This work was supported by the Engineering and Physical Sciences Research Council [grant number EP/M028739/1]. RSE was supported by a Santander Postgraduate Research Award 2015.

注意:请咨询生物安全协议，并在使用前的所有相关材料安全数据表（MSDS）。在这些实验中采用的激光是一个3B类激光;遵守一个安全系统的使用局部规则是必需的。进行激光光谱测量包括使用个人防护装备（护目镜）时，请使用所有适当的安全措施。 从对安乐死按照欧盟法规二千零九分之一千零九十九和动物福利（屠宰或杀害）规例从本地屠宰场获得了1995年牛颈项韧带其他用途7-8周龄Wistar大鼠尾得到筋。 1.样品纤维的制备注:细胞外基质的蛋白纤维可以从各种组织中提取，使用不同的程序。基于广泛的应用程序的协议进行了细化。 <ol><li><stroNG&gt;从鼠尾胶原纤维的提取<ol><li>以100毫克/千克体重钠戊巴比妥的腹膜内注射牺牲大鼠。然后直接在与身体接触的点切断尾部，压下用单刃刀片。包裹的尾部在保鲜膜，并将其存储在-20℃下冷冻直至需要。 </li><li>收集从冷冻尾部，切断为20毫米长的段从近端同时仍冻结，然后将其留在培养皿填充有在RT磷酸盐缓冲盐水（PBS）溶液（pH 7.4）中解冻。 </li><li>一旦尾解冻，用手术刀分裂皮肤做一个切口沿着所述段的长度。然后剥开，露出对尾椎4护套筋束。 </li><li>用细镊子，并注意不要应用任何预应变，轻轻划每根光纤出来的护套，并放置在盛有蒸馏水与0.01％w / v的叠氮化钠小瓶（NAN 3）PR事件的细菌生长，并存储冷藏。单尾产生约三万腱纤维。 </li><li>获得纯纤维状I型胶原蛋白，适用的三部分酶促消化处理10到腱的纤维以除去蛋白聚糖和所有其它非胶原材料。 <ol><li>首先，在200rpm，在pH 8.0浸入纤维在0.125 U / ml的软骨素酶ABC的0.05M Tris缓冲液和0.06 3M醋酸钠（CH 3 COONa）24小时，在37℃在振荡培养箱中。 </li><li>然后，在pH 6.0沉浸在1单位/毫升链霉菌透明质酸纤维在0.05M Tris缓冲液和0​​.15M氯化钠（NaCl）中，并在37℃下返回到振荡孵化24小时。 </li><li>最后，在37℃下浸泡在1毫克/毫升胰蛋白酶的纤维在0.05M磷酸钠（NaHPO 4）和0.15M NaCl的pH 7.2的16小时在振荡培养箱。 </li></ol></li><li>存储包含蒸馏水小瓶冷冻纯化纤维，无线网络次0.01％NaN 3的，以防止细菌生长，直到需要用于测量。 </li></ol></li><li> 从牛项韧带弹性纤维的提取 <ol><li>从屠宰场获得的牛项韧带，在保鲜膜包好，并将其存储在-20°C，直到使用冻结。 </li><li>以产生纯弹性蛋白，收集从冷冻的韧带，允许它在RT在沸水浴根据兰辛程序解冻，使用解剖刀脱脂它和消化韧带，11如下。 <ol><li>制备的0.1M溶液在蒸馏水氢氧化钠（NaOH），并把它添加到脱脂韧带在一个锥形瓶中，覆盖该组织。 </li><li>沸腾的水浴中的烧瓶中在95℃下45分钟。 </li><li>除去从消化烧瓶的韧带和直至获得pH为7.0（用pH计监测）在蒸馏水反复洗涤不溶组织块。 </li><li>从最终除去组织洗溶液和淹没它在蒸馏水（含0.01％NaN 3的混合，以防止细菌生长）在密封容器中，并存储它冷藏。 </li></ol></li><li>收集从冰箱的组织，从大的块（20至50毫米长，直径约 2毫米厚）的距离，注意不要用力过猛和预应变，使用镊子轻轻拉动弹性较小的部分，把它们在培养皿中，用PBS溶液（pH 7.4）。 </li><li>用细镊子，轻轻地挑逗小纤维束周围1mm厚并切成使用手术刀几个毫米的长度。 </li><li>转移的纤维含有蒸馏水的小瓶（含0.01％NaN 3的，以防止细菌的生长），并将其储存冷冻直至需要用于测量。 </li></ol></li><li> 安装光纤到反射主板 <ol><li>使用金​​刚石切割器，切下一块反射硅氧烷滑动。 </li><li>要创建一个水合compartme核苷酸，切封口膜的条带，以适应在硅氧烷滑动与在中心（大到足以容纳一个光纤）的中空切割，并将其放置到硅衬底上。 注:对于干纤维的测量，切断封口膜成U形，使得在步骤1.3.4密封时的四个边中的一个保持打开到空气中。 </li><li>从冰箱中取出纤维，使用一对细镊子从存储溶液收集的单纤维，并将其放置在一个小的培养皿在RT装满纯水5分钟以洗涤样品。然后，收集纤维，并将其转移到硅衬底上的封口膜中空的中心。 小心:避免由该操作期间伸展它损坏光纤，并避免在基片上重新取向的样品，因为这可能会导致机械性能的变化。 </li><li>放置一个薄玻璃盖玻片在光纤和轻轻传递焊加热铁在玻璃表面到下方熔融封口膜密封所述腔室玻璃。 小心:避免通过不使烙铁头太近或过度加热基板损坏光纤。 </li><li>放置在平坦表面上的密封室下一个小的重量和搁置约12小时，以实现样本和硅衬底之间的良好接触避免了对样品的损害，同时。 </li><li>除去的重量和在基片上固定到位的腔室，使用螺钉。 </li></ol></li></ol> 2.设置布里渊实验并获得光纤光谱<ol><li> 样品室的研制 <ol><li>安装在部分1.3中制备的样品上装有一个测角器，以使在面内的样本的旋转，同时保持恒定的散射角（2Φ= 90°， 见图SI-1）的竖直支架和散射体积的位置。 </li><li>上通过文件的样品进行激光的精确焦点调整纳秒。9 小心:激光输出功率可能过高，并产生样品中的烧伤。请确保它被设置足够高，以提供一个良好的灵敏度，但不能过高，以免损坏样品。在这里，我们使用在样品上约 76毫瓦的功率。这足以获得良好的灵敏度不燃烧的样品，也考虑到这是薄且在与有助于消散由激光照射所产生的热量的衬底接触。 </li><li>样品以45°角（Φ），以使用游标刻度入射激光束的位置。通过运行一个测量和在光谱最大化峰的强度（见下文）获得最佳的定位。 </li></ol></li><li> 设置分光计 <ol><li>打开软件收购和数据的操作，并设置了收购样品12的布里渊频谱。这里所描述的过程适用于万亩ltipass串联干涉仪（ 图SI-1A）。 </li><li>对齐两个法布里 - 珀罗（FP）干涉仪独立地改变由控制单元施加到压电的电压。对于这种预对准过程中，观察每个FP反射的光。当由两个的FP反射的强度趋于零，当达到正确的对齐。 </li><li>校准光谱:可访问的频率范围，或自由光谱范围 （FSR），是依赖于第一FP腔的两个反射镜，L通过FSR = C / 2 L，其中c是光的速度之间的距离L由拨号计测量。 </li><li>同步两个FP干涉仪的扫描和光学系统切换到汇接多路径配置。所发射的激光的光强度的反馈控制将在测量过程中自动保持两FP的比对。 </li></ol></li><li> Ø测量˚F布里渊频谱 小心:该布里渊频谱是高度依赖于样本和这些参数，以便仔细控制的温度和水合的关键是获得可再现的光谱。 <ol><li>开始采集样品的布里渊频谱的并运行它，直到一个好的信噪比得以实现。这可能需要根据样品的散射截面，浓度和厚度几分钟。 注意:没有拇指对信噪比的规则，但光谱质量通过基于分析的特定样品的实验者进行检查。有光谱质量和测量的持续时间之间的折衷，因此需要根据具体应用来选择实验参数。 </li><li>用于干试样的测定中，采取连续光谱 - 为每个，在步骤2.3.1 - 在峰的位置，直到没有改变我S测定值。当样品是在与室温气氛平衡并没有进一步的干燥会影响光谱此得以实现。 </li><li>选择的光偏振（VV或VH; V表示垂直和H相对于在散射平面中的光偏振的水平方向）和在每一个角度的纤维轴获取光谱（θ; 图SI-1）通过在样品旋转平面手。 </li><li>保存该布里渊频谱对文件进行后续处理。 </li></ol></li></ol> 3.布里渊频谱分析注:布里渊峰拟合的分析可以使用各种功能来执行。选择了阻尼谐振子（DHO）函数4,13，因为这是从粘弹性阻尼介质声模原峰的有效模式。 <ol><li> 布里渊峰的拟合分析 <ol><li>选择的光谱范围为感兴趣在第峰值Ë布里渊谱。 </li><li>使在合适的基线，如果光谱背景高于零得多。 注:基准可以光谱之间有所不同。确保校正以系统和可重复的方式实施。 </li><li>申请的详细最小二乘使用DHO函数4,13至感兴趣的布里渊峰迭代直至实现收敛并且获得的最佳拟合曲线拟合。然后，保存拟合结果到文件。 </li><li>从各布里渊双峰的两个峰的拟合参数获得的平均值。 </li><li>计算从峰值频率的声波速度（使用下面的表达式）。 </li><li>通过绘制图表， 例如，声波速度与角度纤维轴，拟合结果θ ，并应用相关模型（ 例如，声学各向异性系统9）提取力学量如弹性张量系数。 </li></ol>
	</li>
</ol>

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The authors have nothing to disclose.

布里渊散射光谱是一个独特的工具，通过该蛋白纤维的弹性张量的各个组件可被表征在前所未有的细节。此外，该测量可以在微观尺度进行，从而将提供给我们新的见解的生物结构的微观尺度力学，允许我们，对于第一次，以了解机械，和可能功能的复杂性，意义在矩阵架构和生物化学已揭示在最近几年。 该技术测量在GHz频率范围内的机械性能。这一领域从未有过探索生物大分子结构和它既提高了，并提供了手段来回答有关弹性分子机制的根本问题。 我们所描述的步骤，从动物组织中提取的胶原蛋白和弹性纤维，并测量布里渊scatteri纳克光谱使用反射基底达到光纤生物力学的完整描述。在协议中的关键步骤是那些确保纯化的纤维得到和适当的实验条件到位为纤维蛋白的​​可再现的测量。然而，必须记住，提取程序可以修改的纤维的机械性能承担。 该技术的变型涉及与microfocused布里渊散射和映射光学显微镜的耦合接近13与互补的技术的可能的组合（ 例如，拉曼散射）。该技术目前的应用主要集中在离体生物材料，但重要的进展， 例如，那些基于多个VIPA标准具14，都使得有可能从台式到床边这种技术具有广泛的应用已经妖翻译包括体内应用潜力strated 15,16。 VIPA的方法是什么，我们描述的替代;它具有更快的采集时间，但不一定是在不透明的样品的情况下，适当的如这里进行分析。此外，使用在反射性基材的不在于使用VIPA标准具调校实用的，因为它们的对比度将不足以拒绝准弹性光。有关获得的光谱数据集，该材料的固有弱散射截面的速度的限制可能限制应用到动态生物系统，并从内组织深处的采​​集的数据，但技术精炼可以改善电流的性能。 BLS有望成为生物物理基础研究的细胞外基质的主要工具，从而产生基质生长过程中的新见解的力学性能的演变及其病理损失退化。然而，要记住，测量非侵入，并可能因此在体内进行是重要的。实际上，这已经在角膜16实现，这样的工作可提供新的诊断工具的开发适用范围广的结缔组织病症的平台。 超声弹性和原子力显微镜（AFM）是微机械测量的替代方法，但BLS的技术比前者，不像AFM提供更好的空间分辨率（对亚细胞规模），施加在试样上没有机械力，并且不限制于分析唯一的表面特征。胶原蛋白和弹性模量布里是在GPA的范围，同时从宏观株杨氏模量兆帕的顺序（进一步的细节将另行报道）的。该结果表明与在激励频率的依赖性强的差分弹性模量，由于纤维的粘弹性行为。 BLS可以应用于范围广泛的生物医学科学的问题和材料。它可以在回答有关的生理和生物组织的病理学的问题有所帮助，以及提供的材料和相互作用在分子水平的基本理解的物理工具。

布里渊光散射（BLS）的作用被列昂布里渊于1922年1它包括通过在材料热活化的声学声子的可见光的非弹性散射的发现。在固体物理学，声子在晶格所有原子相干振动。两个交替类型的晶格原子的一维链是表示声学声子，由BLS揭示和光学声子，通过IR吸收和拉曼散射（ 图1）探测之间的差的简单模型。声学声子是同相在链中具有沿传播方向的位移的原子移动（纵向声子），或垂直于传播方向（横声子），而光学声子是原子外的相位变动产生振荡电偶极矩（纵向或横向模式）。 BLS分光镜检查已自1920年以来分析科学被使用;然而，仅80年代以来有高对比度的测量通过使用串联多通路法布里 - 珀罗分光计是不可能的。自那时以来，越来越多的进步在BLS在凝聚态物质（其中光子-光子相互作用被利用）分析应用2-4和磁性材料（通过光子磁振子相互作用）5已经带来的。上的生物医学应用6-8开创性工作铺平到的各种方法的发展途径，其中包括一个在这里施加并且在片状结构使用反射衬底实现的弹性张量的完整描述前面所述9所述一个一个样品。 在目前的工作中，我们应用BLS光谱外基质的结缔组织的基本成分，纤维蛋白弹性蛋白和I型胶原。 ŧYPE I型胶原是刚性的，三螺旋分子，其横向和纵向组装具有广泛的交联，以形成组织中基本刚性纤维，例如筋。胶原的网络通常具有弹性的网络并存，一个蛋白，其，异常，通过熵和与其环境的疏水性相互作用的组合产生长范围的弹性，并且是组织如肺和皮肤的功能是必不可少的。两种纤维在当前研究使用六方晶系模型建模。9在部分1，我们描述了协议来提取从动物组织的纤维和以制备用于光谱测量的样品。在部分2，用于设置的布里渊装置和获得从纤维光谱的程序提出。部分3给出施加到的布里渊频谱，提取包含在其中的相关的机械信息的数据分析的细节。然后，代表性的结果都和discusseð。

<table border="0" cellpadding="0" cellspacing="0" width="762">
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			<td style="width:119px;">Fisher Scientific</td>
			<td>S608-500</td>
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			<td style="width:119px;">Sigma-Aldrich</td>
			<td>P4417</td>
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			<td style="width:119px;">Fisher Scientific</td>
			<td>S2002</td>
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			<td style="width:119px;">Sigma-Aldrich</td>
			<td>H1136-1AMP</td>
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			<td style="width:119px;">Fisher Scientific</td>
			<td>S7653</td>
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			<td style="width:119px;">Sigma-Aldrich</td>
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			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:151px;">S9638</td>
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			<td style="width:119px;">Fisher Scientific</td>
			<td>S320-500</td>
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			<td>Produced In-House&#160;</td>
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			<td>Produced In-House from water still</td>
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			<td>241-7311 &#38; 241-7201</td>
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			<td>No catalogue number. Order upon request</td>
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			<td>631-1571</td>
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			<td>214-1175</td>
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			<td style="width:119px;">VWR</td>
			<td>213-0469</td>
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			<td height="21" style="height:21px;width:204px;">Measuring Cylinder</td>
			<td style="width:119px;">VWR</td>
			<td>612-3838</td>
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			<td style="width:119px;">VWR</td>
			<td>548-0051 &#38; 548-0863</td>
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			<td style="width:119px;">Swann Morton Ltd</td>
			<td>&#160;0914 &#38; 0308</td>
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			<td height="21" style="height:21px;width:204px;">Diamond Scribe</td>
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			<td>394-217</td>
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			<td>231-5332</td>
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			<td height="21" style="height:21px;width:204px;">Fine Forceps</td>
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			<td style="width:119px;">Hanna Instruments</td>
			<td style="width:151px;">HI-2210-02</td>
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			<td height="22" style="height:22px;width:204px;">Orbital Shaker</td>
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	</tbody>
</table>

preparation of extracellular matrix protein fibers for brillouin spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

在该实验中（ 图SI-1A）中使用的布里渊光谱设备先前已描述9它采用单一模式532纳米的固态激光器在样品76毫瓦的输出功率。将20cm消色差透镜聚焦激光光到样品和从在一个反向散射几何样品收集散射光。一种串联多通路法布里 - 珀罗干涉仪用于过滤的散射光，然后由一个低噪声光电二极管检测器检测到。这种方法非常给通过标准具自调心压电扫描的高对比度（ 约 120分贝）和稳定性。偏振器和分析器介绍来选择入射光和散射光的偏振。光谱通常与偏振保持固定选择入射光偏振的垂直（V）方向和分析器选择备选的垂直（V）所得的O散射光偏振中的R水平（H）方向。在这种配置中，纵向和横向的声模分别检测。 一个典型的布里渊频谱具有强烈中央峰由于弹性散射和同样移位的峰，或布里渊双峰 ，这是样品的力学的签名的一个或多个集合。在这些测量中，散射光既可以从散装声子行进的准正交到样品，并在样品基片接口的入射光反射后，从散装声子平行于表面（PS模式）行驶起源9 图2示出了在0.2千兆赫的分辨率与VV极化得到的干燥和水合胰蛋白酶消化的胶原纤维的BLS光谱，用30千兆赫的自由光谱范围和大约10分钟的收集时间每个谱。每个光谱对应于旋转一个特定的角度，θ （ 图SI-1C）。在θ= 0°干胶原纤维，纵模产生的堆积峰（18.92±0.02）GHz的同时PS模式是在（9.85±0.03）GHz的（ 图2A）。在PS峰转移到较低频率为θ从0°（声子探测纤维的轴向方向）变到90°（声子探测径向取向），而在本体峰仅轻微红移时在同一范围内改变θ （声子探测整个旋转的准径向方向上）。在湿胶原纤维，两峰由于纵向声子在整个实验基本上保持不变，以在约 10.5千兆赫的散装峰，并在4.9千兆赫（ 图2B）在PS峰。这表明在峰值频率的80到100％的减少（相对于18.92所述数据和9.85千兆赫，分别），因此材料的刚度，由于水合作用。需要注意的是水合胶原的体积和PS模式在于频率的纯水的模式接近，这表明它的弹性常数是水和纤维的贡献的组合，与由水起到了主导作用。 图3显示了在θ=与VH结构偏振30°测定干胰蛋白酶消化的胶原纤维的频谱;的VV极化的泄漏使得仍然可以观察到的PS和散装峰。横模占在（4.1±0.2）千兆赫（θ= 0°），其略微蓝移从0°到90°θ变化的峰。对于横向和PS峰都拟合结果也示。提取峰值参数和声波速度被衍生为V L = vλ/√2，其中<eM&gt; v是由峰的曲线拟合分析，λ获得的模式的频率是激发波长，532纳米。注意，在该几何形状中，不需要的材料的折射率的知识来获得声模速度（由于散射几何学，Q S = 2 K I SIN（Φ）; 图SI-1b中，c）中 ，因此使这种方法特别有利的。 图4是作为角度θ的函数从纵向和横向模式中得到的声波速度（PS和T峰）的曲线图 。拟合分析以六角形对称弹性固体7的模型-方程A1和A2之下-提供干燥胰蛋白酶消化型的弹性张量的五个组件I型胶原纤维（ 表1）。 9给出<img alt="式（1）" src="/files/ftp_upload/54648/54648eq1.jpg" /> ，（A1） <img alt="公式（2）" src="/files/ftp_upload/54648/54648eq2.jpg" /> ， （A2） 其中ρ是材料的密度，以及c 11，C 33，C 44和C 13是具有六边形对称性表征系统的五个弹性常数中的四个。第五常数，c 12，可以从近似关系C 12〜C 11衍生- 2 C 44 7 系数是类似于先前从未纯化的胶原fibe得到的那些RS 9一个明显的区别出现的位置系数C 13被反射到弹性模量Ëǁ和E相似的价值观<img alt="垂直" src="/files/ftp_upload/54648/54648fig_flippedT.jpg" /> （约7.2和7.7 GPA）的纯化的胶原蛋白。 图5是湿的胶原蛋白与θ的纵向声波速度的阴谋 。在这种情况下，在频率没有周期性变化被观察到，给误差内的等速; 图6示出了在θ= 0°测量干燥和水合弹性纤维的光谱。对于这些样品中未检测到横模。在干弹性，堆积峰在16.8 GHz的同时PS模式出现在8.2千兆赫9（13比干胶原的相应峰值降低了20％）。湿弹性纤维呈现出散pEAK在（12.30±0.01）GHz的（在频率比干燥弹力的散装峰值低37％）。湿弹性的PS模式，是因为在这些频率弹性峰尾紧张不是在频谱明显。另一方面，在大约 7.5千兆赫的峰归因于大体积的水。 图7示出在对θ干弹性纤维声波速度的依赖性。从这些数据中，弹性张量分量（和机械模数），得到（ 表1）。9如湿胶原，存在水合弹性蛋白纤维的机械模量各向同性的证据。这些结果表明激布里渊光谱如何给上刚度，组成和材料的结构方面的相关信息。 <img alt="图1" src="/files/ftp_upload/54648/54648fig1.jpg" /> 网络连接古尔1. 声学和原子的一维链的光学声子。在一维双原子链的声学和光学振动示意图。原子具有质量m 1和m 2的交替。箭头指示的原子位移。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54648/54648fig2.jpg" /> 图2. 胰蛋白酶和纯化的类型的布里渊频谱I型胶原从大鼠尾腱的纤维。（A）的干纤维和（B）的光谱水合从VV测量纤维在不同的角度，以纤维轴θ，以度。纯净蒸馏水频谱也同时显示。谱归一化到散装峰的强度（高度）。<html>标签B和PS分别表示有关体积和并行到表面模峰。误差线表示的标准误差（计数数的平方根）。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54648/54648fig3.jpg" /> 图3. 布里渊干胰蛋白酶纯化型谱胶原蛋白从鼠尾腱纤维。频谱从θ的VH测量 = 30°。标签T，聚苯乙烯和B表示的横向，平行于表面和批量模式，分别相关峰。使用T和PS模式的阻尼谐振子（DHO）模型拟合分析结果也显示。误差棒表示标准误差（计数的数的平方根）。/54648fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54648/54648fig4.jpg" /> 图4. 叠加在干燥的胰蛋白酶的纯化胶原VS角度纤维轴声波速度从布里渊峰的拟合分析得出的干胶原纤维的纵向和横向声波的速度。数据拟合的六角对称弹性固体模型。红线:公式A1（R 2 = 0.99）;蓝线:公式A2（R 2 = 0.36）。误差条指示从协方差矩阵的列文伯格-马夸特非线性最小二乘法拟合布里渊频谱后对角线元素的平方根获得的标准误差。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig4large.jpg" target="_blank">请点击此处查看大VERSI</a><html>这个数字的。 <img alt="图5" src="/files/ftp_upload/54648/54648fig5.jpg" /> 图5. 情节在潮湿的胰蛋白酶的纯化胶原VS角度纤维轴纵向声波速度从布里渊峰的拟合分析得出水合胶原纤维纵向声波速度。示出的线是眼睛的导并给出在此范围内的声波速度的平均值。误差条指示从协方差矩阵的列文伯格-马夸特非线性最小二乘法拟合布里渊频谱后对角线元素的平方根获得的标准误差。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> JPG"/&gt; 图6. 布里渊牛项韧带弹性纤维的光谱。在θ干燥和水合光纤光谱 = 0°。谱归一化到散装峰的强度（高度）。标签B和PS分别表示有关体积和并行到表面模峰。乙F和BW¯¯分别指的是纤维和水的体积峰。误差线表示的标准误差（计数数的平方根）。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54648/54648fig7.jpg" /> 图7.情节在干燥弹性VS角度纤维轴纵向声波速度。干弹性纤维蛋白原纵向声波速度呃从布里渊峰的拟合分析得出。数据拟合的六角对称弹性固体模型。红线:A1方程式9（R 2 = 0.74）。误差条指示从协方差矩阵的列文伯格-马夸特非线性最小二乘法拟合布里渊频谱后对角线元素的平方根获得的标准误差。 <a href="https://www.jove.com/files/ftp_upload/54648/54648fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="参考图1" src="/files/ftp_upload/54648/54648supfig1.jpg" /> 图SI-1。布里渊设置和BLS散射几何学的示意图。（A）中由固态激光器发射的入射光被通过一个消色差透镜发送到样本。通过批量声子和那些来自REFL产生散射的光在衬底表面上，这是在与样品接触的光的挠度，由透镜收集通过的串联多路径法布里 - 珀罗干涉仪过滤并检测由光电倍增。 FP1和FP2表示构成串联设置两个干涉仪。偏振器选择入射光的偏振，以及分析器，用于选择散射光的偏振。 （B）中的BLS几何与具有反射硅衬底的表面接触的样品。一种玻璃滑动件（未示出）被放置在试样上面以密封隔室，并通过焊盘在基板的角部被施加轻柔的压力。入射光（K I）穿过透镜，则在空气-样本界面（K&#39;ⅰ）折射并聚焦在样品基片接口。通过相同的镜头收集到的散射光（K&#39;S）从相互作用的结果与散装声子（Qb）和样品（Q S的那些行，PS）。 。光的方向和垂直于表面之间的角度被表示为Φ和Φ&#39;（C）的样品和通过坐标系的示意图; ž限定轴线平行的非凡于纤维的方向。角度θ和α是那些声子Q S方向和Q B于z a轴分别之间，K I，K&#39;I，K S，K 的 :事件的波数及散射光; Q B，Q S，体积和PS模式，分别为波矢。 （从REF 9转载） <a href="https://www.jove.com/files/ftp_upload/54648/54648supfig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <p类="jove_content"FO:保持与- next.within页="总是"&gt; 表从声波速度拟合分析得出1.弹性系数张干胰蛋白酶纯化的I型胶原纤维弹性系数张（。这项工作）和弹性纤维（参考9）。 <table border="1" fo:keep-together.within-page="1"><tbody><tr><td> 样品 </td><td colspan="2"> 弹性系数（GPA） </td></tr><tr><td>胰蛋白酶消化的胶原蛋白</td><td> C 33 </td><td> 18.7±0.1 </td></tr><tr><td></td><td> C 11 </td><td> 14.4±0.2 </td></tr><tr><td></td><td> C 44 </td><td> 3.4±0.1 </td></tr><tr><td></td><td> C 12 </td><td> 7.2±0.2 </td></tr><tr><td></td><td> C 13 </td><td> 11.2±0.3 </td></tr><tr><td>弹性</td><td> C 33 </td><td> 11.5±0.2 </td></tr><tr><td></td><td> C 11 </td><td> 10.4±0.1 </td></tr><tr><td></td><td> C 44 </td><td> 1.9±0.2 </td></tr><tr><td></td><td> C 12 </td><td> 6.6±0.2 </td></tr><tr><td></td><td> C 13 </td><td> 6.8±0.3 </td></tr></tbody></table>

Watch this Scientific Journal Video about Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy at JoVE.com

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

preparation-extracellular-matrix-protein-fibers-for-brillouin

Research

JoVE Journal

Bioengineering

10.3K 次观看.  University of Exeter. We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

视频: Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

高速的sub-GHz光谱仪激布里渊散射分析

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...