作者希望感谢迈克尔林格伦和南 Sluzny 关于荧光显微镜的建议, 和阿德里亚诺 aguzzi-dur, 约翰 Bijzet, Bouke Hazenberg, 弗兰克赫普纳, Jucker, 苔蕾丝 Klingstedt, 卡琳马格努森, 克里斯蒂娜 Sigurdson, 丹尼尔 Sjölander,克里斯托弗 Röcken, 古尼拉 Westermark, 每 Westermark, 和库尔特 Zatloukal 的贡献组织部分或显微显示在本出版物。本文所收集的数据由瑞典脑基金会 (Hjärnfonden)、瑞典阿尔茨海默病协会 (Alzheimerfonden)、瑞典研究理事会 (VR)、约兰·佩尔松 Gustafsson 协会、乔治和 #38 的捐款提供资金。奥尔森, 欧盟 FP7 健康项目 LUPAS, 林雪平大学。

注意: 柴油合成已经在我们的实验室进行了十多年。通过对亲芳香取代、钯催化交叉、酰胺偶联和酯水解的协议的基本应用, 我们为不同用途而综合定制探头 5 , 12 . 经过合成、表征和纯化后, 柴油产品在室温下冻干并贮存。一些探测器 (其中 hFTAA) 现在是商用的 (请参见 材料表 ). 
 1. 柴油染色液 注意: 如果 hFTAA 是从商业供应商购买的, 请按照供应商和 #39 的说明操作, 而不是1节. 
<ol> <li> 重2毫米氢氧化钠中的冻干 hFTAA, 以制备1毫克/毫升的储存液。把存货放在 4 #176 的玻璃瓶里。股票可以储存一年. </li>
	<li> 在染色的当天, 通过稀释1:10,000 的磷酸盐缓冲盐水 (PBS) 来制备一个工作溶液. </li>
</ol> 2。组织标本的制备 注意: 许多组织类型可以用 hFTAA 作为淀粉样标记来成像。有关示例, 请参见 图 1 。hFTAA 对骨料构象敏感。因此, 染色最好是对不的聚合体进行, 没有表位暴露。如果将组织固定保持在最低限度, 就能获得最佳的频谱质量。因此, 新鲜冰冻的材料, 轻轻地固定在酒精染色时, 是首选。然而, 有可能检测淀粉样沉积也在组织中已固定与 如 , 福尔马林。hFTAA 通常穿透组织好。选择与预期成像技术兼容的试样厚度. 
<ol> <li> 如果福尔马林固定, parafinembedded 部分使用, deparafinize 在二甲苯过夜。连续浸泡99% 乙醇、70% 乙醇、dH 2 O 和 PBS, 每段10分钟。允许组织切片在环境条件下干燥. 
	注意: 二甲苯总是在化学油烟罩中处理。二甲苯和其他有机溶剂是有害的。
	<ol> <li> 在室温下融化 cryosections。在10% 福尔马林过夜和水化的组织切片, 浸泡在连续的99% 乙醇, 70% 乙醇, dH 2 O, PBS, 每10分钟。允许组织切片在环境条件下干燥. </li>
	</ol> </li> <li> 将 hFTAA 工作解决方案的液滴 (大约200和 #181; L) 添加到组织部分以覆盖它。水滴应该通过表面张力保持在原地。在室温下孵育30分钟以进行染色. </li>
	<li> 用500和 #181 冲洗染色液, 然后用吸管将玻片浸入 pbs 浴中10分钟. 允许该剖面在环境条件下干燥. </li>
	<li> 安装使用荧光安装介质。允许安装介质过夜. 
	注意: hFTAA 染色可以与其他染色方法, 如免疫荧光, 细胞或特定的器官特异标记, 等 结合使用。要执行 co-staining, 运行您的选择的完整染色协议, 并添加 hFTAA 染色在年底, 从步骤2.2 开始。有关示例, 请参见 图 2 。对于免疫荧光, 最好选择一个第二抗体, 是兴奋在 640 nm 或更高。在这个波长范围内, hFTAA 不吸收, 因此不能兴奋, 也不会荧光。这确保了 hFTAA 和抗体之间不会有流血. </li>
</ol> 3。显微镜 注意: 使用带有长通滤波器的荧光显微镜。下面的所有设置都用于在 图 4 中生成图像。可能需要根据淀粉样蛋白类型和组织样本进行调整。虽然 hFTAA 与淀粉样蛋白的结合是稳定的漂白, 它是可取的关闭光源时, 标本是不检查或成像. 
<ol> <li> 他的 注意: 该实验是使用荧光显微镜与长传递发射过滤器和相机头为他 (见 材料表 ) 进行的。
	<ol> <li> 使用标准荧光显微镜, 配有长通滤波器和高光谱相机。确保频谱摄像头校准. </li>
		<li> 在软件中, 命名项目、选择和 #34; 光谱图像和 #34; 开始获取. </li>
		<li> 使用 436 nm 的励磁过滤器, 通过眼睛选择感兴趣的对象, 并将光源路径移到照相机上. </li>
		<li> 在案例数据管理器 (CDM) 中, 选择样本类型光谱, 对样本进行标注, 然后按获取。将会打开 "获取" 窗口. </li>
		<li> 在购置窗口和 #34; 光谱成像和 #34; 打开 "设置" 菜单, 选择 "购置属性", 并将频谱范围设置为 460-700, 速度质量以最大速度, 在气体/激光/窄带过滤器上测量类型。关闭对话框. </li>
		<li> 在 "图像" 菜单中, 选择 "实时满"。在图标栏中, 关闭条纹。选择或调整图像的区域, 使其峰值内存值低于 800 MB。将曝光时间设置为在1000和3000之间提供总图像亮度的值。在图标栏中, 按下彩色照相机。收购将开始。当图像采集完成时, 在 #34 中按下保存; 获取光谱图像和 #34; 对话框和 #34; 新的单元和 #34; 在 "数据管理器" 对话框中. </li>
		<li> 从 CDM 中, 使用 "开始分析" 按钮打开收集的图像。将会打开 "数据分析" 窗口. </li>
		<li> 通过使用 "频谱显示" 对话框选择 roi, 可以从图像的每个像素收集光谱信息。选择和 #34;d efine 和 #34; 从图像的相关区域中选择 roi. </li>
		<li> 使用 Lib 按钮将频谱数据保存为文本文件。可以将保存的 .txt 文件导入到任何选择的分析软件中。. slb 文件可用于数据分析软件中的分析。从整个图像的高光谱数据集也可以导出为原始文件, 作为和 #34; 层作为 tif 和 #34;, 或作为和 #34; 文本格式和 #34 的层; 用于使用外部软件的数据和图像分析应用程序. </li>
	</ol> </li> <li> 共焦显微镜 注: 共焦显微镜装有可调谐激光器作为激发源。所有的荧光发射实验将激光强度设置为 0.2% (对应于平均功率为3和 #181; W), 针孔至1通风单元, 帧大小为 1024 px x 1024 像素, 扫描速度为 7, 平均超过16扫描, 位深度为8位 (见 s阮富仲 > 材料表 )。这些设置需要调整每个单独的共焦系统, 激光源, 和样本类型。
	<ol> <li> 用于发射频谱, 收集使用 lambda 模式的数据和使用氩激光的励磁设置为 488 nm。在32通道喘气探测器中使用22通道收集503和 687 nm 之间的发射。将增益设置为 755. </li>
		<li> 若要实现单个通道图像, 请使用 "智能设置" 选项。对于 FITC (绿色滤镜), 将增益设置为 750, 对于 Alexa 535 (红色滤镜) 将增益设置为 845. </li>
		<li> 收集励磁谱与可调谐的激光, 使用励磁与1毫微米步在490和545毫微米之间, 当收集放射在551和586毫微米之间。将激光强度设置为 2% (对应于平均功率为30和 #181; W), 增益为 774. </li>
		<li> 要在频谱模式下收集 Z 堆栈, 请在0.96 和 #181 的步骤中扫描剖面的深度; m。相同的激励和发射设置, 在步骤3.2.1 可以使用, 但设置增益为 730. </li>
	</ol> </li> <li> 电影 注意: 共焦显微镜装有一个电影单元 (参见 材料表 )。
	<ol> <li> 为共焦显微镜设置以下参数: 针孔, 20; 激发波长, 490 nm; 激光强度, 0.5% (对应于平均功率为7.5 和 #181; W)。在 40 MHz 时使用脉冲激光器. </li>
		<li> 在电影软件中, 设置光子计数超过 550 nm。在显示参数窗口中, 跟随光子计数, 直到最大计数是大约4000光子计数. </li>
		<li> 保存文件并将其导出为 SPC 映像. </li>
		<li> 将数据与电影软件中的2个组件的指数衰减相匹配。给出 #967 的适合; 2 和 #60; 2 是好的。y-axis 上的值是给定生存期的计数数. </li>
		<li> 选择要包括的计数的阈值, 例如 , 100。通过 T1 的颜色代码并选择生存期范围。衰变依赖于 hFTAA 的淀粉样结构。观察到300和 1000 ps 之间的荧光寿命。保存该文件. </li>
		<li> 通过使用导出选项导出原始数据, 并将所需数据保存到新文件夹中. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sipe, J. D., 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=Amyloid+fibril+proteins+and+amyloidosis:+chemical+identification+and+clinical+classification+International+Society+of+Amyloidosis+2016+Nomenclature+Guidelines.">Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines.</a> Amyloid. 23, 209-213 (2016).</li><li>Buxbaum, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+amyloidoses:+are+transgenic+mice+worth+the+time+and+trouble?.">Animal models of human amyloidoses: are transgenic mice worth the time and trouble?.</a> FEBS letters. 583, 2663-2673 (2009).</li><li>Hall, A. M., Roberson, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+Alzheimer's+disease.">Mouse models of Alzheimer's disease.</a> Brain Res Bull. 88, 3-12 (2012).</li><li>Westermark, G. T., Johnson, K. H., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+methods+for+identification+of+amyloid+in+tissue.">Staining methods for identification of amyloid in tissue.</a> Methods in enzymology. , 3-25 (1999).</li><li>Klingstedt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+library+of+oligothiophenes+and+their+utilization+as+fluorescent+ligands+for+spectral+assignment+of+protein+aggregates.">Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates.</a> Org Biomol Chem. 9, 8356-8370 (2011).</li><li>Klingstedt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescent+conjugated+oligothiophenes+for+sensitive+fluorescent+assignment+of+protein+inclusion+bodies.">Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.</a> Chembiochem. 14, 607-616 (2013).</li><li>Sjolander, D., 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=Establishing+the+fluorescent+amyloid+ligand+h-FTAA+for+studying+human+tissues+with+systemic+and+localized+amyloid.">Establishing the fluorescent amyloid ligand h-FTAA for studying human tissues with systemic and localized amyloid.</a> Amyloid. 23, 98-108 (2016).</li><li>Magnusson, K., 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=Multimodal+fluorescence+microscopy+of+prion+strain+specific+PrP+deposits+stained+by+thiophene-based+amyloid+ligands.">Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands.</a> Prion. 8, 319-329 (2014).</li><li>Radde, 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=Abeta42-driven+cerebral+amyloidosis+in+transgenic+mice+reveals+early+and+robust+pathology.">Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology.</a> EMBO Rep. 7, 940-946 (2006).</li><li>Sturchler-Pierrat, 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=Two+amyloid+precursor+protein+transgenic+mouse+models+with+Alzheimer+disease-like+pathology.">Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, 13287-13292 (1997).</li><li>Nystrom, S., 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=Evidence+for+age-dependent+in+vivo+conformational+rearrangement+within+Abeta+amyloid+deposits.">Evidence for age-dependent in vivo conformational rearrangement within Abeta amyloid deposits.</a> ACS chemical biology. 8, 1128-1133 (2013).</li><li>Aslund, 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=Novel+pentameric+thiophene+derivatives+for+in+vitro+and+in+vivo+optical+imaging+of+a+plethora+of+protein+aggregates+in+cerebral+amyloidoses.">Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.</a> ACS chemical biology. 4, 673-684 (2009).</li><li>Nilsson, K. P., Herland, A., Hammarstrom, P., Inganas, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+polyelectrolytes:+conformation-sensitive+optical+probes+for+detection+of+amyloid+fibril+formation.">Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation.</a> Biochemistry. 44, 3718-3724 (2005).</li><li>Mahajan, V., 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=Cross+beta-sheet+conformation+of+keratin+8+is+a+specific+feature+of+Mallory-Denk+bodies+compared+with+other+hepatocyte+inclusions.">Cross beta-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.</a> Gastroenterology. 141, 1080-1090 (2011).</li><li>Sjolander, D., Bijzet, J., Hazenberg, B. P., Nilsson, K. P., Hammarstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+rapid+assessment+of+amyloid+by+oligothiophene+fluorescence+in+subcutaneous+fat+tissue.">Sensitive and rapid assessment of amyloid by oligothiophene fluorescence in subcutaneous fat tissue.</a> Amyloid. 22, 19-25 (2015).</li><li>Arja, K., 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=Enhanced+fluorescent+assignment+of+protein+aggregates+by+an+oligothiophene-porphyrin-based+amyloid+ligand.">Enhanced fluorescent assignment of protein aggregates by an oligothiophene-porphyrin-based amyloid ligand.</a> Macromol Rapid Commun. 34, 723-730 (2013).</li><li>Psonka-Antonczyk, K. M., 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=Nanoscale+Structure+and+Spectroscopic+Probing+of+Abeta1-40+Fibril+Bundle+Formation.">Nanoscale Structure and Spectroscopic Probing of Abeta1-40 Fibril Bundle Formation.</a> Front Chem. 4, 44 (2016).</li><li>Herrmann, U. S., 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=Structure-based+drug+design+identifies+polythiophenes+as+antiprion+compounds.">Structure-based drug design identifies polythiophenes as antiprion compounds.</a> Sci Transl Med. 7, 299ra123 (2015).</li><li>Schutz, A. K., 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+amyloid-Congo+red+interface+at+atomic+resolution.">The amyloid-Congo red interface at atomic resolution.</a> Angewandte Chemie (International ed). 50 (26), 5956-5960 (2011).</li><li>Hammarstrom, 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=A+fluorescent+pentameric+thiophene+derivative+detects+in+vitro-formed+prefibrillar+protein+aggregates.">A fluorescent pentameric thiophene derivative detects in vitro-formed prefibrillar protein aggregates.</a> Biochemistry. 49, 6838-6845 (2010).</li><li>CORDIS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Final Report Summary - LUPAS (Luminescent polymers for in vivo imaging of amyloid signatures). , (2013).</li><li>Hahn, K., 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=Establishing+and+validating+the+fluorescent+amyloid+ligand+h-FTAA+(heptamer+formyl+thiophene+acetic+acid)+to+identify+transthyretin+amyloid+deposits+in+carpal+tunnel+syndrome.">Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome.</a> Amyloid. , 1-9 (2017).</li><li>Johansson, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+azide+functionalized+oligothiophene+ligand--a+versatile+tool+for+multimodal+detection+of+disease+associated+protein+aggregates.">An azide functionalized oligothiophene ligand--a versatile tool for multimodal detection of disease associated protein aggregates.</a> Biosens Bioelectron. 63, 204-211 (2015).</li><li>Nordeman, 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=(11)C+and+(18)F+Radiolabeling+of+Tetra-+and+Pentathiophenes+as+PET-Ligands+for+Amyloid+Protein+Aggregates.">(11)C and (18)F Radiolabeling of Tetra- and Pentathiophenes as PET-Ligands for Amyloid Protein Aggregates.</a> ACS Med Chem Lett. 7, 368-373 (2016).</li></ol>

PH PN MB SN 是埃生物技术的小股东, 市场化 hFTAA 在品牌名称 Amytracker545。

aberrantly 折叠蛋白在淀粉样蛋白中的沉积是许多疾病过程的重要事件。在阿尔茨海默病患者的大脑中, 由 hyperphosphorylated tau a 肽和胞内纤维缠结组成的胞外淀粉样斑块被发现。一系列不同的蛋白质,例如, 蛋白 (TTR), 血清淀粉样蛋白 A 组分 (升), 和 IgG 轻链错误和表现为淀粉样沉积在组织外的中枢神经系统。虽然淀粉样沉积物已被认识和研究了一个多世纪, 我们仍然缺乏详细的知识如何开始在分子水平的淀粉样蛋白沉积, 以及可以做些什么来防止这个过程。对于阿尔茨海默氏病, 有必要证实淀粉样变性的过程是如何与神经相关的。治疗淀粉样变性的一个关键任务是开发新的 fine-tuned 工具来监测淀粉样蛋白的起始、进展和回归;因此, 靶向淀粉样蛋白检测是关键。从长远看, 临床诊断将受益于提高淀粉样染色方法的灵敏度和选择性7,15。目前在这方面的挑战是巨大的, 是一个非常积极的研究领域。临床发展和试验的一个主要问题是预后诊断。这些疾病很可能在症状出现之前就开始, 但从治疗开始, 需要对疾病进行鉴别。在这里, 灵敏度是新方法的一个关键方面。此外, 这个问题更复杂, 因为一些蛋白质聚集物是有毒的, 有些是保护性的, 有些是中性的。因此, 有能力监测特定的聚集 morphotypes 是必不可少的, 因为存在不同的聚集物种已被建议解释的异构表型报告的多样性神经退行性蛋白聚集疾病。例如, 朊蛋白是一个典型的例子, 一个相同的主要氨基酸序列如何可以错误成不同的聚合 morphotypes, 从而产生特定的朊病毒菌株。同样的多态性也报告了 a 肽, α-核, 和 tau。在这方面, 已被证明是不同的聚合 morphotypes 光学分配的杰出工具。由于构象引起的光学现象, 在不同类型的系统性淀粉样变性, 以及多态性 a 和 tau 聚合体中发现了朊蛋白-菌株特异的蛋白质聚集物。
淀粉样蛋白沉积是发生在难以穿透生物化学或生物物理方法的分子精确度的组织中的事件。探测事件的可能性ex 体内,在体内, 和在体外使用相同的柴油分子设置的阶段, 以解开事件发生在体内使用的技术, 只可能适用于ex 体内或体外示例11,17。最近报告的高分辨率结构模型表明, 柴油 pentameric 自由贸易区 (pFTAA) 绑定在由平行的侧链形成的腔中, 并与纤维轴并行, 在寄存器内并行测试链18, 这表明它是可能获得有关柴油目标实体的原子解析知识。实质上, pFTAA 的结合腔和结合模式与刚果红19相似, 由一个带有重复的正电荷的赖氨酸侧链的凹槽所决定。与刚果红的亲和力似乎更好, 因为链的灵活性和强大的范德华相互作用的硫原子的噻吩环对疏水腔。prefibrillar 物种的检测 (在响应之前)5,20出现依赖于重复的β板料, 由寄存器平行β-股组成, 其中 hFTAA 比 pFTAA 更长的两个噻吩单位跨度跨越8β-股。
染色协议需要注意以下步骤的问题: (i) 固定: 组织样品的广泛固定可以扰乱淀粉样结构, 并限制检测荧光光谱变化的可能性。hFTAA 分子的构象畸变。从新鲜冷冻材料中 cryosections 的轻度固定是获得最佳光谱分辨率的首选。然而, hFTAA 会在固定组织中染色和荧光淀粉样蛋白, 但功效降低, 光谱变异较少。(ii) 表位暴露: 由于淀粉样蛋白结构的紊乱, 组织的前处理以达到表位抗体结合的接触可能会偶尔降低 hFTAA 的结合能力。如果这是一个问题, 和表位暴露是一个关键步骤的抗体染色协议, 使用连续的部分抗体和 hFTAA, 分别可以考虑。(iii) Overstaining: hFTAA 是非常敏感的。工作解决方案应保持在低 nM 浓度。如果背景染色是一个问题, 则降低 hFTAA 的浓度。如果束缚 hFTAA 的元素在组织中是稀疏的, 过量的 hFTAA 可以聚集并积聚在安装介质中。这是公认的荧光, 不是在焦点平面的组织本身。如果出现这种情况, 请将安装的幻灯片浸入 pbs, 直到盖板滑动到剖面上, 用 pbs 清洗, 然后重新装入新的安装介质, 并重新盖上盖子。(iv) 基于滤波器的成像: 本文主要介绍利用长通滤波器或多个检测通道进行光谱分辨荧光成像。hFTAA 也可以使用短传递过滤器进行监控。请注意, 这将减少对比度, 并取消检测多种颜色的可能性。
在过去的几年中, 越来越明显的是, 作为研究工具, 荧光管, 特别是 hFTAA 显示了非常有希望的性质。结果已经证明了大量的蛋白质和疾病状态, 从 hFTAA 检测的聚集细胞内 (tau, 包涵体肌炎), 系统性淀粉样蛋白 a, 蛋白, 免疫球蛋白轻链 (卡帕和λ) seminogelin 1, 朊蛋白 (包括临床样品)21, 胰岛淀粉样多肽, 和胰岛素7,15。作为诊断工具实施 hFTAA 的一个限制因素是, 荧光显微镜在常规的淀粉样蛋白病理实验室中没有被广泛使用。此外, 他在诊所也不容易得到。然而, hFTAA 淀粉样染色和荧光显微镜已用于临床实验室在一个过滤器为基础的荧光显微镜, 并应被认为是一种免费的诊断方法。这是最近在腕管活检与蛋白淀粉样变性使用的基本设置荧光在一个自动化的时尚22。
此外, 除了各种蛋白质外, hFTAA 荧光还能识别出大量的纤维类型和 pre-fibrillar 淀粉样的聚合体,例如, 检测到了通路纤维物种并进行了定性。在本刊物中, 我们已经展示了一个柴油的各种样本类型使用三显微镜技术。受控合成的使用也允许开发用于生物分子目标的多用途检测,例如, 通过表面等离子体共振 (SPR)23和 radiolabeling 的相互作用记录正电子发射断层扫描 (PET)24。
到目前为止, 已有超过25不同的已发布。增加对淀粉样沉积物的影像学的使用将增加对这些疾病相关的聚合体如何聚集、分解和干扰它们所居住的器官和个体的认识和理解。

淀粉样蛋白在组织中的沉积是一些疾病的病理特征, 如阿尔茨海默氏病、帕金森氏病、系统性淀粉样变性和朊病毒病。尽管与淀粉样蛋白相关的疾病普遍存在, 而且到目前为止, 近40种不同的蛋白质被归类为人类1中的淀粉样前体, 但对淀粉样蛋白沉积与疾病表型之间的关系知之甚少。从人类病人的标本组织学已经被用于诊断和科学目的。已经建立了大量的动物模型来研究淀粉样蛋白负荷与行为、寿命和其他一些疾病进展的表型读出之间的相关性2,3。在药物发现和设计方面也作出了巨大的努力, 以抗击我们最害怕的广泛疾病。然而, 对基因型、表型、淀粉样斑块负荷与药物管理之间的关系的评价并不直接。用于组织中淀粉样蛋白染色和成像的工具通常是钝性的, 并提供低分辨率的淀粉样蛋白的形成和结构信息。
刚果红双折射, 和荧光是典型的方法, 检测和分析组织样本中的淀粉样的负担, 从病人活检和验尸样本, 和动物模型的疾病4。这些技术已经使用了几十年 (自二十世纪二十年代以来的刚果红和自二十世纪六十年代以来的衍生品和衍生物), 虽然仪器已被提炼, 并允许详细分析, 染色程序和分析仍在执行在几乎一个世纪前的同样的方式。
在本文中, 我们描述了一种高度敏感的新的淀粉样蛋白染料的利用, hFTAA5, 这使得检测小未成熟蛋白质沉积高准确度, 以及检测成熟淀粉样。与传统染料相比, hFTAA 已被证明可以检测到更广泛的疾病相关蛋白聚集5,6,7。此外, hFTAA 还可以应用于不同的聚合 morphotypes8的光学分配。在这里, 我们描述 hFTAA 染色和分析的组织从建立的朊病毒疾病和淀粉样β前体蛋白 (APP) 转基因小鼠的设计, 以模拟斑块发育阿尔茨海默病的研究9,10.我们还展示了对系统性淀粉样变性患者的诊断和尸检样本的分析。这些有内在属性, 使他们能够报告在一个牌匾的构象差异;并结合两个不同的属性与绑定和荧光的性质, 差异更显着11。一种荧光显微镜, 装有长通滤波器和高光谱相机头, 用于实现光谱特性的客观分类和显微谱分析的记录。以可调谐激光为激发源的共焦荧光显微镜对淀粉样蛋白的三维性质进行了更详细的评价。可调谐激光可以收集的激励频谱和一步少选择发射波长的显微镜下, 使 co-imaging 的柴油荧光和免疫荧光, 以确定共存的目标蛋白和淀粉样。电影提供了前所未有的敏感性, 对构象差异强加给了污染和揭示的差异, 可能无法检测的荧光发射光谱。
所述染色方法的主要目的是促进对小淀粉样沉积物的灵敏检测, 并在淀粉样沉积物中表征构象多态性。这些知识对于蛋白质聚集疾病的基本理解是非常重要的。

<table>
	<tbody>
		<tr>
			<td>hFTAA/Amytracker545</td>
			<td>Ebba Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dako fluorescene mounting medium</td>
			<td>Agilent technologies</td>
			<td>GM304</td>
			<td></td>
		</tr>
		<tr>
			<td>LeicaDM6000</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lumen 200</td>
			<td>Prior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectraview system</td>
			<td>ASI spectral imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectraview software</td>
			<td>ASI spectral imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM780</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen 2010b v6.0 software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FLIM system</td>
			<td>Becker &#38; Hickl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ar/ML 458/488/514</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tunable Laser In Tune</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging amyloid tissues stained with luminescent conjugated oligothiophenes by hyperspectral confocal microscopy and fluorescence lifetime imaging

在全身的组织中沉积为淀粉样蛋白的蛋白质可能是大量疾病的起因或后果。其中, 我们发现神经退行性疾病, 如阿尔茨海默氏症和帕金森病, 主要困扰中枢神经系统, 和系统性淀粉样变性, 血清淀粉样蛋白 A, 蛋白和 IgG 光链作为淀粉样蛋白在肝脏, 腕隧道, 脾脏, 肾脏, 心脏, 和其他周围组织。淀粉样蛋白已被称为和研究了一个多世纪, 通常使用淀粉样蛋白的特定染料, 如刚果红和 Thioflavin 吨或 Thioflavin (这)。本文以 heptamer-甲酰基噻吩乙酸 (hFTAA) 为例, 对这些染料进行了新的补充, 称为发光共轭吩。hFTAA 易于使用, 并与 co-staining 免疫荧光或其他细胞标记相容。广泛的研究已经证明, hFTAA 检测的疾病相关的蛋白质总量比传统的淀粉样染料的范围更广。此外, hFTAA 也可用于光学分配的不同的聚合 morphotypes, 以允许研究淀粉样纤维多态性。虽然应用的成像方法是可选的, 我们这里演示高光谱成像 (他), 激光扫描共聚焦显微镜和荧光寿命成像 (电影)。这些例子显示了一些成像技术, 在那里, 可利用 amyloids 作为工具, 以获得更详细的知识的形成和结构的性质的。对该技术的一个重要限制是, 就所有传统光学显微镜技术而言, 对集料的微观尺寸的要求, 以允许检测。此外, 骨料应包括一个重复的β表结构, 以允许 hFTAA 约束力。过多的固定和/或表位接触, 修改的聚合结构或构象, 可以呈现较差的 hFTAA 结合, 因此对精确成像的限制。

从人类患者和实验动物的许多不同组织类型的大量蛋白质中, 对淀粉样蛋白的敏感和选择性染色对临床诊断和科学研究至关重要。在本文中, 我们展示了如何可以实现这一点, 通过 hFTAA 的例子, 为染色和多模态成像的淀粉样蛋白从选择的组织类型。自从十年前的 thiophene-based 淀粉样体配体第一次发表13, 在各种组织中由多种蛋白质组成的淀粉样沉积物已被图像使用了6、7、11, 14,15,16 (图 1)。与抗体或其他组织学标记相结合, 该方法为诊断和科学目的提供了优秀的工具 (图 1a,图 2)。通过适当选择荧光标记, 可以在同一剖面 (图 1a、hFTAA 和 DAPI、图 2a hFTAA 在免疫荧光设置中) 对多个荧光进行成像。还可以使用明和荧光 (图 2b, hFTAA 和抗体染色) 连续的部分染色。最近, hFTAA 已被证明是一个非常有前途的补充刚果红染色在临床诊断7,15 (图 3)。
在过去的几十年中, 成像技术的发展增加了对淀粉样蛋白的需求, 这些染料可以在一分钟内辨别, 但同时在数量上对淀粉样沉积物的深度差异。柴油内的共轭系统为它提供了一种独特的能力来报告其装订方式的变化。分子的扭曲会导致共轭系统中的结合角失真, 并阻碍电子的传输 (图 4a)。这反过来又反映了柴油的荧光性质, 无论是在激发和发射波长和发射寿命。我们使用他在一个直立的荧光显微镜, 装有长通滤波器的排放。对于共焦显微镜和电影中的激发和发射谱, 我们使用脉冲激光 LSM780。为了举例说明不同的技术, 我们成像一个对象 (β淀粉样蛋白 (a) 斑块在 APP 转基因鼠标) (图 4b-e)。高光谱荧光光谱 (图 4b) 允许对斑块不同区域内的光谱位移和强度变化进行评估。共聚焦显微镜 (图 4c) 的激发谱可用于确定下游实验的最佳激发波长,如, 组合染色与几种荧光。这种方法也可以被证明是有用的, 以检测的构象差异的结合模式的柴油。共焦模式的发射光谱可用于测定 hFTAA 或多种荧光组合实验中定位的荧光发射特性, 并结合几种柴油和免疫荧光组合.hFTAA 的荧光寿命受到 hFTAA 结合方式的微小变化的影响。因此, 电影是区分绑定目标 (图 4d) 对 hFTAA 施加的差异的强大工具。这可以用于例如不同的朊病毒菌株之间的区别8。共聚焦成像和处理 Z 栈成三维图像是一个有用的工具, 探索的整体形状的淀粉样蛋白聚合 (图 4e)。再次, 使用额外的荧光可以帮助理解存款的组成。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56279/56279fig1.jpg"> 
图 1: 各种组织类型和蛋白质聚集物显示 h 自由贸易区染色: (a) 马洛里-Denk 体组成的角蛋白聚集在肝脏 (1-4 与 DAPI), (b) p62-positive r 夹杂物在散发包涵体肌炎 (s-IBM) 肌肉组织, (c) 胰岛淀粉样多肽在人的胰腺, (d) 的免疫球蛋白轻链淀粉样蛋白在人的肠道, (e) 绵羊病 (朊) 在小鼠的大脑中, (f) 慢性消瘦症 (朊) 在老鼠脑中, (g) a 斑在 APP23 鼠脑, (h) a 病理在 APP/PS1 鼠脑, 和 (i) 脂肪活检涂片从诊断样本的蛋白淀粉样蛋白在人类患者等级 1-4 根据标准刚果红 (CR) 评分。黄色区域显示 hFTAA 染色的淀粉样沉积物和蓝色是来自脂肪组织的自发荧光。在每个面板中指定刻度线长度。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56279/56279fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56279/56279fig2.jpg"> 
图 2: 带有抗体的 co-staining 的示例.(a) 用抗淀粉样蛋白 a (aa) 免疫荧光和 hFTAA 人 aa 淀粉样在同一切片上进行联合染色。上部左: AA 抗体放射640毫微米;右上 hFTAA 在 488 nm;底部面板覆盖图像显示定位在黄色。(b) 抗体染色和 hFTAA 荧光连续切片。顶部面板: AA 抗体民建联染色, 底部面板: hFTAA 成像与 longpass 发射过滤器。缩放栏: 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56279/56279fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56279/56279fig3.jpg"> 
图 3:荧光与短通滤波器对人心脏蛋白淀粉样蛋白的比较染色与迈尔的苏木精/刚果红色 (a-d) 和迈尔的苏木精/hFTAA (e)。(a) 明图像, (b) 明 + 荧光, (c) 明 + 交叉偏振, (d) 明 + 荧光 + 交叉偏振, (e) hFTAA 荧光405nm + 480 nm 励磁 (连续的部分到a-d)。缩放栏: 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56279/56279fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56279/56279fig4.jpg"> 
图 4: 用 hFTAA 和几项技术成像的同一个物体用一个 a 的 APP23 老鼠的斑块来演示.(a) hFTAA 的荧光性质取决于其构象状态。给出了 hFTAA 在平面和扭转状态下的结构。(b) 用于评估连续发射谱的高光谱成像。下面板中的光谱是根据图像中有兴趣的盒装区域进行颜色编码的。(c) (i) 用于励磁成像的共焦成像和 (ii 和 iii) 光谱和发射成像在过滤模式或 (iv) 谱模式。(d) 荧光寿命成像检测淀粉样蛋白的构象差异, 在 a 斑块的外围发现较短的生存期, 而在斑块的中心处的寿命更长。下面板中的直方图显示整个斑块的生命时间分布。图像根据直方图下的刻度进行着色。(e) 在筛选模式中收集的共焦 Z 堆栈, 以显示对象的三维结构。在每个面板中指定刻度线长度。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56279/56279fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging at JoVE.com

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

淀粉样蛋白沉积是不同疾病的标志, 并影响许多不同的器官。本文介绍了荧光结合吩荧光染色技术在光学显微镜下的应用。这种染色方法是一种强有力的工具, 用于检测和探索蛋白质集在临床和科学的设置。

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

imaging-amyloid-tissues-stained-with-luminescent-conjugated

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.3K 次观看.  Linköping University. 淀粉样蛋白沉积是不同疾病的标志, 并影响许多不同的器官。本文介绍了荧光结合吩荧光染色技术在光学显微镜下的应用。这种染色方法是一种强有力的工具, 用于检测和探索蛋白质集在临床和科学的设置。

视频: Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

科研

生物化学

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and sensitivity for DNA. In order to determine if the DNA molecules are full length after the completion of the experiment, a method is required to determine if the stained molecules are full length by digesting DNA with restriction enzymes. However, stained DNA may inhibit the enzymes, so a method is needed to determine what enzymes one could use for fluorochrome stained DNA. In this method, DNA is stained with a cyanine dye overnight to allow the dye and DNA to equilibrate. Next, stained DNA is digested with a restriction enzyme, loaded into a gel and electrophoresed. The experimental DNA digest bands are compared to an in silico digest to determine the restriction enzyme activity. If there is the same number of bands as expected, then the reaction is complete. More bands than expected indicate partial digestion and less bands indicate incomplete digestion. The advantage of this method is its simplicity and it uses equipment that a scientist would need for a restriction enzyme assay and gel electrophoresis. A limitation of this method is that the enzymes available to most scientists are commercially available enzymes; however, any restriction enzymes could be used.

Staining DNA molecules for fluorescence microscopy allows a scientist to view them during an experiment. In the method presented here, DNA molecules are pre-stained with fluorescent dyes and digested with methylation and non-methylation sensitive restriction enzymes.

用限制性酶来测定染有菁染料的 DNA

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

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

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

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

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

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

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