This work was supported in part by the National Institutes of Health (P41-EB015903, R21EY023043, K25EB015885), National Science of Foundation (CBET-0853773) and Human Frontier Science Program (Young Investigator Grant).

注意:布里渊频谱分析需要单纵模激光（〜10毫瓦的样品）。对于调整的目的，使用激光束（&lt;0.1毫瓦）的强烈衰减部分。 纤维1.初始设置和EMCCD（电子倍增电荷耦合器件）相机<ol><li>识别约1600 mm的自由空间对准了在光学平台上的光谱仪。 </li><li>安装EMCCD相机在自由空间对准的结束。 <ol><li>用螺丝将相机连接到岗位。拧紧后持有人的职位所需的光轴高度。拧紧后持有人使用上表夹在光学平台。 </li></ol></li><li>打开EMCCD相机。要留意避免摄像头饱和。 <ol><li>实验室的计算机上安装摄像头软件，然后将相机连接到电脑。 </li><li>禁用增益设置为低积分时间（〜0.01秒）。开始获取EMCCD图像。观察摄像机图像•计算机屏幕上。 </li></ol></li><li>装入约1600毫米，镜头前的光纤准直器。 <ol><li>放置在光纤准直适配器的光纤准直器。 </li><li>安装适配器到一个职位。拧紧后成柱支架在光轴（摄像机的高度）的近似高度。柱支架拧紧到使用表夹子光学平台。 </li></ol></li><li>对准沿着光轴的输出光束。 <ol><li>使用固定螺钉连接标准虹膜后。拧紧后成柱座。 </li><li>放置虹膜在光纤准直器（&lt;50毫米）的前部。调整光圈的光束的高度。移动沿着期望光轴虹膜，应该直接点到相机。 </li><li>直接的激光输出，如果相机饱和之后，插入一个光密度滤光片。使用光纤准直器的调整螺钉安装到对准沿着光轴的光束。一旦实现这一点，该束将干净地穿过沿整个光路的光圈。 </li></ol></li><li>安装一个匹配的消色差透镜对中（f = 30mm）的在照相机的前面。 </li></ol>光谱仪2.水平阶段<ol><li>挂载水平面具。 <ol><li>使用固定螺钉的水平掩模附着到一个职位。拧紧后成柱座。柱支架安装到一个平移载物台，使掩膜水平滑动进出光束路径。 （图1） </li><li>用有头螺钉拧紧在光学平台上的平移阶段，使得掩模急剧成像到CCD照相机在消色差透镜对的焦点距离（f = 30毫米）。 </li></ol></li><li>安装并对准球面透镜S1（F = 200毫米）600个毫米在水平掩模的前面。 <ol><li>用螺丝安装S1到柱座。拧紧后成柱座。 </li><li>将两片虹膜如1.5.1所述。 placi前纳克S1中的光束路径中，插入前1虹膜和所需的S1位置后的一个光圈（600毫米的水平掩模的前面）。调整虹膜的高度，使得所述光束干净地穿过两者。 </li><li>放置S1 600个毫米的水平掩模的前面（ 图1）。调整高度和S1的角度，直到两个，背反射离开透镜和出未来束，干净地穿过虹膜。拧紧柱支架使用表夹子保持S1到光学平台上。 </li></ol></li><li>安装VIPA2在球面透镜的焦平面（200毫米S1的前面）。 <ol><li> VIPA的支架安装到使用附带的支架螺丝后。拧紧后成柱座。柱支架拧紧到横向平移台。 （图1） </li><li>与入射狭缝垂直定向的VIPA持有人小心地VIPA2。 （图1） </li><li>精细调整位置Of显示VIPA安装到恰好在S1的焦平面。 （通过跟踪束腰带白卡检查。） </li><li>用有头螺钉拧紧平移阶段到光学表和滑动VIPA2用该平移阶段的自由度的光束。 </li></ol></li><li>安装并对准球面透镜S2（F = 200毫米）200毫米的VIPA后和200毫米的水平掩模的前面。按照2.2.1-2.2.3描述的过程。柱支架附着到光学表代替，把它安装到与自由沿光轴取向的其程度的平移阶段。 （图1）使用有头螺钉拧紧平移阶段到光学表。 </li><li>使用水平平移阶段（2.3.4）至VIPA2入口滑入光束路径。观察摄像机图像上的垂直线。 <ol><li>细调整S2使用平移阶段安装在2.4直到在摄像机图像上的垂直线出现尖锐。 </li></ol></li><li>调整与自由对VIPA保持器的水平方向倾斜程度和平移阶段的频谱。使用自由的水平平移度来调整光束的入口位置到标准具。使用自由的水平倾斜程度来调节光束的输入角进标准具。 </li><li>测量技巧和吞吐量。 <ol><li>按F5键或者通过点击在左上角"采集设置"，然后选择该选项，在拍照软件"取图像"拍摄图像。 </li><li>右键点击图片并选择选项"线图"。水平拖动光标在图像生成线图。释放光标来观察产生的线图。 </li><li>使用线图来测量技巧。通过半最大值（FWHM）全宽分隔两个峰之间的距离。瞄准&gt; 30。 </li><li>确定由measurin吞吐量摹与之前和VIPA2后，功率计的功率。目的是为&gt; 50％。 </li><li>调光束的输入角与自由对VIPA支架（2.6）的程度的标准具和观察技巧和吞吐量之间的权衡。 </li></ol></li><li>如果精细度和吞吐量都不能令人满意回去精细调整对齐。确保VIPA2是S1的焦平面。重复步骤2.3.3 -2.7。 </li></ol>光谱仪3.垂直阶段<ol><li>滑动VIPA（VIPA2），排列在第2部分，用该平移阶段（2.3.4）的光束。光谱仪的水平阶段现在将表现为1:1的成像系统。 </li><li>安装垂直面具。 <ol><li>用螺丝将附加的垂直面具到一个职位。拧紧后成直角钳后适配器。拧紧第二次后进入直角适配器，放入柱座。柱支架安装到一个垂直平移阶段，allowi纳克的掩模进出的光束路径的垂直滑动。 （图1） </li><li>使用有头螺钉拧紧垂直平移阶段到光学表，使得垂直方向的荫罩急剧成像到EMCCD照相机200毫米S1的前面。 </li></ol></li><li>安装和对准圆柱透镜C1（F = 200毫米）600个毫米的垂直掩模的前面。 <ol><li>柱面透镜支架螺丝到一个职位。拧紧后成柱座。小心地C1到物镜保持器，并拧紧螺丝固定到位。 </li><li>将两片虹膜如1.5.1所述。前放置C1在光束路径中，插入前1虹膜和所需的S1位置后的一个光圈（600毫米的垂直掩模的前面）。调整虹膜的高度，使得所述光束干净地穿过两者。 </li><li>地方的C1 600个毫米的垂直掩模的前面（ 图1）。仔细调整高度，倾斜，和C1至横向位置两者，背反射离开透镜和出到来的光束，被集中到虹膜。拧紧后持有人持有C1到使用表夹在光学平台。 </li></ol></li><li>安装VIPA1在柱面透镜的焦平面（200毫米的C1的前方）。 <ol><li> VIPA的支架安装到使用附带的支架螺丝后。拧紧后成柱座。柱支架拧紧到垂直平移台。 （图1） </li><li>小心地将VIPA1与入射狭缝水平方向的VIPA持有人。 （图1） </li><li>精细调整VIPA的位置安装以精确地放置VIPA1在C1的焦平面。 （通过跟踪束腰带白卡检查。） </li><li>使用有头螺钉拧紧垂直平移阶段到光学表并滑动VIPA1用该平移阶段的自由度的光束。 </li></ol></li><li>安装和对齐圆柱透镜C2（F = 200毫米）200毫米VIPA1后和200毫米的垂直掩模的前面，以下在3.3.1-3.3.3中描述的方法。柱支架附着到光学表代替，把它安装到与自由沿光轴取向的其程度的平移阶段。 （图1）使用有头螺钉拧紧平移阶段到光学表。 </li><li>使用平移台（3.4.4）至VIPA1入口滑入光束路径。观察摄像机图像上的水平线。 <ol><li>使用平移阶段安装在3.5直到在摄像机图像上的水平线出现尖锐精细调整C2。 </li></ol></li><li>调整与自由对VIPA保持器的垂直倾斜程度和平移阶段的频谱。使用自由的垂直平移度来调整光束的入口位置到标准具。使用自由的垂直方向倾斜程度来调节光束的输入角进等人上。 </li><li>测量技巧和吞吐量。 <ol><li>按照步骤2.7.1 -2.7.2在整个相机屏幕的图像垂直生成线图。 </li><li>使用线路积除以两条水平线之间的距离通过全宽度半最大值（FWHM）来测量手腕。瞄准&gt; 40。 </li><li>通过之前和VIPA1后测量与功率计的功率测量吞吐量。目的是为&gt; 30％。 </li><li>调光束的垂直输入角与自由对VIPA支架（3.7）的垂直方向倾斜程度的标准具。观察技巧和吞吐量之间的权衡。 </li></ol></li><li>如果精细度和吞吐量都不能令人满意回去精细调整对齐。确保VIPA1是C1的焦平面。重复步骤3.4.3-3.8。 </li></ol> 4.组合两个阶段和最后的对的<ol><li>滑动VIPA2。观察水平和垂直方向间隔开的点。这些点的重的单频率激光器的光谱特征。调整东哲行的平移阶段，直到点是大家关注的焦点。 </li><li>测量精细度谱仪和吞吐量。 <ol><li>按照步骤2.7.1.-2.7.2产生斜对面的两个激光点的线图。 </li><li>使用线路积除以两个点之间的距离对角通过全宽度半最大值（FWHM）来测量手腕。瞄准&gt; 30。 </li></ol></li><li>建立一个黑盒子包围光谱仪。 <ol><li>使用施工护栏打造的箱框架，其中应包含整个光谱仪从光纤准直器的CCD摄像机（63英寸×9×15英寸）。 </li><li>覆盖盒子骨架遮光布，然后用胶带紧的角落。确保口罩和东哲行都很方便。 </li></ol></li><li>光纤到一标准的光学探头，连接诸如反射共聚焦显微镜4。标准光学p用于收集背散射光长袍将携带一个布里渊信号。 </li></ol> 5.测量布里渊移<ol><li>接近垂直和水平掩模，直到激光签名消失。相应地移动翻译阶段。 </li><li>使增益和增加相机尽可能的积分时间，而不饱和EMCCD相机。 </li><li>观察样品的布里渊偏移。 <ol><li>放置一个样品中的共焦显微镜（或其它光学探测器）的焦点。的空间分辨率将取决于在共聚焦显微镜中使用的物镜。使用塑料盘或试管液体。使用甲醇为第一测量。 </li></ol></li><li>为了优化分光计吞吐量，打开一个掩模的时间和通过分光计订单通过倾斜VIPA和调整其平移台扫描。发现在该信号出现的最强的顺序。再次关闭屏蔽，直到日Ë激光信号消失。重复与其他掩模和VIPA（在2.6和3.7中描述）。 </li><li>取样品的测量。 <ol><li>取频谱的图像以下步骤2.7.1。 </li><li>通过利用水和玻璃（或与已知的布里渊移其他样品）的光谱的图像获取校准测量。通过单击左上角的"文件"，并选择选项"另存为"保存图像。保存在"的.sif"格式的图像，如果在相机软件进行数据分析。保存图像"的.tif"格式，如果在另一计算软件程序进行数据分析。 </li></ol></li></ol> 6.校准布里渊频谱分析和<ol><li>确定自由光谱范围（FSR）和EMCCD像素中的布里渊光谱仪的光学频率转换比（PR）。 <ol><li>装载数据在相机软件或另一computa其它软件程序。 </li><li>跟随步骤2.7.2以产生跨越所述水校准图像的光谱的线图。 </li><li>适合用洛伦兹曲线对光谱的两个峰，以确定在像素位置（P 水S，P 水-AS）而言峰的位置。另外，手动读取出的峰值位置，采取了最高点的峰值。 </li><li>与玻璃校准图像的频谱重复步骤6.1.2-6.1.3。 </li><li>另外，以6.1.1-6.1.4，同时添加EMCCD框架和适合所有四个峰一次。 （图2） </li><li>用公式1.插入对于p 水AS和P 玻璃AS中6.1.3和6.1.4确定的值计算公关。 Ω 玻璃-AS是已知的29.3 GHz的。在这种情况下，由于自由光谱范围仅为20就会出现混叠为9.3千兆赫的频率偏移千兆赫。为简单起见使用9.3 GHz适用于Ω 玻璃AS。使用7.46 GHz的为Ω水AS。 <img alt="式（1）" class="jove_content" p="" src="/files/ftp_upload/53468/53468eq1.jpg" /></li><li>用公式2.插入对于p 玻璃-S，P 玻璃-AS和 PR，在6.16计算值计算FSR。使用9.3千兆赫的Ω 玻璃AS。 <img alt="公式（2）" class="jove_content" src="/files/ftp_upload/53468/53468eq2.jpg" /></li></ol></li></ol><img alt="图2" class="jove_content" src="/files/ftp_upload/53468/53468fig2.jpg" /> 图2.光谱仪的校准 。从校准样品（A）EMCCD相机帧获得。 （二）洛伦兹曲线拟合（红色）测得的数据（蓝色）。 <a href="https://www.jove.com/files/ftp_upload/53468/53468fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>确定的布里渊移一个样品。 <ol><li>跟随步骤2.7.2以产生跨越所述样品的光谱行扫描。 </li><li>适合用洛伦兹曲线对光谱的两个峰，以确定在像素位置而言的峰值位置。另外，手动读取出的峰值位置，采取了最高点的峰值。 </li><li>使用下面的等式4和5与先前计算的值，FSR及PR来计算样品的布里渊偏移。 <img alt="公式3" class="jove_content" p="" src="/files/ftp_upload/53468/53468eq3.jpg" /></li></ol></li></ol>

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H. <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+Biomechanical+Mapping+of+Normal+and+Keratoconus+Corneas.">In Vivo Biomechanical Mapping of Normal and Keratoconus Corneas.</a> Jama Ophtalmol. , (2015).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <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+Measurement+of+Age-Related+Stiffening+in+the+Crystalline+Lens+by+Brillouin+Optical+Microscopy."> In Vivo Measurement of Age-Related Stiffening in the Crystalline Lens by Brillouin Optical Microscopy.</a> Biophys. J. 101 (6), 1539-1545 (2011).</li><li>Antonacci, G., Foreman, M. R., Paterson, C., T&#246;r&#246;k, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+broadening+in+Brillouin+imaging.">Spectral broadening in Brillouin imaging.</a> Appl. Phys. Lett. 103 (22), 221105 (2013).</li><li>Meng, Z., Traverso, A. J., Yakovlev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Background+clean-up+in+Brillouin+microspectroscopy+of+scattering+medium.">Background clean-up in Brillouin microspectroscopy of scattering medium.</a> Opt. Exp. 22 (5), 5410-5415 (2014).</li><li>Reiss, S., Burau, G., Stachs, O., Guthoff, R., Stolz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+resolved+Brillouin+spectroscopy+to+determine+the+rheological+properties+of+the+eye+lens.">Spatially resolved Brillouin spectroscopy to determine the rheological properties of the eye lens.</a> Biomed. Opt. Exp. 2 (8), 2144-2159 (2011).</li><li>Scarcelli, G., Kling, S., Quijano, E., Pineda, R., Marcos, S., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brillouin+microscopy+of+collagen+crosslinking:+noncontact+depth-dependent+analysis+of+corneal+elastic+modulus.">Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus.</a> Invest. Ophthalmol. Vis. Sci. 54 (2), 1418-1425 (2013).</li></ol>

Giuliano Scarcelli and Seok H. Yun hold patents related to Brillouin spectroscopy technology.

这种分光计的配置的一个关键的设计特点是，两个阶段可以独立地对准。当一个VIPA标准具滑出光路，分光计阶段的其余镜片形成1:1的成像系统，使来自每个阶段的频谱图案被成像到CCD照相机。因此，它是直接回到任一个阶段以提高其性能，而不会影响其他级的对准。该组中的协议建议的平移阶段和自由度遵循这一理念，保持独立优化光谱仪的两个阶段的能力。 这是很难装入V...

布里渊散射，首先由Leon布里渊1于1922年描述的，是光从在固体和来自液体或气体的热密度波动的热声模的非弹性散射。的散射光的谱移，通常在子GHz的范围，提供了有关该入射光和与样品中的声子之间的相互作用的信息。其结果是，它可以提供关于被检查材料的粘弹性能的有用信息。 在其自发版本，布里渊散射通常具有横截面在拉曼散射的顺序，产生了非常弱的信号。此外，布里渊频移的幅度比拉曼位移较小的订单。因此，弹性散射光（来自瑞利和米氏散射），杂散光，而反向反射在样本上都可以很容易地掩盖了布里渊光谱特征。故，布里渊光谱仪不仅需要实现的sub-GHz光谱分辨率也高光谱对比度或灭绝。 在传统的布里渊光谱仪这些要求是由扫描光栅单色器，光学打浆方法，以及最普遍地，多遍扫描法布里-珀罗干涉仪2满足。这些方法测量每个频谱分量顺序。这种方法导致了采集时间为单个布里渊频谱范围从几分钟到几个小时，这取决于仪器和在样品上。两阶段VIPA光谱仪，使用该协议建立，具有收集所有频谱分量的内不到一秒，同时提供足够的消光（&gt; 60 dB为单位），以有效地抑制其它杂散信号2的能力。 VIPA的标准具的整合是本光谱仪的关键要素。一个VIPA是一个坚实的标准具有三个不同的C浮动区域:在所述前表面，一个窄抗反射涂层条允许光进入VIPA，而表面的其余部分设有一个高反射（HR）涂层;在后表面的部分反射涂层允许的光的一小部分（〜5％），以进行发送。当聚焦到稍微倾斜VIPA的狭窄的入口时，光束被反射到子组件与VIPA 2内固定的相位差。子组件之间的干扰达到渴望的高光谱色散。在交叉轴配置对两个东哲行依次介绍了正交方向3光谱色散。在正交方向上的光谱色散空间上分离从不需要的串扰，这使得有可能仅拾取布里渊信号的布里渊峰。 图1显示的是两级VIPA分光计的示意图。下面的光学元件中的箭头指示的度自由稀土元素，其中的平移阶段应侧重。 <img alt="图1" class="jove_content" p="" src="/files/ftp_upload/53468/53468fig1.jpg" /> 图1.纯设置。一种光纤开出布里渊散射进入分光光度计。一个柱面透镜C1（F = 200 MM）将光聚焦到第一VIPA（VIPA1）的入口。另一个圆柱透镜的C2（F = 200毫米）映射的光谱角色散成空间分离C2的焦平面。在该平面中，垂直掩模用于选择光谱的所需部分。类似的结构如下，倾斜90度。光束通过球面透镜S1（F = 200毫米）传递并聚焦到第二VIPA（VIPA2）的入口狭缝。甲球面透镜S2（F = 200毫米）产生在其焦平面，其中另一条水平掩模被放置在二维光谱上分离的图案。该HORizontal掩模成像到使用的消色差透镜对的EMCCD相机。 <a href="https://www.jove.com/files/ftp_upload/53468/53468fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 本科生与一些光学课程和基本取向的经验应该能够建立和使用两个阶段的光谱仪。分光计最近已证明是与各种标准的光学探针的3,4,5（例如，共焦显微镜，内窥镜，裂隙灯检眼镜）兼容。这里，光谱仪被连接到共聚焦显微镜。激光被整合90:10分束器后对准成标准研究倒置显微镜系统。来自样品的背散射光被耦合进单模光纤，使得共焦显微镜。

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high speed sub-ghz spectrometer for brillouin scattering analysis

该协议的目标是建立一个平行的高灭绝和高分辨率光学布里渊光谱仪。布里渊光谱是可以用来获得粘弹性材料属性直接读数的非接触测量方法。它已经在材料特性，结构的监测和环境传感的有用工具。在过去，布里渊光谱已经通常使用扫描法布里 - 珀罗标准具来执行频谱分析。这个过程需要高亮度功率和长采集时间，使得该技术不适合生物医学应用。最近推出的新型光谱仪通过采用两个东哲在交叉轴配置克服了这一难题。这一创新使具有内生物组织的安全界限亚秒采集时间和照明电源子千兆赫（GHz）的分辨率频谱分析。通过这种改进促进了多种新的应用程序立方米rrently正在探索在生物研究和临床应用。

<html> 图3示出有代表性的布里渊频谱和其配合为不同的材料。的东哲行两者的厚度为5毫米，导致大约20千兆赫的FSR。的积分时间这些测量是100毫秒。 100测量是平均。一个校准测量之前获得的光谱进行了拍摄。 <img alt="图3" src="/files/ftp_upload/53468/53468fig3.jpg" /> 图不同材质的3布里渊谱 。 洛伦兹曲线拟合（红?...

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High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

在这里，我们提出了一个协议，以建立一个快速的布里渊光谱仪。级联几乎成像相控阵（VIPA）标准具实现了测量速度比传统扫描法布里 - 珀罗光谱仪快1000倍以上。这种改进提供了用于组织和生物材料在低功率水平的体内布里渊分析。

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 ...

high-speed-sub-ghz-spectrometer-for-brillouin-scattering-analysis

Research

JoVE Journal

Bioengineering

14.9K 次观看.  University of Maryland. 在这里，我们提出了一个协议，以建立一个快速的布里渊光谱仪。级联几乎成像相控阵（VIPA）标准具实现了测量速度比传统扫描法布里 - 珀罗光谱仪快1000倍以上。这种改进提供了用于组织和生物材料在低功率水平的体内布里渊分析。 

视频: High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

指南结构照明显微镜TIRF在高速，多种颜色

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

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.

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 ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

实现干扰反射显微镜,实现无标签、高速微管成像

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

使用高速摄像机和图像分析对快速移动微泡区域进行成像和定量

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.