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 ...

multimodal-nonlinear-hyperspectral-chemical-imaging-using-line

Research

JoVE Journal

Chemistry

871 ビュー.  UC San Diego. 生体組織などのメゾスコピック的に不均質な自己組織化試料を研究し、その化学組成や分子配列を明らかにすることを目指しています。私たちは、これらの自己材料の微視的な配置とメゾスコピックな形態が、それらの微視的な性質とどのように関連しているかを探求しています。高開口数と反射型顕微鏡対物レンズの進歩により、ソフトマテリアルの画像を記録する?...

ビデオ: 著者スポットライト:メゾスコピック不均一自己組織化構造の研究におけるVSFG顕微鏡法の可能性の解明 

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.

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synthesis and characterization of self-assembled metal-organic framework monolayers using polymer-coated particles

Author Spotlight: Exploring Self-Assembled MOF-Polymer Composites

A protocol for the synthesis and characterization of self-assembled metal-organic framework monolayers is provided using polymer-grafted, metal-organic framework (MOF) crystals. The procedure shows that polymer-grafted MOF particles can be self-assembled at an air-water interface resulting in well-formed, free-standing, monolayer structures as evidenced by scanning electron microscopy...

Self-Assembly of Polymer-Grafted Metal Organic Framework Monolayers

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transgene expression in cultured cells using unpurified recombinant adeno-associated viral vectors

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

generation of self-assembled vascularized human skin equivalents

Generation of Self-assembled Vascularized Human Skin Equivalents

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research...

formation of ordered biomolecular structures by the self-assembly of short peptides

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based...

kinase inhibitor screening in self-assembled human protein microarrays

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is...

preparation of mechanically stable self-assembled peptides hydrogels

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward...

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

the third channel-assisted unilateral biportal endoscopic technique for lumbar spinal stenosis combined with contralateral disc herniation

Author Spotlight: Unveiling the Potential of TUBE Technique in Spinal Surgery

Here, we present the third channel-assisted UBE technique, which allows for the vertical removal of herniated disc fragments. This technique can effectively address the limitations of traditional UBE techniques. This article will systematically elaborate on this...

A 3-Channel Endoscopic Technique for Lumbar Stenosis and Contralateral Herniation

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antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the...

facile preparation of internally self-assembled lipid particles stabilized by carbon nanotubes

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug...

covalent binding of bmp-2 on surfaces using a self-assembled monolayer approach

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell...

preparation, characteristics, toxicity, and efficacy evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

microbiota of attine ants' gardens: visualizing a microbial landscape by scanning electron microscopy

Author Spotlight: Unveiling the Microbial Biofilm Structure in Fungus-Growing Ant Colonies

We propose an optimized Scanning Electron Microscopy protocol for visualizing highly heterogeneous and delicate samples containing plant and fungal biomass, together with microbiota and biofilm. This protocol allows describing the spatial dimensions of the microbiota...

 Microscale SEM Analysis of Microbial Communities in Fungus-Growing Ant Cultures

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polymerization of methyl acrylate from uio-66-ddmat for the synthesis of self-assembled metal-organic framework monolayers

Polymerization of Methyl Acrylate from UiO-66-DDMAT for the Synthesis of Self-Assembled Metal-Organic Framework Monolayers

surface modification of uio-66 with cat-ddmat for the synthesis of self-assembled metal-organic framework monolayers

Surface Modification of UiO-66 with cat-DDMAT for the Synthesis of Self-Assembled Metal-Organic Framework Monolayers

construction, characterization, and regenerative application of self-assembled human mesenchymal stem cell aggregates

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone...

quantifying the heterogeneous distribution of a synaptic protein in the mouse brain using immunofluorescence

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based...

Synthesis and Characterization of Self-Assembled Metal-Organic Framework Monolayers Using Polymer-Coated Particles

advanced self-healing asphalt reinforced by graphene structures: an atomistic insight

Advanced Self-Healing Asphalt Reinforced by Graphene Structures: An Atomistic Insight

Graphene-modified asphalt nanocomposite has shown an advanced self-healing ability compared to pure asphalt. In this protocol, molecular dynamics simulations have been applied in order to understand the role of graphene in the self-healing process and to explore the self-healing mechanism of asphalt components from the atomistic...

harvest and primary culture of leptomeningeal lymphatic endothelial cells

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of...

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

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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 Data Collection and Analysis

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.