我们感谢为教育和研究的进步而捐献遗体的个人。我们感谢 OneLegacy 基金会，它为获得供体心脏进行研究奠定了基础。我们还感谢加州大学洛杉矶分校转化研究成像中心（放射学系）的 Anthony A. Smithson 和 Arvin Roque-Verdeflor 在 CT 数据采集方面的支持。该项目得到了加州大学洛杉矶分校 Amara Yad 项目的支持。我们感谢 Kalyanam Shivkumar 博士和 Olujimi A. Ajijola 博士建立和维护人类心脏研究管道。我们感谢我们的研究运营经理 Amiksha S. Gandhi 对支持我们项目的奉献精神。这项工作得到了NIH赠款OT2OD023848和P01 HL164311和Leducq赠款23CVD04给Kalyanam Shivkumar的支持，美国心脏协会职业发展奖23CDA1039446给PH，以及UCLA Amara-Yad项目（https://www.uclahealth.org/medical-services/heart/arrhythmia/about-us/amara-yad-project 的支持。本研究中使用的 GNEXT microPET/CT 扫描仪由 NIH 动物研究资助共享仪器 （1 S10 OD026917-01A1） 资助。

这项研究使用了从非失败的供体人类心脏中收集的去识别化组织样本，并获得了加州大学洛杉矶分校 （UCLA） 机构审查委员会的批准。样本来自被拒绝移植的未衰竭心脏。对心脏进行压力灌注，用 4% 多聚甲醛 （PFA） 固定，并在组织处理前按照以下方法进行成像。 图 1 总结了研究顺序的流程图。材料 表中列出了研究中使用的试剂和设备的详细信息。
1. 微观检查
<ol><li>使用改良免疫标记的溶剂透明化器官 3D 成像 （iDISCO+） 方案进行组织透明化。<ol><li>用手术刀解剖 4% PFA 固定的组织，使其适合 3 mm × 16 mm × 25 mm 的共聚焦显微镜室。为了对较厚的组织进行成像，可以在载玻片上堆叠额外的腔室和/或垫片。</li>
		<li>使用分级甲醇 （MeOH） 系列（20%、40%、60%、80% 和 100% MeOH 在去离子 H2O [体积/体积]）下在室温 （RT） 下搅拌每个样品 1 小时。</li>
		<li>在 RT 下用 100% MeOH 洗涤 1 小时，然后在 RT 下浸入 66% 二氯甲烷/33% MeOH 中搅拌过夜。</li>
		<li>第二天，在 RT 下用 MeOH （100%） 洗涤两次 1 小时，在 4 °C 下冷却，并在 4 °C 下用 5% H2O2 在 MeOH （vol/vol） 中处理过夜。</li>
		<li>用分级的 MeOH 系列（80%、60%、40% 和 20% MeOH）再水化，并在 0.01 mol/L PBS 中洗涤 1 小时，每次在 RT 下搅拌。</li>
		<li>在 RT 下，在 0.01 mol/L PBS 和 0.2% Triton X-100 中洗涤组织两次，持续 1 小时。</li>
		<li>在 0.01 mol/L PBS、20% 二甲基亚砜 （DMSO）、0.2% Triton X-100 和 0.3 mol/L 甘氨酸中透化 2 天，在 37 °C 下搅拌，制备免疫标记。</li>
		<li>在 0.01 mol/L PBS 中，含 10% DMSO、0.2% Triton X-100 和 5% 正常驴血清，在 37 °C 下搅拌再封闭 2 天。</li>
		<li>使用与偶联荧光团的 MeOH 相容的一抗进行标记，该荧光团在 0.01 mol/L PBS 中稀释，其中含有 10 mg/mL 肝素 （PTwH）、0.2% 吐温-20、5% DMSO 和 3% 正常驴血清，在 37 °C 下搅拌 3-4 天。</li>
		<li>补充抗体溶液，并在 37 °C 下搅拌再孵育 3-4 天。</li>
		<li>在一抗溶液中孵育 1 周后，在 RT 下用 PTwH 洗涤 4 至 5 次过夜。</li>
		<li>与在 PTwH、3% 正常驴血清中稀释的荧光团偶联的二抗在 37 °C 下搅拌孵育 3 天。</li>
		<li>补充二抗溶液，在 37 °C 下搅拌再孵育 3 天。</li>
		<li>在二抗溶液中孵育 6 天后，在 RT 下用 PTwH 洗涤 4-5 次过夜。</li>
		<li>用分级的 MeOH 系列（20%、40%、60%、80%、100% 和 100% MeOH）脱水。样品可以在 RT 中储存过夜。</li>
		<li>在 RT 下在 66% 二氯甲烷/33% MeOH 中孵育 3 小时，同时搅拌。</li>
		<li>在 RT 下用 100% 二氯甲烷洗涤两次，每次 15 分钟，并搅拌。</li>
		<li>将标本孵育并储存在苄基醚中。填充试管以尽量减少空气氧化样品。</li>
	</ol></li>
	<li>对组织透明化标本进行成像<ol><li>将含有粘合剂的腔室固定在载玻片上，并在腔室周边涂抹指甲油。对于较厚的组织，可以在载玻片上堆叠额外的腔室和/或垫片。</li>
		<li>将透明化的组织放入腔室中，填充苄醚，并盖上盖玻片。</li>
		<li>在盖玻片周围涂上指甲油以形成密封。</li>
		<li>使用带有 5 倍或 10 倍镜头的正置激光扫描共聚焦显微镜获取 tilescan 和 Z 堆栈图像，以在镜头工作距离的深度成像。</li>
		<li>使用适用于所用荧光团发射光谱的激光线以 1024 x 1024 的分辨率成像。使用 488 nm 激光线可以看到肌肉自发荧光。</li>
		<li>确保 z 轴步长与基于指定物镜11 的数值孔径的奈奎斯特采样相称。</li>
		<li>拼接图像并使用软件进行 3D 可视化。</li>
		<li>使用单个和合并通道的 Z 堆栈的最大强度投影 （MIP） 图像创建图形（图 2）。</li>
	</ol></li>
	<li>免疫组化 注：在组织石蜡包埋22 后，使用以下程序创建用于免疫组织化学研究的载玻片。<ol><li>制备折射率匹配溶液 （RIMS）。<ol><li>向去离子 H2O 中加入 10.9 g Na2HPO4（无水）和 3.1 g NaH2PO4（一水合物），制备 0.1 mol/L 磷酸盐缓冲液，总体积为 1 L （pH 7.4）。过滤消毒溶液并将其储存在 RT 中。</li>
			<li>将磷酸盐缓冲液稀释至 0.02 mol/L。</li>
			<li>用磁力搅拌棒在最终储存瓶中搅拌溶液 10 分钟，将 Histodenz 溶解在 30 mL 0.02 mol/L 磷酸盐缓冲液中，该储存瓶可以密封以最大限度地减少蒸发和污染。</li>
			<li>加入叠氮化钠至总浓度为 0.01% （w/v），并用 NaOH 将 pH 值调节至 7.5。</li>
			<li>通过改变 Histodenz 的最终浓度来调整 RI。</li>
			<li>将 RIMS 在 RT 中存放数月。如果发现微生物污染，请丢弃。 注意：请勿高压灭菌任何含有叠氮化钠的溶液。</li>
		</ol></li>
		<li>制备用于免疫组织化学研究的载玻片<ol><li>用切片机创建 5 μm 厚的切片。将组织切片应用于带电的载玻片。</li>
			<li>通过将载玻片在 >75% 二甲苯中孵育 10 分钟来去除石蜡。将玻片移至装有二甲苯的第二个容器中再放置 10 分钟。</li>
			<li>将载玻片浸入 100% 乙醇中 10 分钟，然后在 95% 乙醇中浸泡 5 分钟，在 70% 乙醇中浸泡 5 分钟，去除二甲苯。</li>
			<li>用去离子 H2O 冲洗载玻片 5 分钟。</li>
			<li>将玻片在 90-95°C 下浸入抗原修复缓冲液中 25 分钟。</li>
			<li>让容器在搅拌下冷却至 RT 1 小时。</li>
			<li>将载玻片浸入浸泡缓冲液 （0.01 mol/L PBS + 0.4% Triton X-100） 中，在 4°C 下浸泡 30 分钟。</li>
			<li>用 PAP 笔圈住组织。将 PBS 添加到每张载玻片中，并将其放入加湿室中以防止干燥。</li>
			<li>在 RT 下用 PBS 洗涤载玻片，搅拌 5 分钟。</li>
			<li>用封闭缓冲液（0.01 mol/L PBS + 10% 驴血清 + 0.1% TX-100）封闭 1 小时，同时搅拌。</li>
			<li>与在封闭缓冲液中稀释的一抗在 4°C 下孵育过夜。</li>
			<li>第二天，让载玻片升温至 RT 15 分钟。</li>
			<li>用 0.01 mol/L PBS + 0.2% TritonX-100 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>与在封闭缓冲液中稀释的二抗在 RT 下搅拌孵育 1 小时。</li>
			<li>用 0.01 mol/L PBS + 0.2% TritonX-100 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>用 0.01 mol/L PBS 洗涤载玻片 3 次，每次 5 分钟。</li>
			<li>用滴管滴入 1 滴 RIMS 并盖上盖玻片。</li>
			<li>在盖玻片周围涂上指甲油以形成密封。</li>
			<li>作为阴性对照，在没有一抗的情况下运行样品，以证明不存在特异性染色。</li>
		</ol></li>
		<li>免疫染色玻片成像<ol><li>使用带有 10 倍、20 倍和 40 倍物镜的激光扫描共聚焦显微镜对载玻片进行成像。</li>
			<li>使用适合所用二抗发射光谱的激光线以 1024 x 1024 的分辨率成像。</li>
			<li>使用单个和合并通道的 Z 堆栈的最大强度投影 （MIP） 图像创建图形（图 3）。</li>
		</ol></li>
	</ol></li>
</ol>2. 中尺度检查
<ol><li>立体解剖<ol><li>使用带夹的放大台灯、手术望远镜或体视显微镜，对微小或薄的结构进行精细解剖，例如房室结、房室结动脉和心脏神经丛（亚毫米到毫米级）。</li>
	</ol></li>
	<li>显微 CT 扫描 注意：在压力灌注和固定后以及使用微正电子发射断层扫描 （PET）/CT 扫描仪在解剖的任何阶段进行 CT 成像（图 4）。<ol><li>在样品成像之前，将 CT X 射线源预热 25 分钟。</li>
		<li>将心脏样本放在扫描仪床上。</li>
		<li>将扫描仪床移动到 544 mm 的水平位置和 14 mm 的垂直位置，以使心脏在 CT 视野 （FOV） 中居中。</li>
		<li>以 80 kVp、150 μA 的速度获取 CT 图像，在 1 分钟的扫描时间内以 200 μm 的空间分辨率进行 720 次投影。</li>
		<li>使用 12 厘米 x 12 厘米 x 10 厘米的视野和 600 x 600 x 500 体素的矩阵重建 CT 数据，并另存为 DICOM 文件。</li>
	</ol></li>
</ol>3. 宏观考察
<ol><li>加压灌注和固定 注：作者修改了先前描述的压力灌注和固定技术，并将其应用于拒绝移植的非失败人类心脏 14,19,20,21。<ol><li>使用高流量泵进行灌注固定。使用 100% 乙醇14、4% PFA 或 10% 福尔马林作为固定剂。 注意：通过尽可能远端切除升主动脉、肺干、腔静脉和肺静脉来恢复心脏，并在威斯康星大学解决方案23 中输送。</li>
		<li>使用两个 20-24 Fr 手术插管进行右心和左心灌注。对于右心灌注，插管上腔静脉，并用半切 12-30 mL 大小的塑料注射器在肺干或肺动脉处放置一个通气口，该注射器带有连接到三通旋塞阀的 Luer-Lock 尖端。</li>
		<li>放入适当大小的锁定半切塑料注射器或 1.5-5.0 mL 离心管后，用麻线封闭下腔静脉和另一条肺动脉。<ol><li>对于左心的顺行灌注，将其中一条肺静脉插管，并使用半切 12-30 mL 大小的塑料注射器在主动脉的远端放置一个通风口，该注射器带有连接到三通旋塞阀的 Luer-Lock 尖端。</li>
			<li>对于左心的逆行灌注，将其中一个主动脉弓分支插管，并在主动脉弓的另一个分支处放置一个通风口。将插管的尖端放在两个心室中。</li>
		</ol></li>
		<li>插入适当大小的锁定半切塑料注射器或 1.5-5.0 mL 微量离心管后，用麻线堵塞其他血管孔。用薄纱布盖住注射器/管子/插管的插入部分，以防止渗漏和滑落。使用缝合、绑扎或结块修复大渗漏。允许少量泄漏。</li>
		<li>将心脏悬在塑料容器中。</li>
		<li>将 22-24 Fr 软塑料管连接到每个套管上，并将管子的另一端插入装满固定剂的容器中。</li>
		<li>使用高流量泵在右心和左心回路中循环固定剂，右心和左心分别达到约 100-300 mL/min 和 200-400 mL/min，分别在右心室达到约 20 mmHg 和左心室约 80 mmHg。</li>
		<li>在 4 °C 维持灌注 24 小时。</li>
		<li>用 0.01 mol/L PBS 洗涤心脏 30 分钟，搅拌 4 次。</li>
		<li>将心脏储存在 4 °C 的 0.01 mol/L PBS/0.02% 叠氮化钠中。 注意：压力灌注固定仅对新鲜心脏有效，对从防腐尸体中恢复的心脏无效。</li>
	</ol></li>
	<li>大体解剖<ol><li>在解剖的每个阶段使用照片记录进行渐进式解剖。</li>
		<li>为了保持临床相关性，请特别注意避免扭曲/变形任何结构，以维持心脏的生理形态。</li>
		<li>使用临床相关方向（例如右前斜方向）对目标结构进行成像。</li>
	</ol></li>
	<li>摄影<ol><li>将压力灌注和固定的心脏放在三脚架上，该平台安装有多个叉子，并能够 360度旋转。</li>
		<li>使用数码单镜头反光相机（图 5）24 拍摄心脏，同时使用 C 型支架上的多个发光二极管灯板和宽大的黑色羽绒被背景布。</li>
		<li>使用长焦距 （200 mm） 镜头拍摄 4-6 英尺的工作距离，以最大限度地减少拍摄对象的失真14.</li>
	</ol></li>
	<li>浮雕<ol><li>要显示立体图像，请从 CT 数据集重建一对照片或体积渲染图像，在水平面上的旋转角度相差 10°。</li>
		<li>使用免费软件16 将这些二维 （2D） 图像（称为立体图）转换为立体图。</li>
		<li>要查看立体图，请使用红色/青色眼镜。</li>
	</ol></li>
	<li>摄影 测量 注：摄影测量是从以不同角度拍摄的多张二维照片中生成三维表面渲染重建的应用科学17。<ol><li>将样品悬挂在 C 型支架上或将其放在旋转台上，以使用智能手机获得数百张多向照片。</li>
		<li>使用市售软件以 FBX 格式生成 3D 模型。</li>
	</ol></li>
	<li>CT 扫描 注意：CT 扫描可以在压力灌注和固定后以及解剖的任何阶段进行。<ol><li>将心脏样本悬浮在容器顶部的杆上。为防止心脏在扫描过程中摆动，请用固定在容器底部的塑料尖头支撑心脏底部。因此，空气将起到负对比的作用。</li>
		<li>使用市售的多探测器行 CT 扫描仪进行 CT 扫描，参数如下：管电压为 120 kV，管电流为 800-900 mA，机架旋转为 280 ms。剂量长度产品一般为 500-1200 mGy.cm。</li>
		<li>使用以下参数重建轴向图像数据：截面厚度，0.6 毫米;增量间隔，0.3 毫米;视野，尽可能小（通常为 100-200 毫米）;和一个矩阵，512 × 512。</li>
	</ol></li>
	<li>虚拟解剖<ol><li>使用市售软件分析 CT 扫描图像以生成虚拟解剖图像。 注意：虚拟解剖是体积渲染过程的修改，其中焦点转移到心腔和血管壁18。在此过程中，手动阈值实际上从原始数据集中删除了增强的腔室。</li>
		<li>通过虚拟解剖可视化非增强的壁、隔膜和瓣膜，以生成类似于大体解剖的图像。与心脏标本的粗略解剖不同，虚拟解剖过程中的切割平面实际上是无限的。几乎任何视图都可以根据需要重新创建以可视化感兴趣的结构。</li>
	</ol></li>
	<li>3D 打印<ol><li>在 3D 打印机软件中打开心脏样本的兼容文件。</li>
		<li>在 3D 打印机的打印设置中使用 0.10 mm Fast DETAIL 的配置文件，并将打印速度降低到 20 mm/s。启用 Generate support material（生成支持材料）。</li>
		<li>在 3D 打印机中将 TPU 线材的配置文件 用于“线材设置”。</li>
		<li>将 Original Prusa MK4 Input Shaper 0.4 喷嘴 的配置文件用于打印机中的“打印机设置”。</li>
		<li>切片完成后，将 BGCODE 文件保存在 USB 闪存驱动器中进行 3D 打印。</li>
		<li>使用 1.75 毫米 TPU 细丝进行 3D 打印人体心脏样本。在 3D 打印之前，使用细丝干燥器将 TPU 细丝干燥 6 小时。</li>
		<li>为了减少 3D 打印过程中的线丝张力，请将线轴放在带有内置轴承的线轴支架上，以方便线轴旋转。使用市售的 3D 打印机和纹理粉末涂层钢板进行 3D 打印。</li>
		<li>3D 打印完成后，小心地去除支撑材料。</li>
	</ol></li>
</ol>

从微观到宏观尺度的人类心脏综合 3D 分析

<ol>
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本研究展示了分析从全人类心脏获得的样本的综合管道。代表性结果显示，对单个心脏进行常规的微观到宏观解剖检查。由于人类心脏样本非常珍贵，因此多尺度方法是理想且有效的，这样就不会通过为各种目的应用多种方案来浪费样本的任何部分，包括科学研究中的发现、临床程序的改进和解剖学教育，同时保持解剖学相关性在整个心脏的背景下。
关于微量检查，免疫染?...

由于功能服从形式，了解心脏的结构是欣赏其生理学的基础。尽管大量研究揭示了从微观到宏观的心脏解剖结构 1,2,3，但多个问题仍未解决，尤其是与人类心脏解剖学相关的问题。这部分是因为专注于功能解剖学的基础研究通常使用动物心脏 4,5,6，这通常与人类心脏 1,7,8 不同。此外，每项单独的研究，即使是那些使用人类心脏样本的研究，也往往侧重于非常具体的结构，这使得很难在整个心脏的背景下应用这些发现。如果聚焦结构位于微尺度或中尺度，例如 perinexus9 和 ganglionated plexus10，则更是如此。
在这种情况下，对被拒绝移植的人类心脏的系统结构研究提供了一个独特而难得的机会，可以获得微观和宏观尺度上聚焦的心脏结构的综合图谱11。显微镜检查方案包括组织透明化（溶剂透明化器官的改良免疫标记启用三维 （3D） 成像，iDISCO+）12,13 和免疫组织化学染色。中镜检查方案包括立体解剖、宏摄影和显微计算机断层扫描 （CT）。肉眼检查方案包括大体解剖14、摄影（包括立体画和摄影测量）15、16、17、CT、虚拟解剖18 和物理或虚拟解剖或整个心脏的 3D 打印17。为了准备肉眼检查，进行压力灌注固定以保持心脏的 3D 结构和生理相关形态 14,19,20,21。这些技术的组合应用是独一无二的，对于在人体心脏的 3D 结构背景下关联不同的解剖特征至关重要。
由于获得非病理性人类心脏样本的机会极其有限，因此本文描述的多尺度方法可以最大限度地利用样本。通过应用下面描述的各种程序，具有代表性的结果将向读者说明如何将这些发现用于多种目的，包括科学研究11 中的发现（心脏神经支配的综合分析、神经节神经丛的分布）、临床程序的改进（手术和介入方法的模拟）和解剖学教育（心脏解剖学的真实 3D 演示）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Viewer</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>647 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>711-605-152</td>
			<td></td>
		</tr>
		<tr>
			<td>647 AffiniPure Donkey Anti-Sheep IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>713-605-147</td>
			<td></td>
		</tr>
		<tr>
			<td>AF Micro-NIKKOR 200 mm f/4D IF-ED lens</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Actin, &#945;-Smooth Muscle - Cy3 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C6198</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen Retrieval Buffer (100x EDTA Buffer, pH 8.0)</td>
			<td>Abcam</td>
			<td>ab93680</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PGP9.5 (protein gene product 9.5)</td>
			<td>Abcam</td>
			<td>ab108986</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TH (tyrosine hydrox ylase)</td>
			<td>Abcam</td>
			<td>ab1542</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-VAChT (vesicular acetylcholine transporter)</td>
			<td>Synaptic Systems</td>
			<td>139 103</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>108014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cheetah 3D printer filament (95A), 1.75 mm</td>
			<td>NinjaTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip, 22 mm x 30mm, No. 1.5</td>
			<td>VWR</td>
			<td>48393 151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>Decon laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>GNEXT PET/CT</td>
			<td>SOFIE Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>Sigma-Aldrich</td>
			<td>D2158-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>H1009-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Zeiss</td>
			<td>ZEN (black edition)</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Oxford Instruments</td>
			<td>Imaris 10</td>
			<td></td>
		</tr>
		<tr>
			<td>iSpacer</td>
			<td>Sunjin Labs</td>
			<td>iSpacer 3mm</td>
			<td></td>
		</tr>
		<tr>
			<td>KIRI Engine</td>
			<td>KIRI Innovation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>LEAD-2 - Vertical &#38; Multi-channels Peristaltic Pump</td>
			<td>LONGER</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lightview XL&#160;</td>
			<td>Brightech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (Certified ACS)</td>
			<td>Fischer Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon D850</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NinjaTek NinjaFlex TPU @MK4</td>
			<td>NinjaTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Original Prusa MK4 3D printer</td>
			<td>Prusa Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycam</td>
			<td>Polycam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrusaSlicer 2.7.1</td>
			<td>Prusa Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SARA-Engine</td>
			<td>pita4 mobile LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scaniverse</td>
			<td>Niantic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Gold Antiface Mountant</td>
			<td>Invitrogen</td>
			<td>S36936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide, 5% (w/v)</td>
			<td>Ricca Chemical Company</td>
			<td>7144.8-32</td>
			<td></td>
		</tr>
		<tr>
			<td>SOMATOM Definition AS</td>
			<td>Siemens Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Field Surgi-Spec Telescopes,&#160;</td>
			<td>Designs for Vision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope System SZ61</td>
			<td>OLYMPUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>StereoPhoto Maker</td>
			<td>Free ware developed by Masuji Suto</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides, Precleaned</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziostation2</td>
			<td>Ziosoft, AMIN</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pipeline for multi-scale three-dimensional anatomic study of the human heart

对排斥移植的未衰竭人类心脏的详细研究为在微观和宏观尺度上进行结构分析提供了独特的机会。这些技术包括组织透明化（对溶剂透明化器官进行改良免疫标记的三维 （3D） 成像）和免疫组织化学染色。中镜检查程序包括立体解剖和显微计算机断层扫描 （CT）。肉眼检查程序包括大体解剖、摄影（包括立体和摄影测量）、CT 和物理或虚拟解剖或整个心脏的 3D 打印。在肉眼检查之前，可以进行压力灌注固定以保持心脏的 3D 结构和生理相关形态。将这些技术相结合来研究人类心脏是独特且至关重要的，对于在心脏 3D 结构的背景下理解不同解剖特征（如冠状动脉系统和心肌神经支配）之间的关系至关重要。该协议详细描述了方法，并包括代表性结果，以说明人类心脏解剖学研究的进展。

微量检查 应用组织透明化允许使用共聚焦显微镜对更大体积的组织进行 3D 成像。在心脏中，可以观察到包含心脏神经元的神经节和心肌神经支配的神经模式（图2）。 图 3 显示了神经和平滑肌细胞免疫染色的人左心室心肌的共聚焦图像。观察到血管穿过心肌，并鉴定出许多神经纤维，既与血管相关又独立于血管。
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作者聚焦：通过心脏多尺度分析推进人类心脏解剖学

该协议提供了一个全面的管道，用于分析从人类心脏获得的样品，这些样品跨越微观和宏观尺度。

用于人类心脏多尺度三维解剖学研究的管道

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...

pipeline-for-multi-scale-three-dimensional-anatomic-study-human

Research

JoVE Journal

Biology

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

251 次观看.  David Geffen School of Medicine at UCLA. 了解心脏解剖结构是了解心脏生理学的基础。功能解剖学的研究已经跨越了微观到宏观尺度，但主要使用动物心脏。对排斥移植的供体人类心脏的系统研究为开发涵盖微观到宏观尺度的综合图谱提供了独特的机会。通过对不同尺度的人类心脏的研究，我们展示了心脏的精细神经支配，确定了心脏解剖结构以改进手术和介入方法，并开发了解剖学...

视频: 作者聚焦：通过心脏多尺度分析推进人类心脏解剖学

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.