这项工作得到了波兰国家科学中心资助的Sonata Bis 9项目（2019/34/E/ST5/00276）的支持。

使用偏振敏感双光子显微镜表征球晶

<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>

作者没有利益冲突需要披露。

偏振敏感双光子显微镜是研究淀粉样蛋白超结构内部原纤维局部有序的宝贵工具，只需对标准多光子设置进行微小修改。由于它对非线性光学现象起作用，因此与单光子激发荧光显微镜方法相比，可以减少角度光选择并增强轴向分辨率。此外，与单光子激发荧光显微镜技术相比，它可降低光散射、降低光毒性和更深的样品穿透力19,20。正如我们已经证明...

在过去的几十年中，尽管许多用于蛋白质及其聚集体生物成像的荧光显微镜技术已经取得了重大发展1，但只有少数技术被用于解析它们在样品中的局部排序 2,3。采用荧光寿命成像显微镜4研究了淀粉样蛋白超结构-球晶的内在结构异质性。此外，可以使用偏振敏感方法解决复杂和致密的生物结构（如球晶）内部局部顺序的定量测定3。然而，具有浅层组织穿透的标准荧光技术是有限的，因为使用紫外可见分光度在体内激发荧光团会导致高组织光散射5。此外，这种成像通常需要设计特定的荧光探针并将其结合到目标生物分子上，从而增加了执行成像所需的成本和工作量。
最近，为了解决这些问题，我们的团队采用了偏振敏感双光子激发荧光显微镜 （ps-2PFM） 对生物结构进行无标记成像 6,7。Ps-2PFM允许测量双光子荧光强度对激发光束线性偏振方向的依赖性，并分析发射荧光的偏振8。该技术的实施需要用半波板补充标准多光子显微镜设置激发路径（图1）以控制光偏振平面。然后，从两个雪崩光电二极管收集的信号中创建极坐标图，描述双光子激发荧光强度对激发激光束偏振的依赖性，从而收集荧光偏振的两个相互垂直的分量。
最后一步是数据分析过程，考虑到光学元件（如二向色镜或高数值孔径物镜）对偏振的影响。由于双光子过程的性质，这种方法既减少了角度选择，又提高了轴向分辨率，因为焦平面外荧光团的双光子激发在概率上受到限制。还证明，类似的方法可以成功地用于近红外探针 （NIR） 的体内成像，用于深部组织成像9。Ps-2PFM先前已应用于细胞膜10和DNA11,12中的成像荧光团，以及生物系统的非标准荧光标记物，例如金纳米颗粒13。然而，在所有这些例子中，有关生物分子组织的信息都是间接获得的，并且需要荧光团和生物分子之间预定义的相互取向。
在我们最近的一篇论文中，我们已经证明 ps-2PFM 可以成功地用于确定淀粉样蛋白超结构的自发荧光的局部极化和硫黄素 T（一种淀粉样蛋白特异性染料）与球晶中的淀粉样蛋白原纤维结合的荧光6。此外，在另一项研究中，我们已经证明 ps-2PFM 可用于检测亚微米尺寸范围内淀粉样蛋白球晶内的淀粉样蛋白原纤维取向，这通过将其与透射电子显微镜 （TEM） 成像相关联来证实7.这一结果的实现得益于:i）球晶-淀粉样蛋白的内在自发荧光，当用一个或两个光子激发时，表现出内在的自发荧光，最大发射波长位于450至500nm范围内，双光子吸收截面与标准荧光染料相当14，ii）前面介绍的数学模型，用于描述如何将标记生物膜和DNA结构的染料的ps-2PEF应用于球晶和与其结合的染料表现出的荧光 8,11,15。因此，在进行分析之前，我们强烈建议您仔细阅读我们关于该主题的第一篇论文的正文和支持信息中描述的所需理论6.在这里，我们介绍了如何应用ps-2PFM技术对牛胰岛素球晶进行无标记淀粉样蛋白结构表征的方案。

<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

与单光子激发相比，双光子激发有利于生物成像实验，因为它具有较低的光毒性、更深的组织穿透性、在密集系统中的高效操作以及减少荧光团的角度光选择。因此，与基于线性光学过程的标准成像方法相比，在双光子荧光显微镜 （2PFM） 中引入偏振分析可以更精确地确定样品中的分子组织。在这项工作中，我们重点研究了偏振敏感的2PFM（ps-2PFM）及其在复杂生物结构-淀粉样蛋白球晶中分子有序测定中的应用。阿尔茨海默氏症或帕金森氏症等神经退行性疾病通常通过检测由于蛋白质错误折叠过程受损而形成的淀粉样蛋白-蛋白质聚集体来诊断。探索它们的结构可以更好地了解它们的产生途径，从而开发更灵敏的诊断方法。本文介绍了适用于测定牛胰岛素球晶和球形淀粉样蛋白聚集体内部局部原纤维顺序的ps-2PFM。此外，我们证明了所提出的技术可以解决球状体内部原纤维的三维组织。

所提出的方案通过制备用于 ps-2PFM 测试的淀粉样蛋白超结构、微观系统的构建和适当样品的测量提供了分步指导。然而，在进行最终一组测量之前，将APD与各向同性参考正确对齐至关重要，这应该会导致在两个探测器上收集形状和强度相似的对称信号（图4C）。在进一步的测量中，特别是在分析过程中，应将检测器在X轴和Y轴上测量的强度之间的最小差异作为适当的校正因子...

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

作者聚焦:使用偏振敏感双光子显微镜对复杂生物结构进行无创成像 

本文描述了如何应用偏振敏感双光子显微镜来表征无标记淀粉样蛋白超结构-球晶中的局部组织。它还描述了如何制备和测量样品、组装所需的设置以及分析数据以获取有关淀粉样蛋白原纤维局部组织的信息。

偏振敏感双光子显微镜用于无标记淀粉样蛋白结构表征

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

我们的目标是开发新的光学标记物和技术，用于检测称为淀粉样蛋白的蛋白质聚集体。他们涉及一系列疾病，如阿尔茨海默病和二型糖尿病。因此，为了治愈这些疾病，我们需要有可视化淀粉样蛋白的技术。我们证明了双光子显微镜可用于检测淀粉样蛋白超结构内的淀粉样原纤维取向。它不仅可以使用淀粉样蛋白特异性染料来实现，还可以通过利用淀粉样蛋白的自发荧光来实现。由于我们的技术适用于非线性光学现象，因此与单光子荧光显微技术相比，它可以实现减少角度光选择、增强轴向分辨率、降低光散射、降低光毒性和更深的样品穿透力。偏振敏感双光子荧光显微镜是一种很有前途的工具，可以对各种复杂生物结构的内部结构进行成像，不仅是蛋白质聚集体，还有DNA囊泡和脂质膜。

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

Research

JoVE Journal

Chemistry

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

1.0K 次观看.  Wrocław University of Science and Technology. 我们的目标是开发新的光学标记物和技术，用于检测称为淀粉样蛋白的蛋白质聚集体。他们涉及一系列疾病，如阿尔茨海默病和二型糖尿病。因此，为了治愈这些疾病，我们需要有可视化淀粉样蛋白的技术。我们证明了双光子显微镜可用于检测淀粉样蛋白超结构内的淀粉样原纤维取向。它不仅可以使用淀粉样蛋白特异性染料来实现，还可以通过利用淀粉样蛋白的自发荧?...

视频: 作者聚焦:使用偏振敏感双光子显微镜对复杂生物结构进行无创成像 

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.

生产二硫键稳定的跨膜肽复合物的结构研究

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

科研

化学

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.

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

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).

折叠的DNA折纸一个生物反应机器人与表征

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.

一个反分析方法化工运输的油漆表征

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.

功能化的合成与表征金属 - 有机骨架

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.

镉免费的InP /硫化锌量子点的合成适合生物医学应用

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.

超分子胶体的合成与表征

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).

低能量的阴极射线（氧）氮化物荧光粉

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.

C 型60/石墨烯混合纳米结构的制备与表征

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