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

摘要

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。

摘要

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。

引言

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。

研究方案

1. 高光谱线扫描VSFG显微镜

1. 激光系统
   1. 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。
   2. 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）。将MIR调谐到感兴趣的频率（图1A）。
      注：在本研究中，MIR 以 3450 nm ± 85 nm （~2900 ± 72 ^cm-1） 为中心，脉冲持续时间为 ~290 fs，脉冲能量为 ~6 μJ，包括部分 -CH_x 官能团区域。
2. 上转换光束
   1. 将来自 OPA 的残余 1025 nm 光束通过 Fabry-Perot 标准具（参见 材料表），以产生光谱变窄的上转换光束，其 FWHM 为 ~4.75 ^cm-1。
   2. 用 8 μm 蓝宝石针孔对狭窄的 1025 nm 光束进行空间滤波。
      注意：可以使用近红外卡可视化 1025 nm 光束。
   3. 使用 λ....

讨论

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.......

材料

Name	Company	Catalog Number	Comments
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