作者承认来自澳洲的资金支持 (奖学金1084178和赠款 1087850, 1030469, 1075898 (AMS)), 癌症澳大利亚, Ludwig 癌症研究, 约翰·里德信托, 治疗脑癌基金会, La 筹伯大学和维多利亚癌症机构。澳大利亚维多利亚政府提供的运营基础设施支持计划的资金也得到了承认。

1. 贴壁细胞株在成像实验中的播种
<ol><li>种子 1万 A431 表皮细胞癌细胞每井 (O/N) 在300µL Dulbecco 的改良鹰培养基/营养混合物 F-12 (DMEM/F-12) 培养基中含有10% 胎牛血清, 1% 青霉素-链霉素和 1% GlutaMAX 在8腔内成像适用于高分辨率油浸泡物镜的滑动 (例如, NuncTM 实验室 TekTM 腔 coverglass)。</li>
	<li>在室温下 (RT), 在培养基中添加100毫升的 EGF 配体, 刺激受体在 A431 细胞中的聚集, 10 分钟。</li>
</ol>2. 原发性抗体孵化
<ol><li>用300µL 室温 (RT) 磷酸盐缓冲盐水 (1x PBS) 两次去除介质并冲洗细胞。</li>
	<li>修复细胞与200µL 4% 多聚甲醛在 PBS 为10分钟的 RT。 注意: 为了探索目标聚集的时间变化, 必须省略此固定步骤。</li>
	<li>用300µL 室温 PBS 两次去除粉煤灰和洗涤细胞。</li>
	<li>孵化细胞与200µL 的 PBS 含有主要抗体在10µg/毫升浓度至少2小时4摄氏度。 注意: 为使 ICS 计算有效, 所有在细胞表面存在的受体必须用原抗体标记。这需要初步实验的每一个主要抗体稀释系列 (1:10, 1:50, 1:100, 1:200, 1:500 1:1, 000,等) 将执行。通过荧光强度与簇密度的分析, 确定了最佳主抗体浓度。选定的主要浓度发生时, 簇密度高原随着主要抗体浓度的增加之前的簇密度进一步增加, 由于非特定的抗体结合。在进行时间-过程成像实验时, 血清游离培养基中的原发抗体可以稀释。</li>
	<li>完成所需的潜伏期后, 冲洗细胞与300µL 的 RT PBS 两次。</li>
	<li>在 PBS 中用5% 牛血清白蛋白 (BSA) 阻断样品, 至少在4摄氏度时为2小时。 注意: 固定的样品在一夜之间经常被阻拦 (O/N) 在4°c。</li>
</ol>3. 次生抗体孵化
<ol><li>在 PBS 中准备10µg/毫升荧光标记的二级抗体 (如Alexa 氟 488 IgG)。2µg/毫升的赫斯特33342可能被包括在这个稀释, 以允许改进的样品发现在显微镜上。 注意: 对于时间推移实验, PBS 可以交换为血清自由介质。</li>
	<li>从细胞中取出 5% BSA/PBS, 用二次抗体溶液孵育2小时4摄氏度, 同时保护样品不受环境光线的侵害。</li>
	<li>在二次抗体潜伏期完成后, 用300µL 的 PBS (两次) 冲洗细胞, 并贮存在4摄氏度, 保护环境光线。</li>
	<li>为了防止蒸发, 用 parafilm 将燃烧室的边缘包裹起来。</li>
</ol>4. 样品的共焦显微术
注意: 用于共焦分析的设置将因个别实验中使用的设备和显影而异。现代共焦仪器提供的机制, 以保存在实验中使用的所有成像参数的本机图像文件格式的仪器。必须记录这些设置, 以便在不同的数据收集期间适当地复制实验条件。
<ol><li>在荧光信号的成像过程中, 避免过度饱和。使用图像查找表, 提供像素饱和的假颜色表示, 并减少探测器增益和激光功率相应。在采集软件中, 大多数共焦仪器包括 "范围指示器" 设置或类似选项。</li>
	<li>尽量减少对激光功率荧光的曝光。使用 DNA 标记染色, 如赫斯特33342提供了一个简单的机制, 以检测和定位样品在视野中收集。</li>
	<li>以低分辨率执行示例的初始扫描, 如果软件允许快速扫描速度。这将限制示例的可能漂白。</li>
	<li>在准备收集图像数据时, 增加捕获分辨率并减慢扫描速度。</li>
	<li>如果共焦仪器允许, 使用光子计数模式收集数据。 注: 如果仪器上没有光子计数, 请确保图像采集过程中的灰度是线性的。</li>
	<li>设置每个激光线的针孔, 用于1通风单元 (AU)。</li>
	<li>在图像采集 (x4) 中使用直线或帧平均来减少检测器相关的噪声。 注意: 在这个变型的 ICS 中, 只有一个时间点被捕获的采集时间不是实验限制因素。</li>
	<li>为图像收集选择高放大率, 高数值孔径目标, 如计划复消色差目标, 提供最大分辨率和校正的球面和色差 (例如计划-复消色差100X 或 63X/1.40石油目标)。</li>
	<li>细分采样图像中包含的捕获像素的大小。建议每个像素捕获一个 50 x 50 nm 区域。对于有效的 ICS, 像素大小必须小于 0.1 x 0.1 µm 每像素2 。这是通过放大扫描一个相对较小的区域并选择一个相对较大的图像格式 (例如1024 x 1024 或 2048 x 2048 像素) 来实现的。</li>
	<li>把注意力集中在细胞的顶端或基底表面, 尽可能平坦的区域。在 ICS 分析过程中单独处理的一个图像和感兴趣的区域可以收集多个单元格。</li>
	<li>使用相同的仪器设置来捕获一个无单元格背景区域, 用于示例规范化。</li>
</ol>5. 图像相关谱的计算
注: 在 ImageJ 程序的分发中, 斐济是开源成像处理平台, 需要执行对 ICS 至关重要的自相关和数学功能。ImageJ 的这种分布是推荐的, 因为它包括许多预安装的插件和一个可以跨多个成像实验执行操作的更简单的更新体系结构。
<ol><li>安装斐济程序 (http://fiji.sc/Downloads)。</li>
	<li>加载获取的数据集。</li>
	<li>识别感兴趣的顶端或基底细胞表面膜区, 并复制到像素大小为 2n (256 x 256, 128 x 128, 64 x 64)。"菜单: 图像 > 复制..."</li>
	<li>计算所裁剪区域的平均强度 "菜单: 分析 > 测量"。</li>
	<li>识别背景区域并复制到捕获细胞表面膜的相同像素大小 (2n: 256x256、128x128、64x64)。"菜单: 图像 > 复制..."</li>
	<li>计算感兴趣的背景裁剪区域的平均强度。"菜单: 分析 > 测量。</li>
	<li>从包含感兴趣区域的图像中减去背景的平均强度。"过程 > 图像计算器 > 减法"。</li>
	<li>
执行自相关计算。此计算包含在菜单结构中: "过程 > FTT > FD 数学"。
	<ol>
<li>在 FD 数学对话框中选择: 图像 1: 裁剪图像名称, 操作为相关, 图像 2: 裁剪图像名称, 反向转换。</li>
	</ol>
</li>
	<li>通过除以总像素数来规范化生成的图像。"菜单: 过程 > 数学 > 鸿沟..."(即: 64x64 像素 = 4096)</li>
	<li>通过按规范化裁剪区域平方的平均强度除以, 重新正常化。"菜单: 过程 > 数学 > 鸿沟..." 注意: 这可以通过将图像除以平均强度值两次来实现。</li>
	<li>通过点传播函数绘制一条直线。</li>
	<li>绘制此行的剖面线以计算峰值值。"菜单: 分析 > 剧情简介"。</li>
	<li>通过将峰值值 (结果 5.12) 传输到电子表格并执行以下计算 (Eq 6), 计算每个波束区域的簇数: <img alt="Equation 7" src="/files/ftp_upload/57164/57164eq7v2.jpg">
</li>
</ol>6. 集成电路数据集的批处理
注: 建议建立斐济/ImageJ 宏, 以复制上述协议中描述的程序。这使得在图像相关谱分析中对多个图像文件进行一致和快速的分析。
<ol><li>通过记录 ics 协议中的每个菜单命令建立宏 ics 工作流 "菜单: 插件 > 宏 > 记录..."。</li>
	<li>选择 "创建" 以生成宏。</li>
	<li>选择 "语言 > IJ1 宏"。</li>
	<li>保存宏。</li>
	<li>
将对话框添加到宏的各个步骤中, 为每个函数的运算符提供提醒。对话框还用作暂停分析过程的方法, 以允许在进一步处理之前 (即区域裁剪) 之前进行适当的选择。
	<ol>
<li>对话框宏代码 标题 = "对话框标题"; 味精 = "对话框/协议步骤消息"; waitForUser (标题, 味精); 注意: 可以通过订阅斐济更新程序 (指令 https://imagej.net/BAR) 中的 "条码" 站点来建立额外的效率。这为斐济提供了 "一组广泛适用的例程" 14。一个这样的惯例就是找到顶峰。菜单结构: "条形 > 数据分析 > 查找峰值"。此脚本提供了一种快速计算点扩展函数配置文件中峰值值的方法。</li>
	</ol>
</li>
</ol>7. 协议扩展: 显微镜光束面积的计算
注意: 显微镜的光学传递功能确保即使是分子尺寸的物体在 x-y 平面中的半径约为 200-300 nm 的图像也会出现。ICS 协议允许在亚分辨率荧光珠上执行步骤4-5 来确定点传播函数。特别:
<ol><li>安装亚分辨率 (即< 100 nm 直径) 荧光珠到8室成像幻灯片。</li>
	<li>图像珠, 根据描述的协议, 使用共聚焦显微镜, 用于集成电路实验。</li>
	<li>根据协议步骤 5.1-5.13, 从生成的图像中计算 ICS 函数。</li>
	<li>从绘制的剖面, 计算光束半径 (r) 为全宽的最大值的 ICS 函数的一半。</li>
	<li>计算光束面积 (BA) 为 (Eq 7):<img alt="Equation 8" src="/files/ftp_upload/57164/57164eq8v2.jpg">
</li>
</ol>8. 协议扩展: 计算每单位面积的簇数
<ol><li>计算每个区域单位的簇密度 (CD), 如 (Eq 8): <img alt="Equation 9" src="/files/ftp_upload/57164/57164eq9v2.jpg"> <img alt="Equation 10" src="/files/ftp_upload/57164/57164eq10v2.jpg">
</li>
	<li>除以光束区域的簇 (结果为 5.13) 由梁的面积 (结果为 7.5)。</li>
</ol>

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安德鲁 M 斯科特有 AbbVie 药剂, EMD 雪兰诺和第一惠州市三协 Co 的研究资金支持, 并有顾问和股票所有权的生命科学药物。作者没有其他相关的关联或财务参与任何组织或实体的财务利益或财务冲突的主题或材料讨论的手稿, 除了那些披露。

我们在本协议中描述的图像相关光谱学 (ICS) 技术使用标准共焦显微镜, 无需专门的探测器。ICS 技术描述利用已建立好的免疫细胞化学方法, 提供快速抽样的多种治疗条件, 以增加统计分析。这种方法这样做的绝对精度略有降低, 与替代单分子技术相比, 基于荧光波动的移动分子扩散通过共焦体积, 如荧光相关光谱学 (FCS)15。更专业的 FCS 方法还需要高灵敏度的光子计数探测器, 如雪崩光电二极管 (APD) 或 GaAsP 探测器, 以及专门的专门软件, 而不是常规地在标准共焦仪器上提供。通过对 ICS 精度的综合分析, 确定了该技术能够准确测量激光扫描显微镜7中的光束焦斑中存在 > 1 粒子的簇数。
收集适当的荧光图像对于成功的 ICS 分析至关重要。感兴趣的区域必须包含一个高信号到背景荧光比率与激光和增益强度调整以去除任何信号饱和度。捕获的蜂窝区域必须过度取样, 像素大小约为 50 nm。这个区域必须是均匀的, 小心注意避免细胞边缘或连接点, 这可以增加工件在自相关计算。ICS 方法的一个主要缺陷是对异构蜂窝表面的敏感度。因此, 限制捕获到最大平坦表面的区域, 无论单元格形状如何, 都会提高任何 ICS 分析的准确性。
本文探讨了多种影像学方法, 研究受体配合物的聚集。电子显微镜 (EM) 提供高分辨率的蛋白质聚类空间评估, 但在极高的真空下运行, 不适用于需要时间分辨率的研究。EM 是进一步有限的, 因为它苛刻的样品准备是禁止的活组织调查5。为了克服标准共焦显微镜固有的分辨率限制, Förster 共振能量传递 (烦恼) 可以用来计算标记为重叠的供体和受体显影对的结构的空间组织16.尽管它的敏感性, 然而忧虑不提供关于调查6的合计的大小的信息。各种超分辨率荧光显微技术的发展提供了令人振奋的机会, 以解决细胞对象不可能与标准分辨率共焦方法17,18。这些技术和工具所需的费用和专门知识限制了它们目前的普遍可用性。
随着共焦检测灵敏度的增加和光毒性的降低, 图像相关谱可以扩展, 以探索活细胞中受体聚集在时间推移实验中的时间变化。这可以与增加激光功率, 用于故意诱导个别样品的照片漂白与随后的 ICS 评估 (pbICS), 以取笑分离的高阶聚集目前在一些受体配合物9。进一步的适应, 图像交叉相关光谱学 (ICCS) 提供了分析的两个膜基蛋白的共同定位通过分析图像对每荧光标记蛋白19。这种图像相关光谱学方法的优点是, 它利用了在现场建立的技术, 并提供了一个自由可用的分析管道定量的高动态事件发生在细胞膜上。

治疗性抗体的发展在治疗多种肿瘤类型1中表现出显著的成功。最近的进展, 抗体-药物共轭 (ADC) 作为细胞毒性化合物的传递机制, 扩大了了解抗体动态的要求: 细胞表面的受体相互作用2。在成功地靶向细胞表面受体的抗体后, 这些配合物可以诱导相似的聚集模式对在配体中观察到的那些: 抗体相互作用3。受体聚集的改变会诱发细胞膜的改变, 并导致受体的内化和细胞表面的去除。在抗体-药物共轭的背景下, 这一过程随后将细胞毒性有效载荷释放到内内涵体内, 随后形成细胞质, 从而有效地杀死细胞。
共焦显微镜提供了一种有效的方法来可视化这些重要的相互作用的抗体和他们的目标受体4。探讨靶分子在细胞表面聚集的变化本协议利用空间图像相关光谱学 (ICS) 技术5、6、7 对共焦显微图像进行后处理..
图像相关光谱学的基础是观察到空间荧光强度波动与标记结构的密度和聚集状态的关系。这种关系是在计算被捕获的图像5的空间自相关函数之后建立的。
图像相关光谱学的所有变体都需要对图像自相关进行计算。接着将此函数拟合为二维高斯曲线, 用于提取图像中包含的定量聚合状态参数。简单地说, 图像自相关的计算涉及将图像中包含的所有可能的像素对进行比较, 并计算两者之间相等的亮度。这是可视化为一个函数的距离和方向的像素分离8。
建立了图像相关谱的理论框架, 并由彼得森和智者等来定义。5,6. 在本议定书中, 在斐济/ImageJ 和电子表格应用程序中执行自相关计算, 可将强度波动空间自相关函数的基础描述为(Eq 1):
<img alt="Equation 1" src="/files/ftp_upload/57164/57164eq1v2.jpg">     
其中F代表傅立叶变换;F−1 逆傅里叶变换;F *其复杂共轭;和η的空间滞后变量。在空间集成电路中, 如本协议所述, 可使用2D 快速傅立叶变换算法7、9计算自相关函数。在零空间分离的自相关, 即零滞后, g11(00), 提供了显微镜下每束区域呈现的粒子的逆平均数。它可以通过将空间自相关函数拟合为二维高斯函数(Eq 2)来获得:
<img alt="Equation 3" src="/files/ftp_upload/57164/57164eq3v2.jpg">     
由于图像中捕获的像素包含在集合区域内, 而这些度量值不扩展到无穷大, 因此术语 g∞用作偏移量, 用于计算图像中包含的远距离空间相关性。对于分子大小的聚集体, ω是显微镜的点扩散函数, 由全宽半最大空间自相关函数描述。该仪器的点传播功能范围内, 可通过使用亚分辨率荧光珠校准。
对于本文所描述的图像相关光谱学协议, 使用开放源图像处理平台 (斐济10) 进行 ImageJ 的分布, 完成 ICS 所需的自相关和数学功能。节目11,12。斐济/ImageJ 在 FFT 数学函数中利用预安装的快速傅立叶变换。此函数通过在每个维度13中减小数据的范围, 从而减少了计算所需的计算时间。当2D 自相关函数在 x、y 轴上近似对称时, 通过自相关图像的单行剖面图可以用来测量原始自相关作为空间滞后的函数。在进一步计算之前, 任何零滞后噪声都将被移除, 由此产生的自相关振幅 (峰值、 g(0)T) 在表达式(Eq 3)的背景下得到修正:
<img alt="Equation 4" src="/files/ftp_upload/57164/57164eq4v2.jpg">     
其中 Ib是来自背景区域的平均强度 (不包括单元格)。簇密度或荧光物体的密度是由(Eq 4)定义的:
<img alt="Equation 5" src="/files/ftp_upload/57164/57164eq5v2.jpg">    
在本文所描述的协议中, 我们进一步简单地计算了聚类密度 (CD), 假设是在观测的基础上, 一个归一化的自相关函数将衰减到一个值接近1.0 的空间滞后。与最大空间滞后, 不再有任何相关性的荧光强度值, 因而没有相关的计算在这个区域计算的强度乘以这个强度, 随后除以那强度的正方形, 按定义是相等的到1.0。因此, 从归一化自相关函数的聚类密度可以计算在取其倒数(Eq 5)之前, 从规范化自相关函数中减去 1.0:
<img alt="Equation 6" src="/files/ftp_upload/57164/57164eq6v2.jpg">     
可以对光束区域进行进一步的标定, 以定量在该仪器的点传播函数区域内的簇数。这种校准必须使用相同的光学条件在图像相关光谱学分析中使用。

<table border="0" cellpadding="0" cellspacing="0" width="1408">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:561px;">Nunc Lab-Tek II Chambered Coverglass - 8 well</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:103px;">155409</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPBS, no calcium, no magnesium</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10099141</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:561px;">TrypLE Express Enzyme (1x), no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>12604021</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:561px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A11013</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:561px;">TetraSpeck Fluorescent Microsphere Standards 0.1&#181;m</td>
			<td>ThermoFisher Scientific</td>
			<td>T7279</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cetuximab</td>
			<td>Merck Serono</td>
			<td>3023715501</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Parafilm M 38mx100mm</td>
			<td>Merck Millipore</td>
			<td>BRND701605</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>ProSciTech</td>
			<td>C004</td>
			<td style="width:561px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Human EGF</td>
			<td>R&#38;D System</td>
			<td>236-EG</td>
			<td></td>
		</tr>
	</tbody>
</table>

confocal microscopy reveals cell surface receptor aggregation through image correlation spectroscopy

共焦显微术提供了一个可访问的方法, 以捕获的亚细胞相互作用关键的表征和进一步发展的临床前药物标记荧光探针。随着最近在抗体基础上的细胞毒性药物传递系统的进展, 了解这些药物诱导在受体聚集和内化领域的变化是至关重要的。该协议利用了 ImageJ 的荧光免疫细胞化学和开放源斐济分布的良好方法, 其内置自相关和图像数学功能, 以执行空间图像相关性光谱学 (ICS)。该协议 quantitates 了标记受体的荧光强度作为共焦显微镜光束区域的函数。这为细胞表面的靶分子聚集状态提供了定量测量。这种方法的重点是静态细胞的特征, 有潜力扩展到时间调查的受体聚集。该协议提出了一种可访问的方法, 用于提供在细胞表面发生的聚类事件的量化, 利用已建立好的技术和非专业的成像设备。

成功的图像相关光谱学实验依赖于治疗控制的正确应用。这些治疗组包括检查无原发抗体和无二级抗体治疗组的荧光。一旦为实验建立了最佳共焦激光设置, 就必须捕捉这些控制组的图像, 以确认样本中缺乏非特异荧光。对于许多贴壁癌细胞系, 适用于 ICS 的区域的识别受到缺乏平坦表面的限制。这是补偿的能力, 捕捉一个大视野的多癌细胞在一个单一的共焦图像。这提供了生成所需取样以在治疗组之间进行统计比较的能力。
在这一典型实验中, A431 egfr 阳性表皮样癌细胞, 在室温下以10分钟的100毫升的 EGF 配体刺激, 诱导 egfr 聚类。ICS 分析对两种表皮生长因子 (图 1A) 和静态 (图 1D) 细胞进行孵化后, EGFR 靶向人性化的单克隆抗体, cetuximab。随后, 利用自相关函数在斐济的开源图像处理平台中, 处理了细胞膜 (图 1B 和1E) 中包含的区域 (黄色盒) 的64x64 像素作物。由此产生的自相关图像在被归一化的感兴趣区域的平均强度平方和被裁剪区域的像素个数平方之前减去背景荧光, 从而对其进行规范化 (图 1C 和1F)。行函数工具用于绘制此图像的点传播函数的轮廓 (图 1G 和1H)。这些配置文件的峰值被转换为电子表格应用程序以进行进一步数据处理。在 cetuximab 免疫细胞化学 (图 1I) 之后, 生成了一个斐济宏, 用于复制这些斐济处理步骤, 以便快速处理来自两个治疗组的一系列细胞 (n=6)。在这个实验中, 在静态细胞群中, A431 刺激细胞的表面上每束面积的5.84 个0.85 簇, 以及每束面积的14.94 到1.62 簇, 都能发现 EGFR 受体。假设所用实验条件限制了快速受体的更替, 刺激 A431 细胞的 EGFR 簇密度下降了2.56 倍。当目标分子翻转受限时, 与同一目标分子的聚集状态进行比较时, 每个光束区域的簇数越少, 则表明目标聚集量增加。在表皮生长因子配体刺激下, EGFR 聚集增加2.56 倍于本系统预期。利用本议定书所述步骤生成斐济宏, 可以对各种治疗或环境差异及其对受体聚集的影响进行统计比较。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57164/57164fig1v2.jpg"> 图 1: 图像相关谱法检测黏附癌细胞细胞表面上的荧光标记簇.共焦显微图像的代表性表皮生长因子刺激 (A) 或静态 (D) 粘附 A431 表皮细胞癌细胞 cetuximab 原抗体靶向细胞表面 EGFR 受体与亚历克斯氟488次抗体。受刺激细胞在室温下以10分钟的100毫升 EGF 治疗, 在固定和免疫细胞化学之前诱导 EGFR 聚类。含有像素尺寸 (64 x 64 [2n]) 的作物区域 (黄色盒) 的细胞膜用于进行 ICS 分析 (B和E)。通过对斐济/ImageJ 自相关函数的归一化, 利用直线工具 (黄色) 测量点扩散函数 (C和F), 绘制该线函数的轮廓以检测峰值值 (G和H).表皮生长因子刺激被观察到诱导2.56 倍减少的 EGFR 簇每束面积5.84 ±0.85 相比, 14.9 5±1.62 静态细胞 (n=6) (I)。假设, 在这个分析中使用的实验条件保持了一定数量的细胞膜受体, 这代表了 EGFR 的聚集后, EGF 的刺激增加。刻度条 = 10 µm (A 和 D);1µm (B-C 和 E F)。用 Tukey 样式显示的方框图, 其表示最大和最小观测值不超过 1.5 x 四分位范围 (IQR)。每个光束区域的簇为平均值的标准误差 (SEM)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57164/57164fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy at JoVE.com

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

与细胞表面上的靶受体结合的抗体可以赋予构象和聚类改变。这些动态变化对表征靶细胞内药物的发展有影响。该协议利用共焦显微和图像相关光谱学通过 ImageJ/斐济, 以量化的程度, 受体聚类在细胞表面。

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

confocal-microscopy-reveals-cell-surface-receptor-aggregation-through

Nanyang Technological University

Research

JoVE Journal

Biology

7.0K 次观看.  Olivia Newton-John Cancer Research Institute. 与细胞表面上的靶受体结合的抗体可以赋予构象和聚类改变。这些动态变化对表征靶细胞内药物的发展有影响。该协议利用共焦显微和图像相关光谱学通过 ImageJ/斐济, 以量化的程度, 受体聚类在细胞表面。

视频: Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

科研

生物学

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...