本研究得到了浙江省自然科学基金资助（Z23H290001，LY19H280001）;国家自然科学基金（82274364、81673607、81774011）;以及湖州市科技基金公益研究项目（2021GY49,2018GZ24）。感谢浙江中医药大学中医科学院医学研究中心公共平台的大力帮助、技术支持和实验支持。

1. 材料准备
<ol><li>在12mL的DMEM培养基（10%胎牛血清和100U / mL青霉素 - 链霉素）中培养2 x 106 Panc02细胞，并在5%二氧化碳和95%空气的气氛中保持在37°C和95%湿度48小时。</li>
	<li>收集Panc02细胞，以28× g 离心2分钟，然后弃去上清液。确保样品具有适当的大小（1 x 107 个细胞），否则，以下固定和脱水步骤将无法正常工作。</li>
	<li>加入1mL的2.5%戊二醛作为固定剂，将新鲜的Panc02细胞固定在4°C的1.5mL微量离心管中过夜。 注意:在以下每个步骤（步骤1.3-1.11）中，样品需要在1,006× g 下离心5分钟。</li>
	<li>吸出固定剂，并用0.1M磷酸盐缓冲盐水（PBS）冲洗样品两次，然后在室温下用双蒸水（ddH2O）冲洗两次，每次10分钟。</li>
	<li>加入50μL含有1%四氧化锇（OsO 4）和1.5%亚铁氰化钾的溶液，比例为1:1，在4°C下1小时。然后，用0.1M PBS冲洗两次，每次10分钟，并用ddH2O冲洗10分钟。</li>
	<li>加入 1 mL 的 1% 硫代碳酰肼 （TCH），在室温下孵育 30 分钟至 1 小时，然后用 ddH2O 冲洗四次，每次 10 分钟。</li>
	<li>加入 50 μL 的 1% OsO4，在室温下固定 1 小时，然后用 ddH2O 冲洗四次，每次 10 分钟。</li>
	<li>在4°C下加入1mL的2%乙酸铀酰过夜，然后用ddH2O冲洗四次，每次10分钟。</li>
	<li>在60°C下加入1mL沃尔顿溶液（0.066g硝酸铅溶解在10mL的0.03mol/L天冬氨酸储备溶液中），再孵育1小时。</li>
	<li>用ddH2O冲洗四次，每次10分钟，然后在分级酒精系列中脱水10分钟，每次:50%酒精，70%酒精，80%酒精和90%酒精1x和100%酒精2x。然后，在100%丙酮中脱水两次，每次10分钟。使用每种溶液 1 mL 进行脱水。</li>
	<li>将 200 μL 丙酮与 Pon 812 环氧树脂以 3:1、1:1 和 1:3 的比例混合。将样品分别在室温下浸泡在树脂中2h，4h和4h，然后取100%Pon 812环氧树脂浸渍过夜。 注意:执行染色和树脂包埋步骤时，重要的是在通风橱中操作，因为这些是有毒物质。此外，在实验过程中必须穿上实验室外套和耐溶剂手套。</li>
	<li>将试样放入包埋模具中，然后置于60°C的烘箱中48小时聚合。使用平面包埋29 来减少样品周围树脂的外观。这简化了试样安装，并减少了充电伪影对后续图像分割的影响。 注意: 为了随后生成水平切割表面，可以将少量树脂添加到模具孔中，注意不要过度填充。</li>
	<li>首先清洗硅片，然后用浓缩的H2SO4 / H 2 O2溶液亲水30分钟来制备硅片。使用超薄切片机在亲水硅晶片上收集厚度为70nm的连续切片条，并在60°C烘箱中干燥10分钟。 注意:当切片漂浮到晶圆表面时，请缓慢捕获切片，以防止切片丢失或变形。</li>
</ol>2. 图像采集与三维重建
<ol><li>在将硅晶圆安装到载物台上之前，用蒸馏水清洗切片三次，然后风干。切割尺寸为1 cm x 0.5 cm的导电碳带，并将其粘贴在硅晶片和SEM样品台之间以完成样品设置（图2E）。</li>
	<li>将 FE-SEM 加速电压参数设置为 2 kV，工作距离为 4 mm（图 3B 和 表 1）。 注意:为了提高信噪比并减少图像中的噪声，请尽可能以低放大倍率进行观察。随着倍数的增加，SEM的扫描范围减小，导致图像上的电荷积累增加。</li>
	<li>单击顶部菜单栏中的 H/L 图标 。首先，在低放大倍率下，定向目标切片条的第一部分，然后再次单击 H / L 选项（图3A）切换到高放大倍率模式;通过调整图像亮度、对比度和放大率，为感兴趣的结构收集适当的图像。</li>
	<li>选择" 项目视图">"开放数据"，然后将要分析的图像文件导入软件。</li>
	<li>通过单击对齐 切片>编辑 对齐图片（图 4B），并通过修改图像透明度来调整左下角菜单栏中的 强度范围 值。在这里，使用半自动对齐策略;具体来说，通过自动对齐，使图片与上一张图片重叠，然后进行手动微调。 注意:在这项工作中，在对齐过程中，使用先前的图像作为参考，并利用移动和旋转操作来最大程度地重叠两个图像的目标结构。单击对齐 当前切片对 可以帮助自动对齐图像，但可能会出现错位。</li>
	<li>选择 "提取子体积"模块，并裁剪对齐的数据集以适合整个堆栈重叠部分的大小。</li>
	<li>在 分割 子部分（图 4C）下，选择重新 采样>分割>保存。选择 魔棒 的阈值和 画笔 工具的大小以选择正确的范围。这里再次采用半自动分割方法，首先使用 魔杖 自动选择合适的大面积区域，然后使用 画笔 准确描述细节。 注意:通过更改遮罩值并使用绘制极限线，可以根据感兴趣结构与周围结构之间存在的像素强度的明显差异，将魔棒应用于分割图像。它无法区分图像采集过程中产生的充电伪影。因此，它可能会导致目标区域的分段不正确。画笔工具可用于手动分割以准确重建生物结构。</li>
	<li>在 "分割>选择"下，单击 + 图标以添加所选区域（图 4C）。</li>
	<li>重复上述步骤（2.8）以在不同的图像中选择相同的目标结构，直到选择完成以重建感兴趣的微观结构。 注意:在线粒体重塑过程中，必须从一组连续图像中间的图片中执行目标对象的选择。如果从第一张或最后一张图片中选择所需区域，则重建结果将无法呈现完整的 3D 可视化。</li>
	<li>完成区域分割后，根据对象大小和图片分辨率的最佳结果生成图像文件。单击 裁剪编辑器，然后在 虚拟滑块 框中输入数字 6（图 4C）。</li>
	<li>在左侧的下拉菜单中，右键单击" 项目 "子部分下的灰色区域，然后选择" 生成曲面>创建>应用"。在生成的文件中，使用曲面视图模块创建 曲面 结构和 3D 表示。</li>
</ol>3. 量化
<ol><li>单击删除 小斑点>应用 （图 4D）。在 "项目 "子部分下，右键单击灰色区域，然后选择 "标签分析">"应用 "（图 4D）。</li>
	<li>自定义列的名称，然后选择度量值组。从"本机测量"框中选择"Volume3d 和 Length3d"。 </li>
	<li>单击屏幕左下角的 "应用 "按钮。复制数据，并在统计软件（如GraphPad）中绘制图形。</li>
</ol>

3D 技术可视化胰腺癌细胞中的线粒体嵴

作者声明不存在利益冲突。

这里介绍的方法是一个有用的分步指南，用于应用3D重建技术，该技术涉及将电子显微镜和图像处理技术应用于从连续超薄切片生成的2D断层扫描图像的堆叠和分割。该协议强调了可以通过细胞器超微结构的3D可视化来解决的2D图像的局限性，该图像具有高分辨率水平上结构的强可重复性和更高的精度的优点。更重要的是，这种3D可视化可以应用于肿瘤细胞，使病理机制的研究更加直接可靠。在这项...

线粒体是细胞中最重要的细胞器之一。它们是细胞生物能量学和代谢的中心枢纽1,2，并在癌症中起着关键作用3。胰腺癌（PC）由于其快速扩散和高死亡率，是最难治疗的癌症之一4。线粒体功能障碍主要由线粒体形态变化3,5,6,7引起，与PC8的疾病机制有关。线粒体也是高度动态的，这反映在其网络连接和嵴结构的频繁和动态变化上9。嵴结构的重塑可直接影响线粒体功能和细胞状态10,11，在肿瘤细胞生长、转移和肿瘤微环境变化过程中发生显著改变12,13。
近年来，科学家们使用EM观察研究了这种细胞器14,15,16,17;例如，研究人员使用3D重建技术分析了线粒体动力学6,7,18,19。电子显微镜图像三维重建的一般概念和方法早在196820年就正式确立，涉及结合电子显微镜、电子衍射和计算机图像处理来重建T4噬菌体尾巴。到目前为止，电子显微镜3D成像技术在图像分辨率21、自动化程度22和处理体积23方面取得了重大进展，并且在生物学研究中的应用越来越广泛，从组织水平到纳米尺度的细胞器超微结构水平24。近年来，电子显微镜3D成像也成为一种前景广阔的技术25,26,27。
对线粒体嵴的日益关注特别说明了超微结构体积成像的基本要求。透射电子显微镜（TEM）已被用于可视化在铜网格（400目）28上收集的样品，电子束穿过该切片。然而，由于铜网格的范围有限，不可能对同一样品29的连续切片进行完全成像。这使得TEM成像过程中靶标结构的研究变得复杂。此外，TEM依赖于耗时且容易出错的手动任务，包括切割和收集多个切片并按顺序成像21，因此它不适用于大体积样品的超微结构重建23。目前，大体积样品成像的高分辨率重建是通过使用专用设备实现的，例如TEM相机阵列（TEMCA）30 或两个第二代TEMCA系统（TEMCA2）31，可以在短时间内实现自动化高通量成像。然而，由于需要定制设备，这种类型的成像不具有易于获得和通用的优点。
与TEM相比，基于SEM 32,33自动生成大面积连续体积图像的方法提高了串行成像的效率和可靠性，并提供了更高的z分辨率34。例如，连续块面扫描电子显微镜（SBF-SEM）和聚焦离子束扫描电子显微镜（FIB-SEM）都使高速，高效和分辨率35,36的超微结构的3D重建成为可能。然而，不可避免地，通过SBF-SEM的金刚石刀或通过FIB-SEM33,37的聚焦离子束进行铣削，块表面被机械剃掉。由于两种方法对样品的破坏性，不可能再次重建相同的目标结构以进行进一步分析38,39,40。此外，很少有研究试图使用EM重建癌细胞的3D细胞器超微结构来观察病理变化12。基于这些原因，为了进一步阐明胰腺癌细胞等癌细胞的病理机制，我们提出了一种使用超薄切片机和场发射扫描电子显微镜（FE-SEM）在嵴水平分析线粒体超微结构的连续切片图像的3D重建新技术;通过这项技术，可以使用高效且可访问的方法获取高分辨率数据。使用超薄切片机制成的系列超薄切片可以半永久地存储在网格盒中，并在几年后多次重新成像41.FE-SEM因其能够提供高分辨率成像，高放大倍率和多功能性而作为各种研究领域的工具受到高度重视42。为了尝试以3D形式显示细胞器的精细结构，使用FE-SEM 43,44产生的背散射电子生成具有有用分辨率的串行2D图像堆栈的技术也可用于实现目标区域或其相关结构的高通量和多尺度成像，而无需特殊设备45.电荷伪影的产生直接影响采集图像的质量，因此保持较短的停留时间尤为重要。
因此，本研究详细阐述了该SEM技术中用于重建线粒体嵴46的3D结构的实验程序。具体来说，我们展示了使用Amira软件实现线粒体区域的半自动分割和数字化3D重建的过程，其中还包括使用传统的OTO标本制备方法44,47制作切片样品，使用超切片机切片完成切片收集，并通过FE-SEM获取顺序2D数据。

<table><tbody><tr><td>(1S,3R)-RSL3</td><td>MCE</td><td>HY-100218A</td><td></td></tr><tr><td>Acetone</td><td>SIGMA</td><td>179124</td><td></td></tr><tr><td>Amira</td><td>Visage Imaging</td><td></td><td>2020.2</td></tr><tr><td>Aspartic acid</td><td>MCE</td><td>HY-42068</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&amp;rsquo;s medium</td><td>Gibco</td><td>11995115</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>100983</td><td></td></tr><tr><td>Ferrostatin-1</td><td>MCE</td><td>HY-100579</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10437010</td><td></td></tr><tr><td>Field emission scanning electron microscope</td><td>HITACHI</td><td>SU8010</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Alfa Aesar</td><td>A10500.22</td><td></td></tr><tr><td>Lead nitrate</td><td>SANTA CRUZ</td><td>sc-211724</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>SANTA CRUZ</td><td>sc-206008B</td><td></td></tr><tr><td>Panc02</td><td>European Collection of Authenticated Cell Cultures&amp;nbsp;</td><td>98102213</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Biosharp</td><td>BL505A</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Biosharp</td><td>BL302A</td><td></td></tr><tr><td>Pon 812 Epoxy resin</td><td>SPI CHEM</td><td>GS02660</td><td></td></tr><tr><td>Potassium ferrocyanide</td><td>Macklin</td><td>P816416</td><td></td></tr><tr><td>Thiocarbohydrazide</td><td>Merck</td><td>223220</td><td></td></tr><tr><td>Ultramicrotome</td><td>LEICA</td><td>EMUC7</td><td></td></tr><tr><td>Uranyl Acetate</td><td>RHAWN</td><td>R032929</td><td></td></tr></tbody></table>

a three-dimensional technique for the visualization of mitochondrial ultrastructural changes in pancreatic cancer cells

了解细胞器超微结构的动态特征，不仅具有丰富的未知信息，而且从三维（3D）角度来看也很复杂，对于机制研究至关重要。电子显微镜（EM）具有良好的成像深度，并允许重建高分辨率图像堆栈，以研究细胞器的超微结构形态，即使在纳米尺度上也是如此;因此，3D重建因其无与伦比的优势而变得越来越重要。扫描电子显微镜（SEM）提供了一种高通量图像采集技术，允许在连续切片中从相同的感兴趣区域重建3D大型结构。因此，SEM在大规模三维重建中的应用，以恢复细胞器的真实三维超微结构变得越来越普遍。在该协议中，我们建议结合连续超薄切片和3D重建技术来研究胰腺癌细胞中的线粒体嵴。该协议以逐步的方式描述了如何执行这些技术的详细信息，包括锇 - 硫代碳酰肼 - 锇（OTO）方法，连续超薄切片成像和可视化显示。

在细胞培养过程中（图1A），我们首先将胰腺癌细胞分为用完整培养基培养的对照组，（1S，3R）-RSL348（RSL3，铁死亡激活剂，100nM）组和RSL3（100nM）加铁抑素-149（Fer-1，铁死亡抑制剂，100nM）组。通过上述实验步骤，扫描电镜分别获取对照组、RSL3组和抑制剂组（RSL3+Fer-1组）的38张（补充图1）、43张（补充图2）和44?...

Watch this Scientific Journal Video about A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells at JoVE.com

作者聚焦:胰腺癌细胞线粒体超微结构变化可视化的三维技术  

该协议描述了如何重建线粒体嵴以实现高精度，高分辨率和高通量的3D成像。

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

本项目旨在通过在亚细胞纳米尺度上改变结构，建立细胞器形态与癌症病理机制之间的联系，来评价药物的抗癌作用。该技术将传统的电子显微镜技术与三维重建技术相结合，无需专用设备。与其他技术相比，它可以提高成像效率和重建灵活性，并随时对目标结构进行现实生活中的重建。因此，它非常简单且通用。这种连续超薄载玻片的三维重建技术可以反复还原真实的立体化学，而无需特定的昂贵设备。它可以随时用于所有细胞器的定性和定量分析。实现了观察的简便操作。

a-three-dimensional-technique-for-visualization-mitochondrial

Research

JoVE Journal

Cancer Research

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

1.6K 次观看.  School of Pharmaceutical Science of Zhejiang Chinese Medical University. 本项目旨在通过在亚细胞纳米尺度上改变结构，建立细胞器形态与癌症病理机制之间的联系，来评价药物的抗癌作用。该技术将传统的电子显微镜技术与三维重建技术相结合，无需专用设备。与其他技术相比，它可以提高成像效率和重建灵活性，并随时对目标结构进行现实生活中的重建。因此，它非常简单且通用。这种连续超薄载玻片的三维重建技术可以反复还原真实?...

视频: 作者聚焦:胰腺癌细胞线粒体超微结构变化可视化的三维技术  

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

科研

癌症研究

使用植入式稳定窗的小鼠胰腺成像

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

作者聚焦:通过先进的活体成像彻底改变胰腺疾病的理解 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

用于小鼠胰腺活体成像的稳定窗口

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

生物工程

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

神经科学

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

生物学

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

医学

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

副本

摘要

探索更多视频

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

人类胰岛神经网络的高分辨率3D 成像

人 Intraepidermal 神经纤维内线粒体三维成像及分析

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

器官型切片培养作为原发性胰腺癌和转移瘤肿瘤微环境的临床前模型

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

副本

摘要

探索更多视频

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

人类胰岛神经网络的高分辨率3D 成像

人 Intraepidermal 神经纤维内线粒体三维成像及分析

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

器官型切片培养作为原发性胰腺癌和转移瘤肿瘤微环境的临床前模型

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口