Name: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy (Video) | JoVE
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This work was supported by Sonata Bis 9 project (2019/34/E/ST5/00276) financed by National Science Centre in Poland.

1. Preparing the microscope slides with fully-grown spherulites
NOTE: See the Table of Materials for details about all materials, reagents, and equipment used in this protocol. All solutions were prepared with deionized water (18.2 M&#937;&#183;cm at 25 &#176;C) obtained from the water purification system.
<ol>
	<li>Incubate the amyloid spherulites based on the protocol described by Krebs et al16. with some modifications, as ...

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. -. 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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>

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.

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

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

Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy (Video) | JoVE

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.

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