hyperspectral line-scanning vsfg microscope data collection and analysis

VSFG 현미경을 사용하여 다중 모드, 신속, 초분광 화학 분석

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.

저자 스포트라이트: Mesoscopically Heterogeneous Self-Assembled Structures 연구에서 VSFG 현미경의 잠재력 공개 

라인 스캐닝 진동 합계-주파수 생성 현미경을 사용한 다중 모드 비선형 초분광 화학 이미징

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

초분광 라인 스캐닝 VSFG 현미경 데이터 수집 및 분석

먼저 하전 결합 장치 또는 CCD에서 VS-FG 신호의 수직선 스펙트럼을 수집합니다. 빔 스캐너 방향에 수직으로 샘플을 스캔하여 비공진 강도 이미지를 수집합니다. MATLAB imaging Toolbox의 초분광 이미징 라이브러리 워크플로를 사용하여 데이터를 스펙트럼 혼합 해제합니다.라이브러리의 hypercube 함수를 사용하여 X와 Y가 공간적이고, Z가 주파수 종속 강도에 대응하고, omega가 주파수인 4차원 하이퍼큐브를 만듭니다. 카운트 엔드 멤버 HFC 함수를 사용하여 오경보 또는 PFA 값이 10에서 음의 7일 확률로 고유한 스펙트럼의 수를 식별합니다. 그런 다음 N-파인더 스펙트럼 불혼합 기능을 사용하여 고유한 스펙트럼을 식별합니다.SID 함수를 사용하여 각 픽셀을 이전에 식별된 고유 스펙트럼 중 하나와 연결합니다. 마지막으로 각 격리된 시트의 합계 데이터를 Voit 함수에 맞춥니다. 커버 슬립에 흩어져 있는 자체 조립 시트의 VS-FG 이미지가 캡처되었습니다.그리고 스펙트럼 식별을 통해 모든 시트를 VS-FG 강도가 높은 것과 강도가 낮은 것의 두 가지 유형으로 분류할 수 있음을 발견했습니다. 광학 이미지를 검사하고 비교한 결과, 이미지 중앙의 큰 시트가 이중 시트를 쌓은 것처럼 보였으며, 따라서 VS-FG 강도가 더 작은 것은 파괴적인 간섭에 기인합니다. 시트 중 2개는 다양한 VS-FG 편광으로 측정되었으며 스펙트럼은 Voit 기능을 사용하여 장착되었습니다.

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

Research

JoVE Journal

Chemistry

Hyperspectral Line-Scanning VSFG Microscope Data Collection and Analysis

146 조회수.  UC San Diego. 먼저 하전 결합 장치 또는 CCD에서 VS-FG 신호의 수직선 스펙트럼을 수집합니다. 빔 스캐너 방향에 수직으로 샘플을 스캔하여 비공진 강도 이미지를 수집합니다. MATLAB imaging Toolbox의 초분광 이미징 라이브러리 워크플로를 사용하여 데이터를 스펙트럼 혼합 해제합니다.라이브러리의 hypercube 함수를 사용하여 X와 Y가 공간적이고, Z가 주파수 종속 강도에 대응하고, omega가 주?...

비디오: 초분광 라인 스캐닝 VSFG 현미경 데이터 수집 및 분석

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 ...

연구

화학

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.

초분광 라인 스캐닝 VSFG 현미경 설정, 정렬 및 보정

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.