hyperspectral line-scanning vsfg microscope setup, alignment, and calibration

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

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.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

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

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 Setup, Alignment, and Calibration at JoVE.com

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.

hyperspectral-line-scanning-vsfg-microscope-setup-alignment

Research

JoVE Journal

Chemistry

178 Görüntüleme.  UC San Diego. 1025 nanometre merkezli darbeli lazer sistemini kurarak başlayın. Orta kızılötesi veya orta IR ışını oluşturmak için tohum lazerin çıkışını ticari bir Optik Parametrik Amplifikatöre veya OPA'ya yönlendirin. Orta IR ışınını ilgilenilen frekansa ayarlayın.Spektral olarak daraltılmış bir yukarı dönüşüm ışını üretmek için OPA'dan kalan 1025 nanometre ışınını bir Fabry-Perot etalonundan geçirin. Daralmış ışını sekiz mikron safir iğne deli?...