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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者没有什么可承认的。
Materials
| Cytonote lens-free video microscope | Iprasense | ||
| Horus acquisition software | Iprasense | ||
| 6-well glass bottom culture plates | MatTek corporation | Part No: P06G-0-14-F | |
| DMEM + GlutaMAX medium | Gibco | ||
| heat-inactivated fetal calf serum | Eurobio | ||
| penicillin and streptomycin | Gibco | ||
| Fibronectin | Sigma Aldrich | ||
| Matlab, image processing toolbox | Mathworks |
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