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

Published: December 1st, 2023



1Department of Chemistry and Biochemistry, UC San Diego, 2State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 3Materials Science and Engineering Program, UC San Diego, 4Department of Electrical and Computer Engineering, UC San Diego

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 spectroscopy technique with a spatial resolution of ~100 µm. 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 µm 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.

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 samples3,4,5,6,7,8,9,10,11,12,13,1....

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1. Hyperspectral line-scanning VSFG microscope

  1. Laser system
    1. Use a pulsed laser system (see Table of Materials) centered at 1025 nm ± 5 nm. The laser is set at 40 W, 200 kHz (200 µJ/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.
    2. Guide the output of the seed laser into a commercial optical parametric amplifier (OPA) to generate a mid-infrare.......

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Figure 5
Figure 5: Molecular structure, morphology and potential orientation of SDS@β-CD. (A) Top-view and (B) side-view chemical structure of SDS@β-CD. (C) Representative heterogeneous sample distribution of the mesoscale sheets on the sample plane. The molecular subunit could have diffe.......

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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 SDS@2β-CD 100 µm by 100 µm sheets, a good resolution image (~1 µm resolution) with a high signal-to-noise ratio would take 20 min. This is al.......

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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).


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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

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