Name: interfacial molecular-level structures of polymers and biomacromolecules revealed via sum frequency generation vibrational spectroscopy
Uploaded: 2019-08-13T13:30:00
Description: Watch this Scientific Journal Video about Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy at JoVE.com

This study was supported by the State Key Development Program for Basic Research of China (2017YFA0700500) and the National Natural Science Foundation of China (21574020). The Fundamental Research Funds for the Central Universities, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University) were also greatly appreciated.

1. SFG experimental
<ol>
	<li>Use a commercial picosecond SFG system (Table of Materials), which provides a fundamental 1064 nm beam with a pulse width of ~20 ps and a frequency of 50 Hz, based on an Nd:YAG laser.</li>
	<li>Convert the fundamental 1064 nm beam into a 532 nm beam and a 355 nm beam by using second and third harmonic modules. Directly guide the 532 nm beam as an input light beam and generate the other input mid-infrared (IR) beam covering the frequency range from 1000 to ...

<ol>
	<li>Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Second+Harmonic+Generation+at+Interfaces.">Optical Second Harmonic Generation at Interfaces.</a> Annual Review of Physical Chemistry. 40, 327-350 (1989).</li><li>Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+properties+probed+by+second-harmonic+and+sum-frequency+generation.">Surface properties probed by second-harmonic and sum-frequency generation.</a> Nature. 337, 519-525 (1989).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Polymer+Surfaces+and+Interfaces+with+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Studying Polymer Surfaces and Interfaces with Sum Frequency Generation Vibrational Spectroscopy.</a> Analytical Chemistry. 89 (1), 466-489 (2017).</li><li>Chen, X., Clarke, M. L., Wang, J., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Molecular+Conformation+and+Orientation+of+Biological+Molecules+at+Interfaces.">Sum Frequency Generation Vibrational Spectroscopy Studies on Molecular Conformation and Orientation of Biological Molecules at Interfaces.</a> International Journal of Modern Physics B. 19 (4), 691-713 (2005).</li><li>Eisenthal, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Interfaces+Probed+by+Second-Harmonic+and+Sum-Frequency+Spectroscopy.">Liquid Interfaces Probed by Second-Harmonic and Sum-Frequency Spectroscopy.</a> Chemical Reviews. 96 (4), 1343-1360 (1996).</li><li>Richmond, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molcular+Bonding+and+Interactions+at+Aqueous+Surfaces+as+Probed+by+Vibrational+Sum+Frequency+Spectroscopy.">Molcular Bonding and Interactions at Aqueous Surfaces as Probed by Vibrational Sum Frequency Spectroscopy.</a> Chemical Reviews. 102 (8), 2693-2724 (2002).</li><li>Wang, H., Gan, W., Lu, R., Rao, Y., Wu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+spectral+and+orientational+analysis+in+surface+sum+frequency+generation+vibrational+spectroscopy(SFG-VS).">Quantitative spectral and orientational analysis in surface sum frequency generation vibrational spectroscopy(SFG-VS).</a> International Reviews in Physical Chemistry. 24 (2), 191-256 (2007).</li><li>Shultz, M. J., Schnitzer, C., Simonelli, D., Baldelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+frequency+generation+spectroscopy+of+the+aqueous+interface:+Ionic+and+soluble+molecular+solutions.">Sum frequency generation spectroscopy of the aqueous interface: Ionic and soluble molecular solutions.</a> International Reviews in Physical Chemistry. 19 (1), 123-153 (2010).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Surface+Hydration+of+Poly(2-hydroxyethyl+methacrylate)+in+Solution+in+situ.">Detecting Surface Hydration of Poly(2-hydroxyethyl methacrylate) in Solution in situ.</a> Macromolecules. 49, 3116-3125 (2016).</li><li>Li, X., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Irreversibly+Absorbed+Layer+Promotes+Dewetting+of+Polystyrene+Film+on+Sapphire.">Evolution of Irreversibly Absorbed Layer Promotes Dewetting of Polystyrene Film on Sapphire.</a> Macromolecules. 51, 6653-6660 (2018).</li><li>Lu, X., Spanninga, S. A., Kristalyn, C. B., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Orientation+of+Phenyl+Groups+in+Poly(sodium+4-styrenesulfonate)+and+in+Poly(sodium+4-styrenesulfonate):+Poly(3,4-ethylenedioxythiophene)+Mixture+Examined+by+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Surface Orientation of Phenyl Groups in Poly(sodium 4-styrenesulfonate) and in Poly(sodium 4-styrenesulfonate): Poly(3,4-ethylenedioxythiophene) Mixture Examined by Sum Frequency Generation Vibrational Spectroscopy.</a> Langmuir. 26 (17), 14231-14235 (2010).</li><li>Lu, X., Clarke, M. L., Li, D., Wang, X., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sum+Frequency+Generation+Vibrational+Study+of+the+Interference+Effect+in+Poly(n-butyl+methacrylate)+Thin+Films+Sandwiched+between+Silica+and+Water.">A Sum Frequency Generation Vibrational Study of the Interference Effect in Poly(n-butyl methacrylate) Thin Films Sandwiched between Silica and Water.</a> Journal of Physical Chemistry C. 115, 13759-13767 (2011).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+Probing+Molecular+Ordering+at+the+Buried+Polymer/Metal+Interface+2:+Using+P-Polarized+Input+Beams.">Directly Probing Molecular Ordering at the Buried Polymer/Metal Interface 2: Using P-Polarized Input Beams.</a> Macromolecules. 45, 6087-6094 (2012).</li><li>Lu, X., Myers, J. N., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Ordering+of+Phenyl+Groups+at+the+Buried+Polystyrene/Metal+Interface.">Molecular Ordering of Phenyl Groups at the Buried Polystyrene/Metal Interface.</a> Langmuir. 30, 9418-9422 (2014).</li><li>Li, B., Lu, X., Ma, Y., Han, X., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+Probe+Glass+Transition+Temperatures+of+Polymer+Thin+Films.">Method to Probe Glass Transition Temperatures of Polymer Thin Films.</a> ACS Macro Letters. 4, 548-551 (2015).</li><li>Li, X., Deng, G., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchain+Overlap+Affects+Formation+of+Silk+Fibroin+Secondary+Structure+on+Hydrophobic+Polystyrene+Surface+Detected+via+Achiral/Chiral+Sum+Frequency+Generation.">Interchain Overlap Affects Formation of Silk Fibroin Secondary Structure on Hydrophobic Polystyrene Surface Detected via Achiral/Chiral Sum Frequency Generation.</a> Langmuir. 34, 9453-9459 (2018).</li><li>Kai, S., Li, X., Li, B., Han, X., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+hydrolysis+of+supported+planar+lipids+was+triggered+by+honey+bee+venom+phospholipase+A2+with+the+right+orientation+at+the+interface.">Calcium-dependent hydrolysis of supported planar lipids was triggered by honey bee venom phospholipase A2 with the right orientation at the interface.</a> Physical Chemistry Chemical Physics. 20, 63-67 (2018).</li><li>Wang, J., Buck, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Protein+Adsorption.">Sum Frequency Generation Vibrational Spectroscopy Studies on Protein Adsorption.</a> Journal of Physical Chemistry B. 106, 11666-11672 (2002).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Amide+I+Signals+of+Interfacial+Proteins+in+Situ+Using+SFG.">Detection of Amide I Signals of Interfacial Proteins in Situ Using SFG.</a> Journal of American Chemical Society. 125, 9914-9915 (2003).</li><li>Nguyen, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Spontaneous+Membrane+Insertion+of+a+Tall-Anchored+Membrane+Protein+by+Sum+Frequency+Generation+Spectroscopy.">Probing the Spontaneous Membrane Insertion of a Tall-Anchored Membrane Protein by Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 132, 15112-15115 (2010).</li><li>Li, X., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Ions+Affect+Water+Molecular+Structures+Surrounding+an+Oligonucleotide+Duplex+as+Revealed+by+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Calcium Ions Affect Water Molecular Structures Surrounding an Oligonucleotide Duplex as Revealed by Sum Frequency Generation Vibrational Spectroscopy.</a> Langmuir. , (2018).</li><li>Sartenaer, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency+generation+spectroscopy+of+DNA+monolayers.">Sum-frequency generation spectroscopy of DNA monolayers.</a> Biosensors &amp; Bioelectronics. 22, 2179-2183 (2007).</li><li>Asanuma, H., Noguchi, H., Uosaki, K., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+Cation-induced+Deformation+of+DNA+Self-Assembled+Monolayers+on+Silicon:+Vibrational+Sum+Frequency+Generation+Spectroscopy.">Metal Cation-induced Deformation of DNA Self-Assembled Monolayers on Silicon: Vibrational Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 130, 8016-8022 (2008).</li><li>Howell, C., Schmidt, R., Kurz, V., Koelsch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency-generation+spectroscopy+of+DNA+films+in+air+and+aqueous+environments.">Sum-frequency-generation spectroscopy of DNA films in air and aqueous environments.</a> Biointerphases. 3 (3), FC47 (2008).</li><li>Walter, S. R., Geiger, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+on+Stage:+Showcasing+Oligonucleotides+at+Surfaces+and+Interfaces+with+Second+Harmonic+and+Vibrational+Sum+Frequency+Generation.">DNA on Stage: Showcasing Oligonucleotides at Surfaces and Interfaces with Second Harmonic and Vibrational Sum Frequency Generation.</a> Journal of Physical Chemistry Letters. 1, 9-15 (2010).</li><li>Li, Z., Weeraman, C., Azam, M. S., Osman, E., Gibbs-Davis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermal+reorganization+of+DNA+immobilized+at+the+silica/buffer+interface:+a+vibrational+sum+frequency+generation+investigation.">The thermal reorganization of DNA immobilized at the silica/buffer interface: a vibrational sum frequency generation investigation.</a> Physical Chemistry Chemical Physics. 17, 12452-12457 (2015).</li><li>Lambert, A. G., Neivandt, D. J., Briggs, A. M., Usadi, E. W., Davies, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Spectra+from+Monolayers+on+Composite+Dielectric/Metal+Substrates.">Interference Effects in Sum Frequency Spectra from Monolayers on Composite Dielectric/Metal Substrates.</a> Journal of Physical Chemistry B. 106, 5461-5469 (2002).</li><li>Tong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+effects+in+the+sum+frequency+generation+spectra+of+thin+organic+films.+I.+Theoretical+modeling+and+simulation.">Interference effects in the sum frequency generation spectra of thin organic films. I. Theoretical modeling and simulation.</a> Journal of Chemical Physics. 133, 034704 (2010).</li><li>McGall, S. J., Davies, P. B., Neivandt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Vibrational+Spectra+of+Thin+Polymer+Films:+An+Experimental+and+Modeling+Investigation.">Interference Effects in Sum Frequency Vibrational Spectra of Thin Polymer Films: An Experimental and Modeling Investigation.</a> Journal of Physical Chemistry B. 108, 16030-16039 (2004).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Fresnel+Coefficients+and+Molecular+Structures+of+Model+Cell+Membranes:+From+a+Lipid+Monolayer+to+a+Lipid+Bilayer.">Interfacial Fresnel Coefficients and Molecular Structures of Model Cell Membranes: From a Lipid Monolayer to a Lipid Bilayer.</a> Journal of Physical Chemistry C. 118, 28631-28639 (2014).</li><li>Zhou, J., Anim-Danso, E., Zhang, Y., Zhou, Y., Dhinojwala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Water+at+Polyurethane-Sapphire+Interface.">Interfacial Water at Polyurethane-Sapphire Interface.</a> Langmuir. 31 (45), 12401-12407 (2015).</li><li>Gautam, K. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-Frequency+Vibrational+Spectroscopy+on+Chiral+Liquids:+A+Novel+Technique+to+Probe+Molecular+Chirality.">Sum-Frequency Vibrational Spectroscopy on Chiral Liquids: A Novel Technique to Probe Molecular Chirality.</a> Physical Review Letters. 85, 4474 (2000).</li><li>Rockwood, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6, 1612-1631 (2011).</li></ol>

To investigate the structural information from a molecular level, SFG has its inherent advantages (i.e., monolayer or sub-monolayer sensitivity and interfacial selectivity), which can be applied to study various interfaces, such as the solid/solid, solid/liquid, solid/gas, liquid/gas, liquid/liquid interfaces. Although the equipment maintenance and the optical alignment are still time-consuming, the payoff is significant in that the detailed molecular-level information at the surfaces and interfaces can be obtained.
...

Development of sum frequency generation (SFG) vibrational spectroscopy can be dated back to the work done by Shen et al. thirty years ago1,2. The uniqueness of the interfacial selectivity and sub-monolayer sensitivity makes SFG vibrational spectroscopy appreciated by a large number of researchers in the fields of physics, chemistry, biology, and materials science, etc3,4,5. Currently, a broad range of scientific issues related to surfaces and interfaces are being investigated using SFG, especially for complex interfaces with respect to polymers and biomacromolecules, such as the chain structures and structural relaxation at the buried polymer interfaces, the protein secondary structures, and the interfacial water structures9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26.
For polymer surfaces and interfaces, thin-film samples are generally prepared by spin-coating to obtain the desired surfaces or interfaces. The problem arises due to the signal interference from the two interfaces of the as-prepared films, which leads to inconvenience for analyzing the collected SFG spectra27,28,29. In most cases, the vibrational signal only from one single interface, either film/substrate or film/the other medium, is desirable. Actually, the solution to this problem is quite easy, namely, to experimentally maximize the light fields at the desirable interface and minimize the light fields at the other interface. Hence, the Fresnel coefficients or the local field coefficients need to be calculated via the thin film model and to be validated with respect to the experimental results3,9,10,11,12,13,14,15,30.
With the above background in mind, some polymer and biological interfaces could be investigated in order to understand fundamental science from the molecular level. In the following, taking three interfacial issues as examples: probing poly(2-hydroxyethyl methacrylate) (PHEMA) surface and buried interface with substrate9, formation of silk fibroin (SF) secondary structures on the polystyrene (PS) surface and water structures surrounding model short-chain oligonucleotide duplex16,21, we will show how the SFG vibrational spectroscopy helps to reveal the interfacial molecular-level structures in connection to the underlying science.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)&#160;</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850355P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>100092680</td>
			<td>&#8805;99.7%</td>
		</tr>
		<tr>
			<td>CaF2 prism</td>
			<td>Chengdu YaSi Optoelectronics Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10005817</td>
			<td>&#8805;96.0%</td>
		</tr>
		<tr>
			<td>deuterated DPPC (d-DPPC)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>860345P-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Electromagnetic oven</td>
			<td>Zhejiang Supor Co., Ltd</td>
			<td>C21-SDHCB37</td>
			<td></td>
		</tr>
		<tr>
			<td>Langmuir-Blodgett (LB) trough</td>
			<td>KSV NIMA Co., Ltd.</td>
			<td>KN 2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium bromide anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>20056926</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q synthesis system</td>
			<td>Millipore</td>
			<td></td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Chengdu Mingheng Science&#38;Technology Co., Ltd</td>
			<td>PDC-MG</td>
			<td>Oxygen plasma cleaning</td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate) (PHEMA)</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>192066 MSDS</td>
			<td>Mw = 300 000</td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>330345 MSDS</td>
			<td>Mw = 48 kDa and Mn = 47 kDa</td>
		</tr>
		<tr>
			<td>Silk cocoons</td>
			<td></td>
			<td></td>
			<td>From Bombyx mori</td>
		</tr>
		<tr>
			<td>Single complementary strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H03596</td>
			<td>5&#39;-CGAAGGCTTCCAGCT-3&#39;</td>
		</tr>
		<tr>
			<td>Single strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H04936</td>
			<td>&#160;3&#162;-end modified by cholesterol-triethylene glycol(Chol-TEG) (5&#162;-GCTTCCGAAGGTCGA-3&#162;)</td>
		</tr>
		<tr>
			<td>Sodium carbonate anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019260</td>
			<td>&#8805;99.8%</td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Institute of Microelectronics of the Chinese Academy of Sciences</td>
			<td>KW-4A</td>
			<td>For the prepartion of ploymer films&#160;</td>
		</tr>
		<tr>
			<td>Step profiler</td>
			<td>Veeco</td>
			<td>DEKTAK 150</td>
			<td>For the measurement of film thickness</td>
		</tr>
		<tr>
			<td>Sum frequency generation (SFG) vibrational spectroscopy system</td>
			<td>EKSPLA</td>
			<td></td>
			<td>A commercial picosecond SFG system</td>
		</tr>
	</tbody>
</table>

interfacial molecular-level structures of polymers and biomacromolecules revealed via sum frequency generation vibrational spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

In the Fresnel coefficient part of Protocol Section, we have shown that, theoretically, it is feasible to selectively detect only one single interface at one time. Here, experimentally, we confirmed that this methodology is basically correct, as shown in Figure 5 and Figure 6.&#160;
Figure 5 shows the buried interfacial PHEMA structure after water intrusion with a ~150 nm PHEMA hydrogel film and <strong c...

Watch this Scientific Journal Video about Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy at JoVE.com

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Sum frequency generation vibrational spectra are fitted to investigate interfaces with interfacial selectivity. However Fresnel coefficient method can help to resolve the interference effect when another interface exists. The main advantage of SFG is the interfacial selectivity and submonolayer sensitivity.This noninvasive optical technique is suitable for investing various interfaces such as solid-liquid, solid-solid, liquid-liquid, solid-gas, and liquid-gas interfaces. We suggest that beginners get a lot of practice operating the SFG system with the help of experts in optics and the SFG. There are not shortcuts.SFG has not been generally recognized as powerful technique, so we would like to attract more audience interest and encourage more researchers to apply this technique. To begin, prepare about five millilters of either a two or 4%by weight solution of poly-2-hydroxyethyl methacrylate or PHEMA in anhydrous ethanol. Keep the solution in the lab three days before use.Next, soak four IR grade calcium fluoride right angled prism in analytical grade anhydrous toluene for at least 12 hours. Then immerse the prisms in 30 millilters of ethanol and wipe the surfaces with degreasing cotton for about 10 minutes. Rinse the prism with ultra pure water for two minutes and dry them with nitrogen gas.Next, place the prisms in an oxygen plasma cleaner and evacuate the chamber. Treat the prisms with oxygen plasma for four minutes and keep them in the chamber until they are used. Film preparation should occur within one hour of plasma treatment.In this study, to selectively detect the barrier interface it is of great importance to choose the appropriate film thickness according to the calculated result of the Fresnal coefficient model. When ready to prepare the PHEMA films, fix a clean prism in a prism holder on a spin coater and apply one drop of the solution of PHEMA in ethanol to the prism. Run the spin coater at 1, 500 RPM for one minute to prepare the PHEMA film.Coat additional plasma treated prisms with PHEMA in the same way. Anneal the films in a vacuum oven set to 80 degrees Celsius for at least 10 hours. To perform the experiment place the PHEMA coated face of the prism in deionized water.Wait 10 to 20 minutes and then evaluate the interfacial PHEMA structure at the water and prism interfaces with some frequency generation spectroscopy. While operating the FGS system do not let the light beam directly come into your eyes. To begin preparing the silk fibroin solution heat three liters of 0.02 molar aqueous sodium carbonate to boiling.Boil 7.5 grams of Bombyx mori silk cocoons in this solution while stirring for 30 minutes. Transfer the fibrous matter to a clean container and stir it in two to three liters of deionized water for eight to 10 minutes three times to wash off the unwanted sericin molecules. Dry the fibrous matter in a vacuum oven at 60 degrees Celsius for at least 15 hours.Next, dissolve one gram of dry, degummed silk fibroin in four millilters of 9.3 molar aqueous lithium bromide. Stir the solution at 60 degrees Celsius for two hours. Then place the silk fibroin solution in a 3, 500 Dalton dialysis bag.Dialyze the solution against one liter of deionized water for three days, changing the water three times per day. Once dialysis is complete, store the silk fibroin solution at four degrees Celsius. Next, use the previously described methods to clean a calcium fluoride prism and coat it with a thin polystyrene film from a 3.5%by weight polystyrene solution.Place the polystyrene coated prism in contact with the silk fibroin solution and examine the polystyrene silk fibroin interface with SFG spectroscopy. To begin preparing the oligonucleotide duplex, dissolve 10 nanomoles of the appropriate single-stranded oligonucleotide in 0.5 millilters of ultrapure water. Do the same for the complimentary oligonucleotide.Combine the solutions to obtain a 10 nanomoles per milliliter duplex oligonucleotide solution. Then combine two milligrams of DPPC, two milligrams of deuterated DPPC, and one milliliter of chloroform to obtain the lipid solution. Next, clean and plasma treat a calcium fluoride prism as previously described.Attach this prism to the sample hold of a Langmuir-Blodgett trough. Fill the trough with deionized water and lower one face of the prism into the trough at one millimeter per minute. Inject several microliters of the lipid solution onto the surface of the water and wait for the surface pressure to stabilize at about 12 millinewtons per meter.Then start compressing the lipid monolayer at five millimeters per minute. When the surface pressure reaches 34 millinewtons per meter lift the prism from the trough at one millimeter per minute. Next prepare 500 microliters of a mixture of the duplex oligonucleotide solution and the lipid solution in a one to 100 molar ratio.Then replace the Langmuir-Blodgett trough with a cylindrical polytetrafluorethylene container filled with deionized water. Inject the oligonucleotide lipid mixture onto the water until the surface pressure reaches 34 millinewtons per meter. Put the lipid monolayer coated face of the prism in contact with the lipid oligonucleotide mixture to form the final sample.Evaluate the chiral and achiral water vibrational signals with FSG spectroscopy. In this first example, the interface between PHEMA hydrogels and the calcium fluoride prism showed clear sharp peaks in the SFG spectrum. This was attributed to the smooth interface between calcium fluoride and the hydrogel.The interface between the hydrogel and the surrounding water had broader, less intense features because water molecules could diffuse into the bulk of the hydrogel. Here when the silk fibroin concentration was above the critical overlapping concentration there were do detected chiral secondary structures at the solution polystyrene interface unless methanol was added as an inducing agent. Below the critical overlapping concentration, chiral SFG signals were detected even without adding methanol indicating that ordered secondary structures formed unassisted at the solution polystyrene interface.In the duplex oligonucleotide anchored lipid bilayer sample varying the calcium ion concentration had no significant effect on the chiral water signal which primarily corresponds to the hydration later of the chiral spine in the minor groove. In contrast, the calcium concentration strong effected the achiral water signals which primarily correspond to the water layer surrounding the duplex chain and the bilayer. Over all this suggested that the chiral spine of the water layer could protect the oligonucleotide from calcium ions.Calculating the Fresnel coefficient and choosing an appropriate film thickness can resolve interference problems. If multi-reflection and refraction are considered the right electric field distribution at the interfaces can be adjusted. There are alternative methods that can be used to prepare thin films with desired thicknesses such as step-coating and the chemical vapor deposition.Detecting the surface and interfacial structures related to adhesion, whiting, friction, and other properties can help researchers understand the underlying mechanisms and develop new functional materials. This method can also be applied to other systems where varied interfaces need to be investigated. However, the medium you wish the light beams propagate must be transparent.

interfacial-molecular-level-structures-polymers-biomacromolecules

Research

JoVE Journal

Biochemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...