This work was supported by Sonata Bis 9 project (2019/34/E/ST5/00276) financed by National Science Centre in Poland.

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

<ol>
	<li>Petazzi, R. A., Aji, A. K., Chiantia, S., Giraldo, J., Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in Molecular Biology and Translational Science. 169, 1-41 (2020).</li><li>Kress, A., 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+orientational+behavior+of+MHC+class+I+protein+and+lipid+probes+in+cell+membranes+by+fluorescence+polarization-resolved+imaging.">Probing orientational behavior of MHC class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging.</a> Biophysical Journal. 101 (2), 468-476 (2011).</li><li>Duboisset, 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=Thioflavine-T+and+Congo+Red+reveal+the+polymorphism+of+insulin+amyloid+fibrils+when+probed+by+polarization-resolved+fluorescence+microscopy.">Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.</a> The Journal of Physical Chemistry B. 117 (3), 784-788 (2013).</li><li>De Luca, G., 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+ensemble+polymorphism+and+single+aggregate+structural+heterogeneity+in+insulin+amyloid+self-assembly.">Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.</a> Journal of Colloid and Interface Science. 574, 229-240 (2020).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Obstarczyk, P., Lipok, M., Grelich-Mucha, M., Samo&#263;, M., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+excited+polarization-dependent+autofluorescence+of+amyloids+as+a+label-free+method+of+fibril+organization+imaging.">Two-photon excited polarization-dependent autofluorescence of amyloids as a label-free method of fibril organization imaging.</a> The Journal of Physical Chemistry Letters. 12 (5), 1432-1437 (2021).</li><li>Obstarczyk, P., Lipok, M., &#379;ak, A., Cwynar, P., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+fibrils+in+superstructures+-+local+ordering+revealed+by+polarization+analysis+of+two-photon+excited+autofluorescence.">Amyloid fibrils in superstructures - local ordering revealed by polarization analysis of two-photon excited autofluorescence.</a> Biomaterials Science. 10 (6), 1554-1561 (2022).</li><li>Le Floc'h, V., Brasselet, S., Roch, J. -. F., Zyss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+orientation+in+molecular+ensembles+by+polarization+sensitive+nonlinear+microscopy.">Monitoring of orientation in molecular ensembles by polarization sensitive nonlinear microscopy.</a> The Journal of Physical Chemistry B. 107 (45), 12403-12410 (2003).</li><li>Chen, C., 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=In+vivo+near-infrared+two-photon+imaging+of+amyloid+plaques+in+deep+brain+of+Alzheimer's+disease+mouse+model.">In vivo near-infrared two-photon imaging of amyloid plaques in deep brain of Alzheimer's disease mouse model.</a> ACS Chemical Neuroscience. 9 (12), 3128-3136 (2018).</li><li>Gasecka, P., Balla, N. K., Sison, M., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids-fluorophores+interactions+probed+by+combined+nonlinear+polarized+microscopy.">Lipids-fluorophores interactions probed by combined nonlinear polarized microscopy.</a> The Journal of Physical Chemistry B. 125 (50), 13718-13729 (2021).</li><li>Mojzisova, H., 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=Polarization-sensitive+two-photon+microscopy+study+of+the+organization+of+liquid-crystalline+DNA.">Polarization-sensitive two-photon microscopy study of the organization of liquid-crystalline DNA.</a> Biophysical Journal. 97 (8), 2348-2357 (2009).</li><li>Olesiak-Banska, 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=Liquid+crystal+phases+of+DNA:+Evaluation+of+DNA+organization+by+two-photon+fluorescence+microscopy+and+polarization+analysis.">Liquid crystal phases of DNA: Evaluation of DNA organization by two-photon fluorescence microscopy and polarization analysis.</a> Biopolymers. 95 (6), 365-375 (2011).</li><li>Olesiak-Banska, 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=Gold+nanorods+as+multifunctional+probes+in+a+liquid+crystalline+DNA+matrix.">Gold nanorods as multifunctional probes in a liquid crystalline DNA matrix.</a> Nanoscale. 5 (22), 10975-10981 (2013).</li><li>Grelich-Mucha, M., Lipok, M., R&#243;&#380;ycka, M., Samo&#263;, M., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-+and+two-photon+excited+autofluorescence+of+lysozyme+amyloids.">One- and two-photon excited autofluorescence of lysozyme amyloids.</a> The Journal of Physical Chemistry Letters. 13 (21), 4673-4681 (2022).</li><li>Brasselet, S., M&#233;ly, Y., Duportail, G., 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=.">.</a> Fluorescent Methods to Study Biological Membranes. , 311-337 (2013).</li><li>Krebs, M. R., 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=The+formation+of+spherulites+by+amyloid+fibrils+of+bovine+insulin.">The formation of spherulites by amyloid fibrils of bovine insulin.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (40), 14420-14424 (2004).</li><li>A&#239;t-Belkacem, D., Gasecka, A., Munhoz, F., Brustlein, S., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+birefringence+on+polarization+resolved+nonlinear+microscopy+and+collagen+SHG+structural+imaging.">Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging.</a> Optics Express. 18 (14), 14859-14870 (2010).</li><li>Sch&#246;n, P., Munhoz, F., Gasecka, A., Brustlein, S., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+distortion+effects+in+polarimetric+two-photon+microscopy.">Polarization distortion effects in polarimetric two-photon microscopy.</a> Optics Express. 16 (25), 20891-20901 (2008).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50 (6), 823-839 (2006).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21 (11), 1369-1377 (2003).</li><li>Ferrand, P., 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=Ultimate+use+of+two-photon+fluorescence+microscopy+to+map+orientational+behavior+of+fluorophores.">Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.</a> Biophysical Journal. 106 (11), 2330-2339 (2014).</li><li>Chipman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization+index+and+the+average+degree+of+polarization.">Depolarization index and the average degree of polarization.</a> Applied Optics. 44 (13), 2490-2495 (2005).</li><li>de Reguardati, S., Pahapill, J., Mikhailov, A., Stepanenko, Y., Rebane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-accuracy+reference+standards+for+two-photon+absorption+in+the+680&amp;#x2013;1050+nm+wavelength+range.">High-accuracy reference standards for two-photon absorption in the 680&amp;#x2013;1050 nm wavelength range.</a> Optics Express. 24 (8), 9053-9066 (2016).</li><li>Mansfield, J. C., Mandalia, V., Toms, A., Winlove, C. P., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+reorganization+in+cartilage+under+strain+probed+by+polarization+sensitive+second+harmonic+generation+microscopy.">Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy.</a> Journal of The Royal Society Interface. 16 (150), 20180611 (2019).</li><li>Duboisset, J., A&#239;t-Belkacem, D., Roche, M., Rigneault, H., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+model+of+the+molecular+orientational+distribution+probed+by+polarization-resolved+second-harmonic+generation.">Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation.</a> Physical Review A. 85 (4), 043829 (2012).</li><li>Loksztejn, A., Dzwolak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+Bifurcation+in+aggregating+insulin:+an+induced+circular+dichroism+study.">Chiral Bifurcation in aggregating insulin: an induced circular dichroism study.</a> Journal of Molecular Biology. 379 (1), 9-16 (2008).</li></ol>

The authors have no conflicts of interest to disclose.

Polarization-sensitive two-photon microscopy is a valuable tool for studying the local ordering of fibrils inside the amyloid superstructures, requiring only small modifications of the standard multiphoton setup. Since it operates on nonlinear optical phenomena, reduced angular photo-selection and enhanced axial resolution can be achieved compared to one-photon excited fluorescence microscopy methods. In addition, it leads to lower light scattering, lower phototoxicity, and deeper sample penetration when compared to the ...

Over the past decades, although there has been significant development of numerous fluorescence microscopy techniques for bioimaging of proteins and their aggregates1, only a few have been used to resolve their local ordering within the sample2,3. Fluorescence lifetime imaging microscopy4 was used to study the intrinsic structural heterogeneity of amyloid superstructures-spherulites. Moreover, quantitative determination of the local ordering inside complex and densely-packed biostructures such as spherulites could be resolved using polarization-sensitive methods3. However, standard fluorescence techniques with superficial tissue penetration are limited since using UV-VIS wavelengths to excite fluorophores in vivo leads to high tissue light scattering5. In addition, such imaging often requires designing and binding specific fluorescent probes to a targeted biomolecule, thereby increasing the cost and amount of work needed to perform imaging.
Recently, to address these issues, our team has adapted polarization-sensitive two-photon excited fluorescence microscopy (ps-2PFM) for label-free imaging of biological structures6,7. Ps-2PFM allows for the measurement of the dependence of two-photon fluorescence intensity on the direction of linear polarization of the excitation beam and analysis of the polarization of the emitted fluorescence8. The implementation of this technique requires supplementation of the standard multi-photon microscope setup excitation path (Figure 1) with a half-wave plate to control the light polarization plane. Then, polar graphs, depicting the dependence of two-photon excited fluorescence intensity on the polarization of the excitation laser beam, are created from signals collected by two avalanche photodiodes, thus collecting the two, mutually perpendicular components of fluorescence polarization.
The last step is the data analysis process, taking into account the impact of the optical elements, such as dichroic mirrors or a high numerical aperture objective, on polarization. Because of the nature of the two-photon process, this method provides both reduced angular photo-selection and enhanced axial resolution, as two-photon excitation of fluorophores outside the focal plane is probabilistically limited. It was also proven that similar methods could be successfully implemented for in vivo imaging of near-infrared probes (NIR) for deep-tissue imaging9. Ps-2PFM has been previously applied to image fluorophores in cell membranes10 and DNA11,12, as well as non-standard fluorescent markers of biological systems, such as gold nanoparticles13. However, in all these examples, the information about the organization of biomolecules was obtained indirectly and required a predefined mutual orientation between a fluorophore and a biomolecule.
In one of our recent papers, we have shown that ps-2PFM could be successfully applied for determining the local polarization of the autofluorescence of amyloid superstructures and fluorescence from Thioflavin T, an amyloid-specific dye, bound to amyloid fibrils in spherulites6. Furthermore, in another one, we have proven that ps-2PFM could be utilized to detect amyloid fibril orientation inside amyloid spherulites in a sub-micron size regime, which was confirmed by correlating it with transmission electron microscopy (TEM) imaging7. The achievement of this outcome was possible thanks to i) intrinsic autofluorescence of spherulites-amyloids, when excited with either one or two photons, exhibit intrinsic autofluorescence with emission maxima located in a range from 450 to 500 nm and two-photon absorption cross sections comparable with standard fluorescent dyes14, ii) mathematical models introduced previously to describe how ps-2PEF of dyes labeling biological membranes and DNA structures could be applied to fluorescence exhibited by spherulites and dyes bound to them8,11,15. Thus, before proceeding with the analysis, we highly recommend looking through the required theory described in both, the main text and supporting information of our first paper concerning this topic6. Here, we present the protocol for how to apply the ps-2PFM technique for a label-free amyloid structural characterization of bovine insulin spherulites.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 24 x 24 mm</td>
			<td>Chemland</td>
			<td>04-298.202.04</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mountant for histology</td>
			<td>Sigma-Aldrich</td>
			<td>6522</td>
			<td>Slide mountant</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock tubes, 1.5 mL, polypropylene</td>
			<td>Chemland</td>
			<td>02-63102</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ThermoMixer&#160;C</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Used for spherulite incubation</td>
		</tr>
		<tr>
			<td>HLP 5UV Water purification system</td>
			<td>Hydrolab</td>
			<td></td>
			<td>Source of dionized water used in sample preparation</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (&#8805;37%, APHA &#8804;10),</td>
			<td>Sigma-Aldrich</td>
			<td>30721-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin powder from the bovine pancreas (&#8805;25 units/mg (HPLC))</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (HPLC grade)</td>
			<td>Sigma-Aldrich</td>
			<td>270474</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides with a concave, 76 x 26 x 1 mm</td>
			<td>Chemland</td>
			<td>04-296.202.09</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX60</td>
			<td>Olympus</td>
			<td></td>
			<td>Polarized Optical Microscope used in Figure 2</td>
		</tr>
		<tr>
			<td>PTFE thread seal tape, 12 mm x 12 mm x 0.1 mm, 60 gm2</td>
			<td>Chemland</td>
			<td>VIT131097</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope ps-2PFM setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ultra II</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FELH0800 - &#216;25.0 mm Longpass Filter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FESH0700 - &#216;25.0 mm Shortpass Filter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDQ100 photon-counting avalanche photodiodes&#160;</td>
			<td>ID Quantique</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton short-pass emission filter 720 nm&#160;</td>
			<td>Semrock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounted Achromatic Half-Wave Plate, 690-1200 nm</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Plan Apo Oil Immersion 100x/1.4 NA</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>piezo 3D stage</td>
			<td>Piezosystem Jena</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polarizing Beamsplitter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S130C - Slim Photodiode Power Sensor, Si, 400 - 1100 nm, 500 mW</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabView 2018</td>
			<td>National Instruments</td>
			<td></td>
			<td>Version 18.0.1f2</td>
		</tr>
		<tr>
			<td>Matplotlib library</td>
			<td></td>
			<td></td>
			<td>Version 3.3.2</td>
		</tr>
		<tr>
			<td>NumPy library</td>
			<td></td>
			<td></td>
			<td>Version 1.19.2</td>
		</tr>
		<tr>
			<td>SciPy library</td>
			<td></td>
			<td></td>
			<td>Version 1.5.2</td>
		</tr>
		<tr>
			<td>Spyder Python 3 IDE</td>
			<td></td>
			<td></td>
			<td>Version 4.1.5</td>
		</tr>
	</tbody>
</table>

polarization-sensitive two-photon microscopy for a label-free amyloid structural characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

The presented protocol provides step-by-step guidance through the preparation of amyloid superstructures for testing with ps-2PFM, construction of the microscopic system, and measurements of the proper sample. However, before the final set of measurements, it is vital to properly align the APDs with an isotropic reference, which should result in collecting a symmetrical signal of similar shape and intensity on both detectors (Figure 4C). Even minimal differences between the intensities measu...

Watch this Scientific Journal Video about Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy at JoVE.com

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

polarization-sensitive-two-photon-microscopy-for-label-free-amyloid

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Chemistry

1.0K Views.  Wrocław University of Science and Technology. This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

Video: Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...