Name: lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture
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1. 细胞培养监测采集
<ol><li>在 DMEM + 谷氨酰胺 (例如, GlutaMAX) 中生长 HeLa 细胞, 辅以 10% (v/v) 热灭活胎小牛血清和1% 青霉素和链霉素。</li>
	<li>涂层6井玻璃底部培养板与纤维连接蛋白 (25 µg/毫升) 为 1 h。然后每个井种子 2 x 104单元格。</li>
	<li>在购置期间, 每3天更换一次培养基。</li>
	<li>对于延时采集, 使用视频无透镜显微镜 (商用)。 注: 这是基于无透镜的计算成像技术, 如欧兹坎<em...

<ol>
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在本文中, 我们表明, 无透镜的视频显微镜可以在孵化器内使用, 以捕获数以千计的细胞的动力学。为了描述整个方法, 我们解释了如何用标准的细胞跟踪算法来分析2.7 天的 HeLa 细胞在培养中的时间推移获取。结果是一个具有 2.2 x 106单元格测量和10584个单元周期轨道的数据集。这项收购是对 Hela 细胞的一种文化进行的, 细胞间距离相对较大 (细胞密度 < 500 细胞/毫米2), 细胞形态学可?...

在几个细胞周期内监测培养的哺乳动物细胞, 准确测量细胞大小和细胞干质量是一项具有挑战性的任务。几个无标签的光学技术能够执行此任务1,2: 移相干涉测量3, 数字全息显微镜 (DHM)4,5,6,7, quadriwave 横向剪切干涉测量8,9和定量相断层扫描10,11。这些方法使人们对哺乳动物细胞周期的认识有了许多新的见解。然而他们很少加上自动细胞追踪算法, 并且他们的吞吐量仍然是有限的, 当测量细胞质量轨道1 (N < 20 分别在3,4,5,6). 因此, 需要用一种新的光学方法来测量大统计量 (N > 1000) 的细胞质量轨迹。
在本文中, 我们展示了无透镜视频显微镜的能力, 同时在孵化室内直接图像数以千计的细胞, 然后量化单个细胞指标沿数以千计的单个细胞周期轨道。无透镜显微镜是一种定量的相位成像技术, 它允许在一个非常大的视野 (通常数十毫米2, 这里29.4 毫米2)12,13 中获取密被包装细胞的相位图像. ,14,15。在单个单元格级别确定了几个度量值,例如、单元格区域、单元格的干质量、单元格粗细、单元格主轴长度和单元格长宽比12、15、每个图像。然后, 通过应用单元跟踪算法, 可以将这些功能绘制为每个单元格, 作为实验时间14、15的函数。此外, 通过检测细胞内的细胞分裂的发生, 有可能提取其他重要信息, 如初始细胞干质量 (刚刚细胞分裂), 最终细胞干质量 (刚刚细胞分裂) 和细胞周期持续时间,即, 两个连续的分区15之间的间隔。所有这些测量都可以计算出非常好的统计数据 (N > 1000), 因为大的视野通常允许分析200到1万细胞在一个单一的无透镜获取。
为了解释这种基于无透镜视频显微镜的方法, 我们描述了2.7 天内对 HeLa 细胞培养进行监测和量化的协议。数据分析是基于多波长全息重建、细胞跟踪、细胞分割和细胞分裂算法的四步过程。这里表明, 空间分辨率和相对较快的帧速率 (每10分钟一次采集) 获得与此无透镜视频显微镜设置兼容标准的细胞跟踪算法。对这个数据集的完整分析结果是在整个细胞周期内测量10584个细胞轨道。
总之, 无透镜视频显微镜是一个强大的工具, 自动监测数以千计的未标记, 不同步, 和未修改的细胞, 每个实验;在多个单元周期中跟踪每个单元格。因此, 我们的测量提供了几个细胞参数的平均值, 但更重要的是, 细胞间的变异超过了大量的细胞。

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Cytonote lens-free video microscope&#160;</td>
			<td style="width:208px;">Iprasense</td>
			<td style="width:156px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horus acquisition software</td>
			<td>Iprasense</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well glass bottom culture plates</td>
			<td>MatTek corporation</td>
			<td>Part No: P06G-0-14-F&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM + GlutaMAX medium&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat-inactivated fetal calf serum&#160;</td>
			<td>Eurobio</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin&#160;</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matlab, image processing toolbox&#160;</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

在这里, 我们表明, 无透镜的视频显微镜, 使我们能够同时捕获数以千计的细胞的动力学, 直接在孵化器内, 并有可能监测和量化的单个细胞沿几个细胞周期。我们描述了用于监视和量化 HeLa 细胞培养2.7 天的完整协议。首先, 用无透镜的视频显微镜进行细胞培养, 然后在四步过程中分析数据: 多波长全息重建、细胞跟踪、细胞分割和细胞分裂检测算法.因此, 我们表明, 有可能收集具有超过1万个细胞周期轨道和超过 2 x 106细胞形态学测量的数据集。

对于全息重建过程, 光场由标量字段a <img alt="Equation 4" src="/files/ftp_upload/56580/56580eq4v3.jpg">描述 (其中是从样本到距离z的平面上的a的复数值, 以及侧面位置和波长λ). <img alt="Equation 5" src="/files/ftp_upload/56580/56580eq5v3.jpg">光传播是由惠更斯-菲涅尔理论建模的, 它提供了一个传递者内核. <img alt="Equation 6" src="/files/ftp_upload/56580/56580eq6v3.jpg"> </img>因此, 探测器平面?...

Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

无透镜的视频显微镜使我们能够在孵化器内直接监测细胞培养。在这里, 我们描述了用于获取和分析2.7 天长时间获取培养的 HeLa 细胞的完整协议, 导致一个数据集 2.2 x 106测量单个细胞形态学和10584细胞周期轨道。

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

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Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...