Momey F, Coutard J-G, Bordy T, Navarro F, Menneteau M, Dinten J-M, Allier C
Univ. Grenoble Alpes, F-38000 Grenoble, France; CEA, LETI, MINATEC Campus, F-38054 Grenoble, France.
Biomed Opt Express. 2016 Jan 15;7(2):512-24. doi: 10.1364/BOE.7.000512. eCollection 2016 Feb 1.
In this paper, we discuss a new methodology based on lensfree imaging to perform wound healing assay with unprecedented statistics. Our video lensfree microscopy setup is a simple device featuring only a CMOS sensor and a semi coherent illumination system. Yet it is a powerful mean for the real-time monitoring of cultivated cells. It presents several key advantages, e.g. integration into standard incubator, compatibility with standard cell culture protocol, simplicity and ease of use. It can perform the follow-up in a large field of view (25 mm(2)) of several crucial parameters during the culture of cells i.e. their motility, their proliferation rate or their death. Consequently the setup can gather large statistics both in space and time. Here we uses this facility in the context of wound healing assay to perform label-free measurements of the velocities of the fronts of proliferation of the cell layer as a function of time by means of particle image velocimetry (PIV) processing. However, for such tissue growth experiments, the field of view of 25 mm(2) remains not sufficient and results can be biased depending on the position of the device with respect to the recipient of the cell culture. Hence, to conduct exhaustive wound healing assays, we propose to enlarge the field of view up to 10 cm(2) through a raster scan, by moving the source/sensor with respect to the Petri dish. We have performed acquisitions of wound healing assay (keratinocytes HaCaT) both in real-time (25 mm(2)) and in final point (10 cm(2)) to assess the combination of velocimetry measurements and final point wide field imaging. In the future, we aim at combining directly our extended field of view acquisitions (>10 cm(2)) with real time ability inside the incubator.
在本文中，我们讨论了一种基于无透镜成像的新方法，用于以前所未有的统计学方式进行伤口愈合分析。我们的视频无透镜显微镜装置是一种简单的设备，仅具有一个CMOS传感器和一个半相干照明系统。然而，它是实时监测培养细胞的有力手段。它具有几个关键优势，例如可集成到标准培养箱中、与标准细胞培养方案兼容、简单易用。它可以在细胞培养过程中对几个关键参数在大视野（25平方毫米）内进行跟踪，即细胞的运动性、增殖率或死亡率。因此，该装置可以在空间和时间上收集大量数据。在此，我们在伤口愈合分析的背景下使用该设备，通过粒子图像测速（PIV）处理，对细胞层增殖前沿的速度进行无标记测量，作为时间的函数。然而，对于此类组织生长实验，25平方毫米的视野仍然不够，并且结果可能会因设备相对于细胞培养容器的位置而产生偏差。因此，为了进行详尽的伤口愈合分析，我们建议通过相对于培养皿移动光源/传感器，通过光栅扫描将视野扩大到10平方厘米。我们已经实时（25平方毫米）和在终点（10平方厘米）进行了伤口愈合分析（角质形成细胞HaCaT）的采集，以评估测速测量和终点宽视野成像的结合。未来，我们旨在将扩展视野采集（>10平方厘米）与培养箱内的实时能力直接结合起来。
