The instrument development is supported by Grant NSF CHE-1828666. ZW, JCW and WX are supported by the National Institutes of Health, National Institute of General Medical Sciences, Grant 1R35GM138092-01. BY is supported by the Youth Innovation Promotion Association, Chinese Academy of Sciences (CAS, 2021183).

1. Hyperspectral line-scanning VSFG microscope
<ol>
	<li>Laser system
	<ol>
		<li>Use a pulsed laser system (see Table of Materials) centered at 1025 nm &#177; 5 nm. The laser is set at 40 W, 200 kHz (200 &#181;J/pulse) with a pulse width of ~290 fs. 
		NOTE: The exact repetition rate can vary, and a high repetition rate laser generally works better for this VSFG microscope.</li>
		<li>Guide the output of the seed laser into a commercial optical parametric amplifier (OPA) to generate a mid-infrare...

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

The most critical steps are from 1.42 to 1.44. It is critical to align the objective lens well for an optical spatial resolution. It is also important to collect the emitted signal, relay, and project the scanning beam as a line at the entrance slits. Proper alignments would guarantee the best resolution and signal-to-noise ratio. For a typical sample, like&#160;SDS@2&#946;-CD 100 &#181;m by 100 &#181;m sheets, a good resolution image (~1 &#181;m resolution) with a high signal-to-noise ratio would take 20 min. This is al...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Camera Por</td>
			<td>Thorlabs</td>
			<td>WFA4100</td>
			<td>connect a camera to a microscope or optical system</td>
		</tr>
		<tr>
			<td>25.0 mm Right-Angle Prism Mirror, Protected Gold</td>
			<td>Thorlabs</td>
			<td>MRA25-M01</td>
			<td>reflect light and produce retroreflection, redirecting light back along its original path</td>
		</tr>
		<tr>
			<td>3&#8221; Universal Post Holder-5 Pack</td>
			<td>Thorlabs</td>
			<td>UPH3-P5</td>
			<td>hold and support posts of various sizes and configurations</td>
		</tr>
		<tr>
			<td>30 mm to 60 mm Cage Plate, 4 mm Thick</td>
			<td>Thorlabs</td>
			<td>LCP4S</td>
			<td>convert between a 30 mm cage system and a 60 mm cage system</td>
		</tr>
		<tr>
			<td>500 mm Tall Cerna Body with Epi Arm</td>
			<td>Thorlabs</td>
			<td>CEA1500</td>
			<td>provide the function of enabling top illumination techniques in microscopy</td>
		</tr>
		<tr>
			<td>60 mm Cage Mounted &#216;50.0 mm Iris</td>
			<td>Thorlabs</td>
			<td>LCP50S</td>
			<td>control the amount of light passing through an optical system</td>
		</tr>
		<tr>
			<td>60 mm Cage Mounting Bracket</td>
			<td>Thorlabs</td>
			<td>LCP01B</td>
			<td>mount and position a 60 mm cage system in optical setups</td>
		</tr>
		<tr>
			<td>Air spaced Etalon</td>
			<td>SLS Optics Ltd.</td>
			<td>Customized</td>
			<td>generate narrow-band 1030 nm light&#160;</td>
		</tr>
		<tr>
			<td>Cage Plate Mounting Bracket</td>
			<td>Thorlabs</td>
			<td>KCB2</td>
			<td>hold and adjust mirrors at a precise angle</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>Andor Technologies</td>
			<td>Newton&#160;</td>
			<td>2D CCD for frequency and spatial resolution</td>
		</tr>
		<tr>
			<td>Collinear Optical Parametric Amplifier</td>
			<td>Light Conversion</td>
			<td>Orpheus-One-HP</td>
			<td>Tunable MID light generator</td>
		</tr>
		<tr>
			<td>Copper Chloride</td>
			<td>Thermo Fischer Scientific</td>
			<td>A16064.30</td>
			<td>Self-assembly component</td>
		</tr>
		<tr>
			<td>Customized Dichroic Mirror</td>
			<td>Newport</td>
			<td>Customized</td>
			<td>selectively reflects or transmits light based on its wavelength or polarization</td>
		</tr>
		<tr>
			<td>Ext to M32 Int Adapter</td>
			<td>Thorlabs</td>
			<td>SM1A34</td>
			<td>provide compatibility and facilitating the connection between components with different thread types</td>
		</tr>
		<tr>
			<td>Infinity Corrected Refractive Objective</td>
			<td>Zeiss</td>
			<td>420150-9900-000</td>
			<td>Refractive Objective</td>
		</tr>
		<tr>
			<td>Infinity Corrected Schwarzschild Objective</td>
			<td>Pike Technologies Inc.</td>
			<td>891-0007</td>
			<td>Reflective objective</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Carbide, Light-Conversion</td>
			<td>C18212</td>
			<td>Laser source</td>
		</tr>
		<tr>
			<td>M32x0.75 External to Internal RMS</td>
			<td>Thorlabs</td>
			<td>M32RMSS</td>
			<td>adapt or convert the threading size or type of microscope objectives&#160;</td>
		</tr>
		<tr>
			<td>M32x0.75 External to M27x0.75 Internal Engraving</td>
			<td>Thorlabs</td>
			<td>M32M27S</td>
			<td>adapt or convert the threading size or type of microscope objectives&#160;</td>
		</tr>
		<tr>
			<td>Manual Mid-Height Condenser Focus Module</td>
			<td>Thorlabs</td>
			<td>ZFM1030</td>
			<td>adjust the focus of an optical element</td>
		</tr>
		<tr>
			<td>Monochromator</td>
			<td>Andor Technologies</td>
			<td>Shamrock 500i</td>
			<td>Provides frequency resolution for each line scan</td>
		</tr>
		<tr>
			<td>Motorized module with 1&#34; Travel for Edge-Mounted Arms</td>
			<td>Thorlabs</td>
			<td>ZFM2020</td>
			<td>control the vertical positon of the imaging objective</td>
		</tr>
		<tr>
			<td>Nanopositioner</td>
			<td>Mad City Labs Inc.</td>
			<td>MMP3</td>
			<td>3D sample stage</td>
		</tr>
		<tr>
			<td>Resonant Scanner</td>
			<td>EOPC</td>
			<td>SC-25</td>
			<td>325Hz resonant beam scanner</td>
		</tr>
		<tr>
			<td>RGB Color CCD Camera</td>
			<td>Thorlabs</td>
			<td>DCU224C</td>
			<td>Brightfield camera, discontinued but other cameras will work just as well</td>
		</tr>
		<tr>
			<td>RGB tube lens</td>
			<td>Thorlabs</td>
			<td>ITL200</td>
			<td>white light collection</td>
		</tr>
		<tr>
			<td>Right Angle Kinematic Breadboard</td>
			<td>Thorlabs</td>
			<td>OPX2400</td>
			<td>incorporate a sliding mechanism with two fixed positions</td>
		</tr>
		<tr>
			<td>Right Angle Kinematic Mirror Mount, 30 mm</td>
			<td>Thorlabs</td>
			<td>KCB1</td>
			<td>hold and adjust mirrors at a precise angle</td>
		</tr>
		<tr>
			<td>Right Angle Kinematic Mirror Mount, 60 mm</td>
			<td>Thorlabs</td>
			<td>KCB2</td>
			<td>hold and adjust mirrors at a precise angle</td>
		</tr>
		<tr>
			<td>SM2, 60 mm Cage Arm for Cerna Focusing Stage</td>
			<td>Thorlabs</td>
			<td>CSA2100</td>
			<td>securely mount and position condensers</td>
		</tr>
		<tr>
			<td>Snap on Cage Cover for 60 mm Cage, 24 in Long,</td>
			<td>Thorlabs</td>
			<td>C60L24</td>
			<td>enclose and protect the components inside the cage</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Thermo Fischer Scientific</td>
			<td>J63394.AK</td>
			<td>Self-assembly component</td>
		</tr>
		<tr>
			<td>Three-Chnnale Controller and Knob Box for 1&#34; Cerna Travel Stages</td>
			<td>Thorlabs</td>
			<td>MCM3001</td>
			<td>control ZFM2020</td>
		</tr>
		<tr>
			<td>Tube lens</td>
			<td>Thorlabs</td>
			<td>LA1380-AB - N-BK7</td>
			<td>SFG signal collection</td>
		</tr>
		<tr>
			<td>Visible LED Set</td>
			<td>Thorlabs</td>
			<td>WFA1010</td>
			<td>provide illumination in imaging setup</td>
		</tr>
		<tr>
			<td>Whitelight Source</td>
			<td>Thorlabs</td>
			<td>WFA1010</td>
			<td>Whitelight illumination source for brightfield imaging</td>
		</tr>
		<tr>
			<td>WPH05M-1030 - &#216;1/2&#34; Zero-Order Half-Wave Plate, &#216;1&#34; Mount, 1030 nm&#160;</td>
			<td>Thorlabs</td>
			<td>WPH05M-1030</td>
			<td>alter the polarization state of light passing through it</td>
		</tr>
		<tr>
			<td>WPLQ05M-3500 - &#216;1/2&#34; Mounted Low-Order Quarter-Wave Plate, 3.5 &#181;m&#160;</td>
			<td>Thorlabs</td>
			<td>WPLQ05M-3500</td>
			<td>alter the polarization state of light passing through it</td>
		</tr>
		<tr>
			<td>X axis Long Travel Steel Extended Contact Slide Stages</td>
			<td>Optosigma</td>
			<td>TSD-65122CUU</td>
			<td>positioning stages that offer extended travel in the horizontal (X) direction</td>
		</tr>
		<tr>
			<td>XT95 4in Rail Carrier</td>
			<td>Thorlabs</td>
			<td>XT95RC4</td>
			<td>mount and position optical components</td>
		</tr>
		<tr>
			<td>X-Y Axis Translation Stage w/ 360 deg. Rotation</td>
			<td>Thorlabs</td>
			<td>XYR1</td>
			<td>precise movement and positioning of objects in two dimensions, along with the ability to rotate the platform</td>
		</tr>
		<tr>
			<td>XY(1/2&#34;) Linear Translator with Central SM1 Thru Hole</td>
			<td>Thorlabs</td>
			<td>XYT1</td>
			<td>provide precise movement and positioning in two dimensions</td>
		</tr>
		<tr>
			<td>Yb doped Solid State Laser</td>
			<td>Light Conversion</td>
			<td>CB3-40W</td>
			<td>Seed laser</td>
		</tr>
		<tr>
			<td>&#946;-Cyclodextrin</td>
			<td>Thermo Fischer Scientific</td>
			<td>J63161.22</td>
			<td>Self-assembly component</td>
		</tr>
	</tbody>
</table>

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.

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.

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65388/65388fig05.jpg" /> 
Figure 5: Molecular structure, morphology and potential orientation of SDS@&#946;-CD. (A) Top-view and (B)&#160;side-view chemical structure of SDS@&#946;-CD. (C) Representative heterogeneous sample distribution of the mesoscale sheets on the sample plane. The molecular subunit could have diffe...

Watch this Scientific Journal Video about Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures at JoVE.com

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 ...