作者感谢吉尔伯特绿色的技术援助。这项工作是由NIAMS /美国国立卫生研究院（R01-AR060484）和国防部（W81XWH-12-1-0555）授予SMY，并从美国国立卫生研究院（U24 CA92871和U54 CA151838）赠款，以支持MGP

所有的动物研究得到遵守约翰霍普金斯大学动物护理和使用委员会的规定。 1。附近骨骼胶原重塑体内的红外（NIR）荧光成像<ol><li> 光激活的笼CMP和尾静脉注射 <ol><li>准备110微升的每个鼠标笼CMP解决方案（20-30克鼠体重）：100微升的配制溶液，用一个额外的10微升缓冲转移和注射过程中可能发生的损失。混合11微升IR680-AHX-NB（GPO）9原液（400μM）与11微升无菌PBS溶液100μM的半胱氨酸;然后使总体积至110微升用1×PBS缓冲液中。最终的肽溶液中含有4纳摩尔IR680-AHX-NB（GPO），9和100微升的1X PBS溶液1纳摩尔半胱氨酸。 </li><li>慢慢withdra瓦特肽溶液注入0.5ml的胰岛素注射器用透明桶和一个小计（28-30 G）针，小心地取出气泡。打开紫外灯，让灯预热2-5分钟。 </li><li>将含肽的注射器直接在紫外灯（365纳米，&gt; 25毫瓦/厘米2）5分钟，以允许光激活。 </li><li>同时，在一个限制器将鼠标下加热灯，与尾部或其他识别方法，如耳标或脚趾纹身永久性记号笔标记鼠标和消毒尾用70％的酒精。 </li><li>紧接在UV活化后，注入100μl的紫外线活化IR680-AHX-NB（GPO）9溶液到尾静脉中，优选在尾部的后部。注射后请将鼠标回到笼子里。 </li></ol></li><li> 胶原重塑活动的近红外荧光成像 <ol><li>集用珍珠冲激2.0软件的脉冲成像器至37℃的摄像床的加热板。 </li><li>把全裸或鼠标剃成含有氧气2％的异氟醚麻醉诱导室。在处理和通过监测呼吸频率（〜1/sec）核实缺少鼠标移动的麻醉平面的深度。 </li><li>鼠标经过麻醉，打开珍珠成像仪抽屉，然后将动物的成像床上。快速去除鼻锥塞，并滑动鼠标的枪口鼻锥内的成像过程中，以保持鼠标在麻醉状态下（在氧气2％异氟烷在2升/分钟通过鼻锥交付）。一旦鼠标到位，关上抽屉。 </li><li>当仪器显示“准备就绪”的图像软件面板上，点击“700路”，“白光”和“85微米”框后面的“获取图像”按钮。近红外光谱（激发685 nm，发射720纳米）的D白色光照片，然后将使用自动对焦和曝光拍摄。 </li><li>当录制完成后，打开抽屉，打开鼠标，并从另一个角度获取图像。鼠标可以放在它的前面（腹），背（背）或朝向相机两侧。磁带窄条可以作为约束伸展四肢得到的利益（ 如肋骨，脚踝和手腕）该地区的最佳观赏。 </li><li>一个适当的时间来观察IR680-CMP的骨骼摄取24-96小时后注射（HPI）之间，根据用量（〜4纳摩尔/鼠标）。为了获得高分辨率的图像，牺牲鼠标颈椎脱位而根据深麻醉后72 HPI，用手术钳和剪刀去除皮肤（和头发），和上述图像是由近红外荧光。 </li><li>分析获取的近红外荧光图像。 </li></ol></li></ol> 2。胶原蛋白的前体可视化0;组织切片<ol><li> 材料准备 <ol><li>制备将1ml 10％（重量/体积）在去离子水中的BSA溶液。解冻1毫升山羊血清在水浴中在室温下进行。切几片封口膜在大约2厘米×5厘米大小。 </li><li>准备10毫升封闭缓冲液通过混合500μl的山羊血清，1毫升的10倍的PBS和8​​.5毫升去离子水。通过稀释50微升10％（重量/体积）牛血清白蛋白4.45毫升去离子水和500微升10X PBS制备5毫升CMP稀释缓冲液。 </li><li>稀释储备液的羧基标记笼的CMP，CF NB（GPO）9（480微米），以使用CMP稀释液5微米。从2.5-30μM的最佳CMP浓度而变化，这取决于组织的类型和组织样品的性质。 100微升卷被推​​荐用于染色的一个组织部分幻灯片。存储制定的CM​​P SOLUT离子在黑暗中。 </li><li>让小鼠角膜组织切片平衡至室温。孵育载玻片在1×PBS缓冲液中5分钟。在此演示中使用的小鼠角膜组织中均带有前缀在PBS溶液中的4％多聚甲醛固定1小时，冷冻保存在Tissue-Tek的十月介质，cryosectioned至8微米的厚度，以及安装在带电载玻片上。 </li></ol></li><li> 组织染色和成像 <ol><li>将玻片在湿盒。每个滑动，申请0.5毫升阻断溶液并孵育载玻片30分钟，在室温下进行。通过印迹在纸巾上的幻灯片除去封闭液。 </li><li>动用约100微升的CF NB（GPO）9解决方案，以覆盖每个幻灯片的组织切片。孵育载玻片，持续2分钟，以允许在CMP溶液渗透到组织中。 </li><li>打开紫外灯。当灯泡预热，露出CMP覆盖的组织本身ctions紫外灯为6分钟（365纳米〜8毫瓦/厘米2）脱保护笼中医。紫外线照射后，覆盖每个滑动用一块封口膜，以防止干燥的组织切片。将载玻片在潮湿的腔室，并培育它们在4℃下2小时。 </li><li>准备1:3,000稀释在1x PBS DAPI的解决方案。 CMP染色后，轻轻地用钳子取出封口膜，涂抹在纸巾上的幻灯片，以去除多余的CMP解决方案。适用于约100μl的稀释的DAPI溶液到每个组织切片后，于室温下放置1分钟。 </li><li>染色后，浸泡在1×PBS缓冲液中的幻灯片染色缸5分钟。重复此3X新鲜PBS洗涤，洗去未结合的染色剂。 </li><li>加安装介质上的组织切片的降低，且用玻璃盖玻片覆盖它，同时避免俘获气泡。放幻灯片在一个纸板滑动托盘，以保护他们免受光。 </li><li>图像中的胶原纤维束（FITC通道）和细胞核（DAPI通道）中使用荧光显微镜的组织切片。 </li></ol></li></ol>

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作者宣称没有竞争的财务权益。

如可在此协议中可以看出，CMP的递送是直截了当的，因为紫外活化的笼中医是唯一的额外步骤，以共同的尾静脉注射协议19。关键是要注入肽探针的出笼和亚稳单链状态。轿厢组防止CMP从自组装体和结合到胶原蛋白（ 图2B），直到它被UV光在该点进行CMP开始折叠成三股螺旋除去。注射制剂含有半胱氨酸，它可以与开裂笼组紫外线照射期间迅速作出反应，以减少毒性。众多的小鼠不同品系（ 如 SKH-1和DR-1），使用这一提法已经过测试，而且我们没有发现这些小鼠的任何行为或其他健康缺陷长达六个月。如果注射不暴露于紫外线后立即跟进，中医师会折叠成三聚体的三螺旋，它们没有什么可杂交能力图2C显示了一个极端的情况下注射前的中医已全部重新组合成三螺旋通过长时间的延迟时间（&gt; 2天）。为了解决这个问题，CMP溶液应在相对 ​​低的浓度（ 例如 〜40微米）来制备，和UV-decaging后立即注射到小鼠中。由于慢三螺旋折叠率稀释条件的中医（半时间的复性：&gt; 50分）和短紫外线照射和注射时间（5〜7分钟计），大部分的中医单进入血液钢绞线表格时，此协议遵循。一旦注入，中医有望保持为单链，随着浓度由20倍的血池减少，从而导致显着减少的重组率18。如果注射被延迟（ 例如 ，由于动物处理）和同源三聚体重折叠被怀疑的部分组装肽可通过加热被重新激活，以高于7076，C解离的中医师为单链，然后迅速冷却和喷射。虽然不太方便，例如热解离中医还将显示预期的体内靶向（ 图2D）的结果。 在这个演示中，裸鼠被用于近红外荧光成像，因为近红外信号的高达50％，可阻止头发。当与头发的小鼠模型（尤其是那些与黑毛）的工作，我们建议使用电动剃须刀或头发卸妆妆鼠标在成像之前感兴趣的区域。在图1中，有从该动物的腹部区域，这是由自体荧光叶绿素在动物饮食引起的假阳性信号。这样的背景信号可通过切换鼠标纯化日粮成像之前约4天显著降低。如可见于图1和图2A所示 ，有强烈的CMP摄取在尾旋Ë。因此，为了避免从不完善尾静脉注射造成伪影，我们建议在尾部的后部注入，使得在注射部位未在荧光图像捕获。 标记CMP使目标的胶原重塑体内 ，很难用其他方法来检测特定位置和成像。例如，在基于ELISA的血液和尿液测定法可用于切割的胶原端肽片段的敏感，但该方法不能定位的胶原周转的来源，因为它针对可溶性抗原。使用近红外光谱的体内成像技术的主要局限性标记中医是深度的穿透衰减和淬灭基汇集红细胞血红蛋白是七百一分之六百八十〇纳米的近红外染料的内源淬灭剂。 CMP的体内成像的未来应用包括使用中医标记直接发射γ射线的放射性核素或正电子EMI SPECT或PET成像tters，用于诊断涉及胶原重构（ 例如，骨质疏松症，关节炎，动脉粥样硬化和纤维化）的疾病状态。这些放射性标记的CMP类似物将消除由于组织的深度和汇集红细胞的存在信号损耗的限制，并允许患病组织的定量测量。此外，比较晚成像时间（ 例如 48-96 HPI）从中医的慢绑定和清拆行动而可能使其难以按照胶原重塑的快速变化。这种限制可以通过结合动力学和CMP显像剂的亲和力进一步的工程来克服。由于CMP结合具有小结构变性胶原中，基于CMP的胶原蛋白成像是相辅相成的二次谐波产生显微镜只能像胶原纤维5。 当染色组织切片与荧光标记的中医师，我们发现它有利于孵育中在4℃下，在UV活化的肽溶液样品，因为三重螺旋杂交变得容易在较低温度下1,20。它也被发现，浸渍在染色缸中新鲜的PBS缓冲液的幻灯片是，洗掉未结合中医，其中工程不是仅仅在样品移液洗涤缓冲液好得多的最有效方法。在CMP-胶原链的杂交，是非常具体和鲁棒的，因此在这个视频呈现的简单染色方案可以容易地修改为costaining额外的生物标记物，以及用于识别在未固定的组织切片的胶原降解。这是一个有效和方便的替代品使用抗胶原抗体检测胶原纤维组织的各种样品中。

胶原蛋白，在哺乳动物中最丰富的蛋白，它在组织发育和再生通过支持细胞增殖和细胞分化的关键作用。而胶原纤维（ 如 I型，II），在结缔组织的组织给予的机械强度，网络状的胶原（Ⅳ型）形成基底膜，其中细胞附着并形成有组织的组织基本支架。胶原重塑既包括胶原降解（蛋白酶）和合成（生长因子促进）。虽然胶原重塑，例如在骨，是正常组织更新过程中，多余的重塑活性，或者其在异常发生的地点的一部分，典型地指示伤口愈合响应损伤或慢性的病理状况，如癌症，骨质疏松症，关节炎，和纤维化1-4。直接图像进行胶原重塑体内的能力，可能导致进度的了解离子这些疾病，以及新的诊断和治疗。例如，实时成像可以提供有关的严重程度和疾病的位置信息，并且还可以用于评估新的治疗剂的疗效。多光子激光扫描显微术和二次谐波的产生已被应用到图像纤维状胶原为在活体小鼠5监测胞外基质重塑的肿瘤。然而，这种技术需要的动物将被安装有透明背皮褶腔室，这是一种侵入性程序。胶原重塑的直接和非侵入性的成像将受益于一个探头，专门针对胶原蛋白进行改造。这样的探针很难制备，因为它需要从完整和成熟胶原，其大量存在于正常组织6区分的重塑胶原。 胶原是由所谓的三螺旋极其罕见的蛋白结构，这是蛋白酶如在胶原重塑基质金属蛋白酶（MMP）裂解。切割的胶原碎片失去其三重螺旋结构，并成为折叠链（明胶），这是由特异性蛋白酶1进一步消化。最近发现的胶原蛋白模拟肽（CMP），其具有的倾向折叠成三股螺旋结构，可以专门针对这是从它的三股螺旋状态由任何热变性或通过酶促降解1,7分离的胶原束。单体中医和变性胶原蛋白链间的结合主要是通过驱动三螺旋杂交。因为中医自我组装成同源三聚体三螺旋在室温下与胶原蛋白杂交，笼子里的小博闻的驱动力[（GPO）的4 注 GPO（GPO）4，指定为NB（GPO）9，O：羟脯氨酸]，被开发，其中包含一个光可切割硝基苄组（NB）连接至肽的中央甘氨酸。毒品调查科笼基立体防止CMP从折叠成三股螺旋，然而，去除网箱组的紫外线照射立即触发的三重螺旋折叠蛋白和胶原蛋白的杂交1。当标记的近红外（NIR）荧光基团的单体中医是全身性地递送到模型小鼠，它们可以特异性地靶向并允许在变性胶原的组织中进行正常的（ 例如，在骨和软骨）和病理（ 例如，在肿瘤）重塑1 体内成像。 荧光标记的中医也可用于在组织学组织切片成像胶原。在组织学研究，收获的组织往往保存了固定，以保持细胞成分，并从整体恶化组织形态。定影的程序，其中包括热，并用有机溶剂处理，以及化学交林肯克的试剂（ 如多聚甲醛），变性胶原8的三重螺旋结构。这种变性生成网站为CMP杂交。它已经表明，荧光标记中医可特异性地结合到胶原中固定的组织切片（ 如皮肤，角膜，骨）甚至比抗胶原抗体，这使得轻便鉴定在肝纤维化组织9病理状况更有效。在CMP针对含有的三螺旋基序的氨基酸序列，这是共同的所有类型的胶原蛋白的变性的胶原束。因此，中医也算是一种广谱胶原蛋白染色剂。在这里，我们提出了详细的实验程序一）使用成像荧光标记笼中医在体内变性的胶原束和体外组织切片II）可视化胶原。一个近红外标记，IR680，被结合到笼中的CMP实时成像，whilë羧基（CF）用于组织染色工作，其与标准荧光显微镜的兼容性。该协议的重点是中医的成像应用程序相关的胶原重塑。方法为CMP合成可以在以前的报告1,7,9-15被发现。在这个视频报告，在正常小鼠和小鼠角膜组织切片成像骨骼组织被选择用于示范目的，但这里介绍的方法，可以容易地应用到许多病理和生物模型涉及胶原重构（ 如肿瘤，伤口愈合），如以及几乎任何前缀的组织样本含有胶原蛋白。

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IRdye 680RD Maleimide (IR680)</td>
			<td style="width:123px;">LI-COR</td>
			<td style="width:137px;">929-71050</td>
			<td style="width:167px;">Used for CMP labeling, protocol presented elsewhere1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5(6)-Carboxyfluorescein</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:137px;">21877-5G-F</td>
			<td style="width:167px;">Used for CMP labeling, protocol presented elsewhere12</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cysteine</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:137px;">YC2200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Isoflurane</td>
			<td style="width:123px;">Butler Veterinary Supply</td>
			<td style="width:137px;">4/4/5260</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat Serum</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:137px;">G9023-10ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Albumin from bovine serum (BSA)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:137px;">A9647</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">DAPI</td>
			<td style="width:123px;">Roche Applied Science</td>
			<td style="width:137px;">10236276001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluoroshield mounting medium</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:137px;">F6182-20ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:643px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes</td>
			<td style="width:123px;">BD</td>
			<td style="width:137px;">329461</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV lamp</td>
			<td style="width:123px;">McMaster-Carr</td>
			<td style="width:137px;">1447T17</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Pearl impulse imager</td>
			<td style="width:123px;">LI-COR</td>
			<td style="width:137px;">9400</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Surgical forceps and scissors</td>
			<td style="width:123px;"></td>
			<td style="width:137px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">EasyDip slide staining system</td>
			<td style="width:123px;">Light Labs</td>
			<td style="width:137px;">M900-12B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">20-place cardboard microscope slide tray</td>
			<td style="width:123px;">Light Labs</td>
			<td style="width:137px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover glasses</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:137px;">12-545-c</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescence Microscope</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:137px;">Eclipse TE2000-E</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides

胶原是细胞外基质的支持组织的形成和维护的主要结构成分。虽然胶原重塑正常组织更新的一个组成部分，涉及肿瘤，关节炎和许多其他病理情况下重塑活动过量。在胶原重塑，胶原分子的三重螺旋结构是由在细胞外环境的蛋白酶破坏。此外，目前许多组织的组织样品中胶原蛋白的一部分被固定和保存过程中变性。因此，这些变性的胶原蛋白链可以服务于生物成像的有效指标。我们以前开发一个笼中的胶原蛋白模拟肽（CMP），可以是光引发的形成三重螺旋结构，这是唯一的胶原蛋白杂交与变性的胶原链。这个程序的总体目标是：i）以图像变性的胶原链Ř从正常重塑活性在体内 esulting，和ii）使用的光引发笼中医形象化在离体的组织切片的胶原蛋白。实现有效的杂交和成功的体内和体外成像，荧光标记的笼中医要么被光激活的立即静脉注射前，或在组织切片中，直接激活。在裸鼠体内胶原蛋白和前缀在小鼠角膜组织切片正常的骨骼重塑胶原蛋白进行成像在此过程中。基于所述CMP-胶原杂交技术这里提供的成像方法可能导致组织重塑过程的深入了解，以及允许具有高胶原重塑活性相关的疾病的新诊断的发展。

图1示出的24-96小时后喷射后的光引发IR680-AHX-（GPO）9如何分布在一个健康的雌性SKH1裸鼠的典型结果。它显示了明显的CMP堆积在骨架后24 HPI，在该点最未结合的肽的已在很大程度上被清除出去。 CMP与重塑胶原链杂交的过程似乎相对较慢，尤其是在与其他肽成像探针（ 如 RGD 16）。这最有可能是因为胶原蛋白的三螺旋17,18的慢折叠率。然而，一旦杂交，在CMP链强烈结合于胶原的组织，由于只有轻微的信号还原后24 HPI（ 图1）所示。在96 HPI，皮肤切除后鼠标的近红外荧光图像清楚地表明IR680-AHX-（GPO）的骨骼摄取9在脊柱和肋骨，以及内部的膝盖，脚踝，手腕和下颌骨（ 图2A）。 在离体组织染色，光引发荧光标记的笼中医特异性杂交的组织切片中变性的胶原束。在图3中，CMP染色清楚地表明在角膜基质，这表明其作为一个具体的胶原蛋白染色剂使用的细平行的胶原纤维。 <img alt="图1" fo:content-width="3.5in" fo:src="/files/ftp_upload/51052/51052fig1highres.jpg" src="/files/ftp_upload/51052/51052fig1.jpg" /> 图1裸鼠的串行近红外荧光图像静脉给药与照片decaged IR680-AHX-（GPO）9显示了为期四天的骨骼吸收。 <a href="https://www.jove.com/files/ftp_upload/51052/51052fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:conten叔宽度=“4英寸”FO：SRC =“/ files/ftp_upload/51052/51052fig2highres.jpg”SRC =“/ files/ftp_upload/51052/51052fig2.jpg”/&gt; 图2。给予照片decaged IR680-AHX-（GPO）的小鼠近红外荧光图像如图9（A），笼IR680-AHX-NB（GPO）9无紫外线照射（B），预先折叠的三螺旋[IR680-AHX-（GPO ）9] 3（C）或热分解单链IR680-AHX-（GPO）9（D）在皮肤去除后96 HPI。骨靶向通过CMP只能在A和D可以观察到。近红外荧光信号都显示在彩虹的规模。 <a href="https://www.jove.com/files/ftp_upload/51052/51052fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/51052/51052fig3highres.jpg" src="/files/ftp_upload/51052/51052fig3.jpg" /> 图3。鼠标共同荧光显微rneal组织切片前缀在多聚甲醛固定，并用DAPI染色和光引发CF（GPO）9使用协议2，显示致密的胶原基质（通过CMP染色，绿色）以及细胞上皮和内皮（通过DAPI染色，在蓝色）（比例尺：100微米）。

Watch this Scientific Journal Video about Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides at JoVE.com

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

本程序演示了在附近的胶原重塑活动在小鼠红外荧光成像以及胶原蛋白的使用笼胶原蛋白模拟肽，可以是光引发的杂交与变性的胶原链组织切片的体外活体染色。

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

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Bioengineering

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

11.8K 次观看.  University of Utah. 本程序演示了在附近的胶原重塑活动在小鼠红外荧光成像以及胶原蛋白的使用笼胶原蛋白模拟肽，可以是光引发的杂交与变性的胶原链组织切片的体外活体染色。 

视频: Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

体外拟态正常与异常的人造血

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距“和”分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Methods Article

成像变性的胶原股