hyperspectral line-scanning vsfg microscope data collection and analysis

使用 VSFG 显微镜进行多模式、快速、高光谱化学分析

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical technique1,2, has been used extensively as a spectroscopy tool to chemically profile symmetry-allowed samples3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22. Traditionally, VSFG has been applied to interfacial systems8,9,10,11 (i.e., gas-liquid, liquid-liquid, gas-solid, solid-liquid), which lack inversion symmetry - a requirement for VSFG activity. This application of VSFG has provided a wealth of molecular details of buried interfaces12,13, configurations of water molecules at interfaces14,15,16,17,18, and chemical species at interfaces19,20,21,22.
Although VSFG has been powerful in determining molecular species and configurations at interfaces, its potential in measuring molecular structures of materials lacking inversion centers has not been fulfilled. This is partly because the materials could be heterogeneous in their chemical environment, compositions, and geometric arrangement, and a traditional VSFG spectrometer has a large illumination area on the order of 100 &#181;m2. Thus, traditional VSFG spectroscopy reports on ensemble-averaged information of the sample over a typical 100 &#181;m2 illumination area. This ensemble averaging may lead to signal cancellations between well-ordered domains with opposite orientations and mischaracterization of local heterogeneities15,20,23,24.
With advances in high numerical aperture (NA), reflective-based microscope objectives (Schwarzschild and Cassegrain geometries), which are nearly free of chromatic aberrations, the focus size of the two beams in VSFG experiments can be decreased from 100 &#181;m2 to 1-2 &#181;m2 and in some cases submicron25. Including this technological advancement, our group and others have developed VSFG into a microscopy platform20,23,26,27,28,29,30,31,32,33,34,35,36. Recently, we have implemented an inverted optical layout and broadband detection scheme37, which enables a seamless collection of multimodal images (VSFG, second harmonic generation (SHG), and brightfield optical). The multi-modality imaging allows quick inspection of samples using optical imaging, correlating various types of images together, and locating signal positions on the sample images. With the achromatic illumination optics and choice of pulsed laser illumination source, this optical platform allows for future seamless integration of additional techniques such as Fluorescence microscopy38 and Raman microscopy, among others.
In this new arrangement, samples such as hierarchical organizations and a class of molecular self-assemblies (MSAs) have been studied. These materials include collagen and biomimetics, where both the chemical composition and geometric organization are important to the ultimate function of the material. Because VSFG is a second-order nonlinear optical signal, it is specifically sensitive to intermolecular arrangements39,40, such as intermolecular distance or twisting angles, making it an ideal tool for revealing both chemical compositions and molecular arrangements. This work describes the VSFG, SHG, and brightfield modalities of the core instrument consisting of a ytterbium-doped cavity solid-state laser that pumps an optical parametric amplifier (OPA), a home-built multimodal inverted microscope and monochromator frequency analyzer coupled to a two-dimensional charged coupled device (CCD) detector27. A step-by-step construction and alignment procedures, and a complete part list of the setup, are provided. An in-depth analysis of an MSA, whose fundamental molecular subunit is comprised of one molecule of sodium-dodecyl sulfate (SDS), a common surfactant, and two molecules of &#946;-cyclodextrin (&#946;-CD), known as SDS@2&#946;-CD herein, are also provided as an example to show how VSFG can reveal molecule-specific geometric details of organized matter. It has also been demonstrated that chemical-specific geometric details of the MSA can be determined with a neural network function solver approach.

作者聚焦：揭示 VSFG 显微镜在研究介观异质自组装结构中的潜力 

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

We aim to study mesoscopically heterogeneous self-assembled samples, such as biological tissues, to reveal their chemical composition and molecular arrangements. We are exploring how the microscopic arrangement and mesoscopic morphology of these self materials relate to their microscopic properties. With advances in high numerical aperture and reflective-based microscope objectives, we as well as others have demonstrated vibrational sum-frequency generation microscope with one square micron resolution, which can simultaneously record images of soft materials and also spatially resolve the spectral, forming the so-called hyperspectral images.Hyperspectral imaging records experimental data in multiple dimensions, two in spaces, one in frequency and possibly one in time. However, quick collection, storage, and analysis of such big data to maximize information remains a challenge. Sampling the image only to collect useful data points is also challenging.By developing a faster line-scanning technology, we could accelerate the data acquisition time by 100-fold. Furthermore, the microscope is compatible with sum-frequency generation or SFG, second harmonic generation or SHG, as well as broad-field imaging. Multimodality imaging allows quick inspection of samples using optical imaging and correlating various image modalities.SHG imaging assists the optical technique of our technique, but without the spectral resolving power, is widely used in biophysics. Our SFG microscope will now be able to provide powerful molecular level insights currently missing in biophysical studies of soft tissues.

Watch this Scientific Journal Video about Hyperspectral Line-Scanning VSFG Microscope Data Collection and Analysis at JoVE.com

高光谱线扫描VSFG显微镜数据采集与分析

首先在带电耦合器件或CCD上收集VS-FG信号垂直线的频谱。通过垂直于光束扫描仪方向扫描样品来收集非共振强度图像。使用 MATLAB 成像工具箱高光谱成像库工作流程对数据进行光谱解混。使用库中的超立方函数创建一个四维超立方体，其中 X 和 Y 是空间的，Z 对应于频率相关的强度，omega 是频率。使用计数端成员 HFC 函数识别唯一光谱的数量，误报或 PFA 值的概率为 10 到负 7。然后使用 N-finder 光谱解混功能识别独特的光谱。使用 SID 函数，将每个像素与先前识别的唯一光谱之一相关联。最后，将每个孤立工作表的总和数据拟合到 Voit 函数。捕获了分散在盖玻片上的自组装板材的VS-FG图像。通过光谱识别，发现所有片材均可分为两种类型，一种是VS-FG强度较高的，另一种是强度较低的。通过检查并与光学图像进行比较，图像中心的大片似乎有堆叠的双片，从而将较小的VS-FG强度归因于相消干涉。其中两片通过各种VS-FG偏振进行测量，并使用Voit函数拟合光谱。

hyperspectral-line-scanning-vsfg-microscope-data-collection

Research

JoVE Journal

Chemistry

Hyperspectral Line-Scanning VSFG Microscope Data Collection and Analysis

146 次观看.  UC San Diego. 首先在带电耦合器件或CCD上收集VS-FG信号垂直线的频谱。通过垂直于光束扫描仪方向扫描样品来收集非共振强度图像。使用 MATLAB 成像工具箱高光谱成像库工作流程对数据进行光谱解混。使用库中的超立方函数创建一个四维超立方体，其中 X 和 Y 是空间的，Z 对应于频率相关的强度，omega 是频率。使用计数端成员 HFC 函数识别唯一光谱的数量，误报或 PFA 值的概率为 10 到?...

视频: 高光谱线扫描VSFG显微镜数据采集与分析

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

科研

化学

Hyperspectral Line-Scanning VSFG Microscope Setup, Alignment, and Calibration

Begin by setting up the pulsed laser system, centered at 1025 nanometers. Guide the output of the seed laser into a commercial Optical Parametric Amplifier, or OPA, to generate a mid-infrared or mid-IR beam. Tune the mid-IR beam to the frequency of interest.Pass the residual 1025 nanometer beam from OPA through a Fabry-Perot etalon to produce a spectrally-narrowed up-conversion beam. Spatially filter the narrowed beam with an eight-micron sapphire pinhole. Control the polarization of the 1025 nanometer pulse with a lambda by two wave plate.Next, guide the mid-IR beam through a delay stage for fine control of the temporal overlap. Control the polarization of the mid-IR with a lambda by two wave plate. Spatially overlap both the up-conversion and mid-IR beams at a customized Dichroic Mirror, or DM, that is transmissive to mid-IR and reflective to near-IR.Use two irises to guide the alignment, one right after the DM and one at the far end. Use a power meter after the iris to determine whether mid-IR is centered, and use a near-IR card to locate near-IR positions. Direct the overlapped beams into an inverted microscope with an integrated 325 hertz, single-axis, resonant beam scanner that is mounted to an integrated two-position scanner, or I2PS.Focus the two spatially-overlapped beams onto the sample with a purely reflective Schwarzschild Objective, SO.Collect the Vibrational Sum Frequency Generation, or VSFG, signal generated by the sample with an infinity-corrected Refractive Objective, RO.Guide the collimated output VSFG signal through a linear polarizer and then through a telecentric tube lens system composed of two focal lenses, TL1 and TL2, each having a 60 millimeter focal length. To switch to the Second Harmonic Generation, or SHG mode, block the IR beam and rotate the grading of the spectrograph to 501.5 nanometers. To switch to bright-field optical imaging.Turn on the white light source. Move the integrated slider, I2PS, to collect bright-field images in the counterpropagating direction. With the imaging objective, RO, acting as the condenser and the condenser objective, SO, acting as the imaging objective.Then use a commercially available two-blends system to form an image of the collimated output of the RO at the sensor plane of an RGB bright-field camera. Use a standard one-micron-thick sample of zinc oxide pattern sputter coated on a coverslip to roughly optimize the position of the sample plane or the nanopositioner z-axis by bringing it into bright-field focus, using the bright-field imaging modality. Turn on the resonant beam scanner to collect a line of images.After the line section of the sample is hyperspectrally imaged, scan the sample in the axis perpendicular to the line scanning axis using the three-dimensional nano positioner. Take vertical slices of the image data and establish the pixel-to-micron ratio.

高光谱线扫描VSFG显微镜的设置、对准和校准

Begin by collecting the spectra of a vertical line of the VS-FG signals on the charged coupled device or CCD. Collect non-resonant intensity images by scanning the sample perpendicularly to the beam scanner direction. To spectrally unmix the data using the MATLAB imaging toolbox hyperspectral imaging library workflow.Use the hypercube function in the library to create a four-dimensional hypercube where X and Y are spatial, Z corresponds to the frequency dependent intensity, and omega is the frequency. Identify the number of unique spectra with the count end members HFC function, with a probability of false alarm or PFA value of 10 to the negative seven. Then identify unique spectra using the N-finder spectral unmixing function.Using the SID function, associate each pixel with one of the previously identified unique spectra. Finally, fit the sum data for each isolated sheet to the Voit function. VS-FG images of self-assembled sheets dispersed on a cover slip were captured.And through spectral identification, it was found that all sheets could be categorized into two types, one with higher VS-FG intensity and the other with lower intensity. By inspecting and comparing with the optical image, the large sheet at the center of the images appeared to have stacked double sheets, thereby attributing the smaller VS-FG intensity to destructive interference. Two of the sheets were measured by various VS-FG polarizations and the spectra were fitted using the Voit functions.