这项工作得到了赠款 NFSC 81170806 的部分支持。作者感谢武汉大学口腔医学院的微 CT 核心设施, 帮助对大鼠股骨进行扫描和分析。

涉及动物主题的程序是按照《动物保育和使用指南》 (NIH 出版, 第八版, 2011) 进行的, 并已由武汉机构动物保育和使用委员会审查和批准。大学.
1. 软件安装
<ol><li>安装 ImageJ 软件。从 https://imagej.nih.gov/ij/下载与64位 Java 捆绑在一起的 ImageJ (版本 1.51p) 软件的 Windows 版本。将下载的软件解压到一个文件夹中, 随后将其称为 "ImageJ 目录"。 注: 小梁分析插件需要64位 java 运行时间 (版本 1.8) 和64位 windows 操作系统, 最好是64位 windows 7 操作系统。</li>
	<li>安装小梁分析插件。请求小梁分析插件从 http://www.bomomics.com, 并解压的内容到 ImageJ 的插件目录, 这是 "ImageJ 目录/插件"。 注: 该插件可以作为一个自由版本获得, 其中5指定相邻切片的测量报告, 或作为商业版本, 其中的切片范围可以指定和测量。</li>
</ol>2. 为小梁分析准备3D 数据集
<ol><li>在标准扫描协议5之后, 用微型 CT 扫描大鼠股骨, 然后以可导入 ImageJ 的格式 (例如) tiff 格式保存数据。如果在单个样本管中同时扫描多个骨样本, 首先将数据导入 ImageJ, 然后使用 ImageJ 的图像处理工具裁剪其他样本, 从而分离出每个骨骼。随后, 将生成的图像保存为可在以后导入 ImageJ 的格式。 注意: 分析中使用的代表性示例图像文件 (辅助文件 1)。</li>
	<li>
模拟标准2D 和3D 对象。
	<ol>
<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击圆圈按钮, 然后在弹出窗口中输入200作为直径, 然后单击OK以生成直径为200像素的模拟圆 (图 3B)。以 tiff 格式保存生成的圆。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击正方形按钮, 然后在弹出窗口中输入200作为侧面长度, 然后单击OK以生成具有200像素的边长 (图 3B) 的模拟正方形。以 tiff 格式保存生成的正方形。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击矩形按钮, 然后在弹出窗口中输入200作为宽度和100作为高度, 然后单击确定生成具有200像素宽度和100像素高度的模拟矩形 (图3 B)。以 tiff 格式保存生成的矩形。 注: 一个圆 (直径: 200 像素), 正方形 (边长: 200 像素) 和一个矩形 (宽度: 200 像素; 高度: 100 像素) 为后续分析保存。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击多维数据集按钮, 然后在弹出窗口中输入30作为侧面长度, 然后单击确定生成模拟多维数据集。最后, 单击插件 | 3D |卷查看器查看生成的多维数据集并以 tiff 格式保存它 (图 3C)。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击长方体按钮, 然后输入80作为长度, 40 作为宽度, 30 作为高度在弹出窗口中, 然后单击确定生成模拟长方体。最后, 单击插件 | 3D |卷查看器查看生成的长方体并以 tiff 格式保存它 (图 3C)。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击球面按钮, 然后在弹出窗口中输入30作为直径, 然后单击确定生成模拟球体。最后, 单击插件 | 3D |卷查看器查看生成的球体并以 tiff 格式保存它 (图 3C)。</li>
		<li>打开 ImageJ 软件。在插件 |BoMomics |模拟对象菜单, 单击钢瓶按钮, 然后在弹出窗口中输入30作为直径和100作为高度, 然后单击确定生成模拟气缸。最后, 单击插件 | 3D |卷查看器查看生成的气缸并以 tiff 格式保存它 (图 3C)。 注: 球体 (直径:30 像素), 立方体 (边长:30 像素), 长方体 (长度:80 像素; 宽度:40 像素; 高度:30 像素) 和圆筒 (直径:30 像素; 高度: 100 像素) 为后续分析保存。</li>
	</ol>
</li>
</ol>3. 性能分析参数
<ol><li>打开 ImageJ 软件, 打开或导入扫描的图像。</li>
	<li>滑动底部滚动条以选择一个切片, 然后单击图像 |调整 |阈值按钮。在 "弹出式阈值" 窗口中, 通过手动检查调整最小和最大阈值, 以确保骨骼与背景分离良好, 并将最小阈值记录为皮质骨阈值。 注: 由于不同的微 CT 制造商使用不同的比例因子来存储图像中的 X 射线衰减系数, 所以实际的阈值应该通过手动检查或按照特定制造商的建议.在我们的实践中, 设置最小阈值的 ISQ 文件生产的微型 CT 机到 6,000-7, 000 可以可靠地分离骨骼的扫描背景。</li>
	<li>单击插件 |BoMomics |Trab 参数分析按钮。在弹出窗口中, 将切片索引设置为代表切片的位置, 并设置皮质骨 ("Cort 骨")、范围 ("范围") 和步骤 ("步长") 值, 用于计算用于分析分割参数的一组皮质门限, 其中皮质骨阈值由步骤 3.2 (图 1) 获取。默认范围和步骤值为2000和400为大多数微 CT 图像工作。将切片索引设置为示例数据集的 5, 并保留其他设置的默认值。 注: "Cort 骨" 是皮质骨的门槛, 用于参数分析的阈值将从最低阈值更改为最高阈值, 并增加步长值, 其中最低阈值是 Cort 的值。骨范围和最高阈值是 Cort 骨 + 范围。如果 Cort 骨骼、范围和步骤的 set 值分别为6000、1000和 500, 则皮质骨骼的最低阈值为6,000-1、000 = 5000, 最高阈值为 6000 + 1000 = 7000, 分析中使用的阈限为 5000,5500、6000、6500和7000。</li>
	<li>设置噪音直径 ("噪音直径"), 步骤 ("步骤"), 和范围 ("范围") 值, 用于指定分析中的一组噪声值, 以及用于计算一组孔值的孔直径 ("孔直径")、步骤 ("步长") 和范围 ("范围") 值。通常, 默认设置适用于大多数微 CT 骨样, 其中噪声、步骤和范围分别为5、5、2、孔径、步长和范围分别为15、5、2。保留示例数据集的默认设置。 注: "噪音直径" 是噪音抑制过滤器的直径, 而 "孔直径" 是皮质骨内孔的直径。用于参数分析的噪声和孔值可以类似于上面描述的皮质阈值, 使用指定的噪声/孔、范围和步长值。由于在采集图像时扫描参数的变化, 噪声和孔参数应根据图像质量进行实证确定, 因为没有一般的噪声范围和孔参数对所有扫描都有好处。阈值、噪音或孔的最低数值必须大于或等于 0, 并且如果使用提供的参数计算出的最低值为负值, 则将特定的最低值设置为0。</li>
	<li>单击确定以执行参数分析。在 "参数分析结果" 窗口中直观地检查分割结果, 然后选择切片图层, 以便准确地勾勒出骨骼外部边界 (图 1B)。随后, 从参数分析结果表中与所选切片层对应的项中检索分析参数 (表 1)。</li>
</ol>4. 小梁分析
<ol><li>
小梁骨的分割
	<ol>
<li>打开 ImageJ 软件, 然后打开或导入扫描的图像。</li>
		<li>单击插件 |BoMomics |Trab 细分按钮, 并填写适当的分析参数。设置 "开始", "轮廓边界", "Trab"。骨骼 "," 降噪直径 "," 孔填充直径 ", 和" 皮质厚度直径 "分别为5、7200、7000、6、12和25。 注: "开始" 和 "结束" 指定选定切片范围的小梁骨分割, "轮廓边界" 是对应的异形 "Cort 骨" 参数, "降噪直径" 到 "噪音直径"。参数, 并 "孔填充直径" 到 "孔直径" 参数。"皮质厚度直径" 是排除外皮质骨的指定厚度。trab.骨骼 "是提取小梁骨的门槛 (图 2), 其中设置是使用分析分析参数命令确定的, 如步骤3.5 中所述。</li>
		<li>单击确定执行小梁分割。在Trab 细分结果窗口 (图 2B) 中直观地检查分割结果。以 tiff 格式 (图 2B) 中的分段小梁骨窗口保存所提取的小梁骨, 可由其他软件进一步分析。</li>
	</ol>
</li>
	<li>
小梁骨分析。
	<ol>
<li>打开 ImageJ 软件, 然后打开或导入扫描的图像。</li>
		<li>单击插件 |BoMomics |Trab 分析按钮, 并填写适当的分析参数, 如 "开始"、"轮廓边界"、"Trab"。骨骼 "," 降噪直径 "," 孔填充直径 ", 和" 皮质厚度直径 "如上所述 (图 3A), 其中设置是使用" 分析分析参数 "命令确定的, 如步骤3.5 中所述。设置 "开始", "轮廓边界", "Trab"。骨骼 "," 降噪直径 "," 孔填充直径 ", 和" 皮质厚度直径 "分别为5、7200、7000、6、12和25。 注意: 在插件的自由版本中, 五个相邻切片从指定的 "开始" 切片索引开始选择用于度量, 而在商业版本中, 用户可以指定任意数量的切片。</li>
		<li>在 "结果报告" 部分中, 为要测量的参数选择一个或多个选项, 其中所选区域的小梁骨量 (BV)、总容积 (TV) 和厚度均测量为二维 (2D) 或三维(3D) 可供选择, 通过三个复选框, 即 "BV 电视", "2D", 和 "3D"。选择复选框 "2D" 和 "3D", 然后单击 "确定" 执行小梁分析 (图 3A,表 2)。 注: 当检查 "bv 电视" 时, 无论 "2D" 和 "3D" 的选择状态如何, 都报告了 bv、电视和强度的原始测量方法, 并将分割后的小梁骨提取并显示在一个新窗口中, 可以保存并进一步分析其他软件.在检查 "2D" 时, 报告了在每个切片级别上用平板模型测量的 BV、电视、强度和厚度的原始测量值。如果检查 "3D", 则在没有任何模型假设的情况下直接计算每个体素的三维厚度, 然后报告每个切片级别取样的 BV、电视、强度和三维厚度的原始度量值。但是, 如果未选择复选框, 则使用上述参数集对小梁骨进行分段, 但不报告任何测量。</li>
	</ol>
</li>
</ol>5. 量化模拟物体
<ol><li>打开 ImageJ 软件, 然后打开一个模拟图像。在这里, 打开模拟球体的直径为30像素的例子。</li>
	<li>选择插件 |BoMomics |Trab 分析按钮并填写相应的分析参数, 如前所述。保留默认值为 "开始"、"结束"、"轮廓边界"、"Trab"。骨骼 ", 并设置" 降噪直径 "," 降噪直径 "," 孔填充直径 ", 和" 皮质厚度直径 "到 0 (图 3)。 注意:对于模拟对象, 没有意外的信号噪声, 也可能没有相应的皮层壳。因此, 应适当地设置这些值的参数 (默认设置为零)。对于 3D quantifications, 在指定的 "开始" 切片之前和之后使用插件的自由版本处理30切片, 而只有由 "开始" 和 "结束" 切片指定的区域中的切片才能通过插件的商业版本进行分析。</li>
	<li>在结果报告部分中, 为要测量的参数选择 "2D" 和 "3D", 然后单击确定对模拟对象执行小梁分析 (表 3)。</li>
</ol>6. 小梁测量和数据表示的校准: 剖面分析区域中小梁的分布
<ol><li>根据微 CT 供应商的说明, 从扫描数据集中获取校准信息。 注意: 只有原始测量的 BV, 电视, 强度和厚度是由插件报告。为了获得与其他软件生成的报告类似的度量值, 需要对结果进行校准。</li>
	<li>打开 "Microsoft Excel", 然后打开 "报告的结果" 表。根据以下表达式计算新 excel 列中校准的骨量 (bv)、总容积 (TV)、骨矿物质含量 (BMC)、骨量分数 (bv/TV) 和骨密度 (BMD)。</li>
	<li>注: 扫描分辨率 (分辨率、µm)、灰度缩放 (缩放)、密度单位 (毫克 HA/cm3)、密度斜率 (斜率) 和密度截距 (拦截) 可以从扫描的微 CT 图像或元文件 (如 Scanco ISQ 文件) 中提取。因此, 校准的措施计算如下: 假设 bv、电视和强度都是原始测量, BVc和电视c是校准值、分辨率、缩放、斜率、截距均来自于微 CT 图像的校准文件。 bvc = BV x 分辨率3 [微米3] 电视c = 电视 x 分辨率3 [微米3] bv/电视 = bv ÷电视 BMC = (强度÷缩放 x 斜率-BV x 拦截) x 分辨率3 x 10-12 [毫克 HA] 骨密度-BMC ÷电视c x 1012 [毫克 HA/cm3]</li>
	<li>使用 Microsoft Excel 软件 (图 4) 创建针对切片层 (X) 的校准度量值 (Y) 的 XY (散点) 图。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Miner Res. 25 (7), 1468-1486 (2010).</li></ol>

提出了一种用于量化物体厚度的底层算法的专利。作者与其他人合作, 注册了 bomomics.com 网站, 托管免费版本插件, 在这里提供咨询和量化图像分析服务的要求。

本研究介绍了一种用于分析小梁骨的 ImageJ 插件, 它具有自动、高效和人性化的功效。该插件还可用于量化任何2D 或3D 对象, 用于逐层度量区域、卷和厚度。目前, 通过标准的显微 CT 分析, 每个样品的每一个小梁参数只有一个测量值, 不能完全代表所选分析区域中测量实体的特征。所述插件报告每个样本的每一个参数的逐层数量, 在选定的分析区域充分保留测量参数的分布信息, 因此更先进和敏感的?...

小梁骨显微 CT 分析是跟踪不同骨丢失情况下动物骨骼形态学变化的标准方法1,2,3, 其中几个变量与报告了骨骼的结构4。但是, 此类参数在长骨骼的骺中不均匀分布5, 并且仅报告每个样本的每个结构变量的汇总值或平均值 (由当前商用微 CT 机6,7, 但单个值不能完全表示分析区域中测量参数的特征。一层一层定量的小梁骨不仅保留了对每个变量的更多信息, 而且还能够在分析区域中对此类变量的分布进行分析, 便于随后对结构进行统计分析。在不同条件下的更改5。因此, 该方法的目的是量化微梁骨的显微 ct 扫描在每个切片水平, 这是没有在任何商业可用的微 ct 分析包目前。
为了有效地分割小梁骨切片, 需要自动分割方法。然而, 目前的显微 CT 分析标准技术是基于人工交互式轮廓, 然后是半自动插补, 将小梁骨从皮质隔间分离出来, 这是劳动密集型、易出错和与大量运算符偏差关联8,9,10。自动分割方法11,12报告, 但这种方法只有在良好的分离小梁骨和皮质骨的区域, 但不是在没有明确的分离区域。另外, 不同的样本12需要不同的分割参数, 通过尝试各种参数组合12, 手动选择适用于骨骼样本组的满意的分割参数是很繁琐的,即使在设置所有相关参数时, 分割过程也是自动的。由于骨外边界与扫描背景有最大的对比, 长骨骼的骺皮质壳在所选择的分析区域中表现出很少的变化, 根据长骨骼外边界轮廓分割方法可以可靠准确地将小梁骨从皮质壳中分离出来。这种分割方法的优点是, 分割是基于背景和骨骼的外边界之间的差异, 而不是小梁和皮质骨骼之间的差异6,12, 13, 因此通常很容易找到一个组合的分割参数, 这是令人满意的一组骨样本, 有助于更可靠地分析不同组之间的小梁变化。
在每个切片级别上, 报告了2D 分析的面积、周长和二维 (2D) 厚度, 而在 3D quantifications 中报告了体积、表面和三维 (3D) 厚度。目前的图像分析工具通常不报告此类信息, 表明所报告的程序可应用于需要此类信息的一般图像。

<table border="0" cellpadding="0" cellspacing="0" style="width:595px;" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td style="width:93px;">NIH</td>
			<td style="width:119px;">imagej</td>
			<td style="width:167px;">Any version with a java 1.8 run time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trabecular analysis plugin</td>
			<td style="width:93px;">Bomomics</td>
			<td style="width:119px;">bomomics</td>
			<td>free or commercial version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro CT scanner</td>
			<td style="width:93px;">Scanco</td>
			<td style="width:119px;">&#956;-50</td>
			<td>micro CT from any vendor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Computer System</td>
			<td style="width:93px;">Lenovo</td>
			<td style="width:119px;"></td>
			<td>any brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Windows Operating System</td>
			<td style="width:93px;">Microsoft</td>
			<td style="width:119px;">Windows 7 x64</td>
			<td>any 64-bit Windows operating system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Office Software</td>
			<td style="width:93px;">Microsoft</td>
			<td style="width:119px;">Office 2010</td>
			<td>any speadsheet software that has xy chart function</td>
		</tr>
	</tbody>
</table>

outer-boundary assisted segmentation and quantification of trabecular bones by an imagej plugin

微计算机断层扫描 (显微 CT) 通常用于评估不同骨质流失情况下小动物骨量和微梁的显微结构特征。然而, 显微 CT 图像小梁分析的标准方法是切片半自动手工轮廓, 这是劳动密集型和易出错的。本文描述的是根据骨的外边界自动分割小梁骨的有效方法, 在适当的情况下, 在合适的情况下, 可自动识别和分割短梁骨。设置了分割参数。为了分析满意的分割参数, 给出了分割结果的图像栈, 将分割参数的所有可能组合按顺序变化, 并通过相关参数的分割结果可以易于目视检查。作为插件的质量控制特性, 模拟标准对象被量化, 在测量量可以与理论值进行比较。该插件报告了小梁性质和小梁厚度的逐层量化, 并能很容易地对所选区域内的这种性质分布进行分析。虽然逐层量化保留了关于小梁骨的更多信息, 并有助于进一步统计分析结构变化, 但此类措施无法从当前商业软件的输出中获得, 只有单一每个示例都报告每个参数的量化值。因此, 所描述的工作流是比较准确、高效地分析小梁骨的较好方法。

小梁分析插件旨在自动分割和量化小梁骨的准确性。最初, 骨外边界被发现和划定后, 一个孔充填手术, 其中任何孔内的骨外皮质壳填补。然后进行侵蚀手术, 排除外皮质骨, 并获得分割小梁骨。最后, 对分段区小梁骨的测量方法进行量化。
由于微 CT 图像固有的噪音, 分割使用预定义的任意参数往往无法准确识别骨骼的外部边界?...

Watch this Scientific Journal Video about Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin at JoVE.com

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

我们提出了一个工作流, 以分割和量化的小梁骨2D 和3D 图像基于骨骼的外部边界使用一个 ImageJ 插件。这种方法比当前手工轮廓方法更有效和准确, 并提供了一层层的 quantifications, 在当前的商业软件中是不可用的。

用 Imagej 插件对小梁骨的外边界辅助分割和量化

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

outer-boundary-assisted-segmentation-quantification-trabecular-bones

Research

JoVE Journal

Bioengineering

9.2K 次观看.  Wuhan University. 我们提出了一个工作流, 以分割和量化的小梁骨2D 和3D 图像基于骨骼的外部边界使用一个 ImageJ 插件。这种方法比当前手工轮廓方法更有效和准确, 并提供了一层层的 quantifications, 在当前的商业软件中是不可用的。

视频: Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

科研

生物工程

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

使用噬菌体显示肽库筛选和识别针对纤维细胞生长因子受体2的小肽

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

机械测试的注入性量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

通过心血管磁共振成像定量小鼠心脏左心室功能、心肌劳损和血流动力学力

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...