compatibility and facilitating the connection between components with different thread types
Infinity Corrected Refractive Objective Zeiss 420150-9900-000 Refractive Objective
Infinity Corrected Schwarzschild Objective Pike Technologies Inc. 891-0007 Reflective objective
Laser Carbide, Light-Conversion C18212 Laser source
M32x0.75 External to Internal RMS Thorlabs M32RMSS adapt or convert the threading size or type of microscope objectives 
M32x0.75 External to M27x0.75 Internal Engraving Thorlabs M32M27S adapt or convert the threading size or type of microscope objectives 
Manual Mid-Height Condenser Focus Module Thorlabs ZFM1030 adjust the focus of an optical element
Monochromator Andor Technologies Shamrock 500i Provides frequency resolution for each line scan
Motorized module with 1" Travel for Edge-Mounted Arms Thorlabs ZFM2020 control the vertical positon of the imaging objective
Nanopositioner Mad City Labs Inc. MMP3 3D sample stage
Resonant Scanner EOPC SC-25 325Hz resonant beam scanner
RGB Color CCD Camera Thorlabs DCU224C Brightfield camera, discontinued but other cameras will work just as well
RGB tube lens Thorlabs ITL200 white light collection
Right Angle Kinematic Breadboard Thorlabs OPX2400 incorporate a sliding mechanism with two fixed positions
Right Angle Kinematic Mirror Mount, 30 mm Thorlabs KCB1 hold and adjust mirrors at a precise angle
Right Angle Kinematic Mirror Mount, 60 mm Thorlabs KCB2 hold and adjust mirrors at a precise angle
SM2, 60 mm Cage Arm for Cerna Focusing Stage Thorlabs CSA2100 securely mount and position condensers
Snap on Cage Cover for 60 mm Cage, 24 in Long, Thorlabs C60L24 enclose and protect the components inside the cage
Sodium dodecyl sulfate Thermo Fischer Scientific J63394.AK Self-assembly component
Three-Chnnale Controller and Knob Box for 1" Cerna Travel Stages Thorlabs MCM3001 control ZFM2020
Tube lens Thorlabs LA1380-AB – N-BK7 SFG signal collection
Visible LED Set Thorlabs WFA1010 provide illumination in imaging setup
Whitelight Source Thorlabs WFA1010 Whitelight illumination source for brightfield imaging
WPH05M-1030 – Ø1/2" Zero-Order Half-Wave Plate, Ø1" Mount, 1030 nm  Thorlabs WPH05M-1030 alter the polarization state of light passing through it
WPLQ05M-3500 – Ø1/2" Mounted Low-Order Quarter-Wave Plate, 3.5 µm  Thorlabs WPLQ05M-3500 alter the polarization state of light passing through it
X axis Long Travel Steel Extended Contact Slide Stages Optosigma TSD-65122CUU positioning stages that offer extended travel in the horizontal (X) direction
XT95 4in Rail Carrier Thorlabs XT95RC4 mount and position optical components
X-Y Axis Translation Stage w/ 360 deg. Rotation Thorlabs XYR1 precise movement and positioning of objects in two dimensions, along with the ability to rotate the platform
XY(1/2") Linear Translator with Central SM1 Thru Hole Thorlabs XYT1 provide precise movement and positioning in two dimensions
Yb doped Solid State Laser Light Conversion CB3-40W Seed laser
β-Cyclodextrin Thermo Fischer Scientific J63161.22 Self-assembly component
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Tags

Multimodal ImagingNonlinear MicroscopyHyperspectral ImagingVibrational Sum-frequency GenerationSum-frequency Generation MicroscopySecond Harmonic GenerationMesoscopic HeterogeneitySoft MaterialsBiophysicsSelf-assembled StructuresMolecular ArrangementChemical Composition

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Wagner, J. C., Yang, B., Wu, Z., Xiong, W. Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy. J. Vis. Exp. (202), e65388, doi:10.3791/65388 (2023).
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