Name: lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture
Uploaded: 2018-02-23T14:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

作者没有什么可承认的。

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et . 所述。16 , 它被修改为在孵化器中以37摄氏度12、15的控制温度执行连续监视。它具有一个互补的金属氧化物半导体 (CMOS) 图像传感器, 像素间距为1.67 µm, 成像面积为 6.4 x 4.6 毫米2。多波长照明由一个多芯片组件发光二极管 (led) 装置提供, 它提供红色、绿色和蓝色照明。波长分别为636、521和 452 nm, 其光谱带宽分别为25、45和 25 nm。红绿蓝 (RGB) 发光二极管位于150µm 针孔的上方, 距离细胞约5厘米。测量接近 LED 的光功率低至10µW 在每个波长和照明时间仅一秒每承购。因此, 在无透镜的视镜下观察细胞培养时, 不会出现光毒性问题。</li>
	<li>将单元格区域性容器与 CMOS 传感器 (图 1) 联系在一起。在底部使用带有玻璃盖玻片的容器, 这对无透镜采集的质量很重要。还可以使用塑料容器, 但由于这些容器的光学质量较差, 无透镜采集将被降级。</li>
	<li>使用采集软件 (商用) 控制视频无透镜显微镜, 既可实现定时采集, 又可进行全息重建。用户在软件界面中需要输入的参数包括帧速率、实验持续时间和细胞培养类型, 即黏附细胞或漂浮细胞。</li>
	<li>设置采集软件的输入参数。每10分钟将帧速率设置为一次获取, 并将单元格区域性类型设置为 "粘附"。 注意: 每10分钟一次获取帧速率是一个很好的折衷, 因为它确保了可靠的单元跟踪, 而结果数据集的大小仍然是合理的。最快的帧速率可实现与这种无透镜显微镜设置是每5分钟一次获得。进一步增加帧率导致 CMOS 传感器过度加热, 这会影响细胞在培养中的生存能力。</li>
	<li>启动延时采集, 全息重建算法 (商用) 将自动处理无透镜采集, 获得细胞培养的相位图像。全息重建算法是基于多波长相位检索算法17 , 并提供一个 RGB 重建相图像的范围内的每一个单一的单元格 (π, + π) 在一个大视野的29.4 毫米2。无透镜在线全息术的原理是在 z=0 上用平面波 (在正常化后的振幅 1) 来照亮一个稀薄的样本, 并在 CMOS 传感器上检测随后的衍射强度图像在短距离 z = z, 通常为1毫米后,示例.在我们的设置中, 光照按顺序切换到3波长 (λ1= 0.450 µm, λ2= 0.540 µm, λ3= 0.647 µm) 来测量同一样本的三衍射模式。</li>
</ol>2. 细胞培养数据分析
<ol><li>对于单元跟踪, 使用 Trackmate 算法, 一个开源斐济插件, 用于自动跟踪单个粒子18。首先, 将全部的时间推移捕获加载到斐济 (加载图像序列命令)。由于数据集的大小很大, 通常 400 RGB 帧为 29.7 Mb, 因此以8位格式加载它们会更快。接下来, Trackmate 斐济插件引导用户通过细胞跟踪算法的几个阶段, 即单元检测阶段、单元跟踪阶段以及用于单元检测和计算轨道的几个滤镜。在每个阶段, 配置算法并立即显示结果, 以便在必要时可以轻松地来回导航以重新调整设置。Trackmate 接口的各种菜单显示在图 2中, 其中的实际设置用于 HeLa 单元实验。</li>
	<li>将采集软件的输入参数设置为如下 (请参见图 2)。将估计的 blob 直径设置为15像素, 探测器阈值为 0.25, 链接最大距离为15像素, 间隙闭合最大距离为15像素, 轨道中的点数为3.5。 注意: 这些设置明显不同于一个实验, 特别是与单元格大小对应的 "估计 blob 直径" (这里给出的像素为1.67 µm)。同样的评论也适用于 "链接最大距离", 它解释了在两个连续帧 (这里给出的像素为1.67 µm) 的最大距离。它的最佳值取决于细胞的运动, 也依赖于细胞密度。</li>
	<li>在单元格跟踪过程的末尾, 以三个文本文件 ("分析" 按钮) 的形式生成结果, 请参见图 2)。最有用的文件, 名为 "轨道统计" 中的 "点", 由一个表列出所有检测到的单元格, 它们各自 (x、y) 位置在获取、帧号和跟踪编号中。根据这些数据, 使用专用算法执行单元格分割和检测单元划分 (请参阅辅助代码文件)。另外两个文件被命名为 "跟踪统计信息" 和 "跟踪统计信息" 中的 "链接", 包括处理轨道的所有结果,例如跟踪工期、检测到的间隙数、跟踪初始帧等。</li>
	<li>使用单元格分割算法 (见补充码文件), 自动提取几个描述细胞形态学从重建阶段图像的指标。这些指标是细胞表面积, 细胞主轴长度 (l), 细胞小轴长度 (l) 和细胞长宽比 (l/升)。从无透镜显微镜中恢复的相位与前述15所讨论的试样层的密度和厚度成正比。</li>
	<li>除了细胞的形态学特征外, 从重建阶段13,19,20提取定量细胞干质量 (CDM) 测量结果 <img alt="Equation 1" src="/files/ftp_upload/56580/56580eq1v3.jpg">情商 (1) 其中φ (x, y) 是重构的相移19, λ波长和α = 1.8 x 10-4 m3/千克与折射率变化有关的特定折射率1, 19。</li>
	<li>用估计细胞折射率与培养基的差异来评估细胞厚度。20给出了单元格厚度h(x、y) 和测量的相移之间的关系: <img alt="Equation 2" src="/files/ftp_upload/56580/56580eq2v3.jpg">情商 (2.1) <img alt="Equation 3" src="/files/ftp_upload/56580/56580eq3v3.jpg">情商 (2.2) 使用Δn(x,y) = n单元格(x,y)- n中 (x,y), 单元格和区域性媒体之间的折射率差。在下面, 假定Δn = 0.025的常量值, 这是从在 HeLa 细胞核 (n = 1.35511) 和磷酸盐缓冲盐水 (PBS) 培养基 (n = 1.33) 中测量的折射率估计出来的。虽然细胞厚度值只是指示性的, 因为它是基于一个假设, 它的相对变化是有意义的, 可以利用来检测分裂细胞。</li>
	<li>使用专用算法 (请参阅辅助代码文件) 检测细胞分裂的发生, 然后提取与检测到的细胞分裂相关联的单元轨道, 以检测细胞分裂, 测量厚度大于8µm 的单元格。首先确定, 然后检查一个新的细胞是否在空间和时间紧密地出现。这样, 细胞分裂的检测依赖于两个健壮的标准。可替代的和更精细的细胞分裂检测方法可以应用到 lensfree 时间推移获取21,22,23。</li>
</ol>

<ol>
	<li>Zangle, T. A., Teitell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+mass+profiling:+an+emerging+approach+in+quantitative+biophysics.">Live-cell mass profiling: an emerging approach in quantitative biophysics.</a> Nat Methods. 11 (12), 1221-1228 (2014).</li><li>Popescu, G., Park, K., Mir, M., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technologies+for+measuring+single+cell+mass.">New technologies for measuring single cell mass.</a> Lab Chip. 14 (4), 646-652 (2014).</li><li>Reed, J., 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=Rapid,+massively+parallel+single-cell+drug+response+measurements+via+live+cell+interferometry.">Rapid, massively parallel single-cell drug response measurements via live cell interferometry.</a> Biophys J. 101 (5), 1025-1031 (2011).</li><li>Mir, M., 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=Optical+measurement+of+cycle-dependent+cell+growth.">Optical measurement of cycle-dependent cell growth.</a> Proc Natl Acad Sci. USA. 108 (32), 13124-13129 (2011).</li><li>Girshovitz, P., Shaked, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+cell+morphological+parameters+based+on+interferometric+phase+microscopy+and+their+application+to+cell+life+cycle+characterization.">Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization.</a> Biomed Opt Express. 3 (8), 1757-1773 (2012).</li><li>Kemper, B., Bauwens, A., Vollmer, A., Ketelhut, S., Langehanenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+quantitative+cell+division+monitoring+of+endothelial+cells+by+digital+holographic+microscopy.">Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy.</a> J Biomed Opt. 15 (3), (2010).</li><li>Mir, M., Bergamaschi, A., Katzenellenbogen, B. S., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+quantitative+imaging+for+monitoring+single+cancer+cell+growth+kinetics+and+drug+response.">Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.</a> PLoS One. 9 (2), 1-8 (2014).</li><li>Bon, P., Savatier, J., Merlin, M., Wattellier, B., Monneret, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+detection+and+measurement+of+living+cell+morphometric+features+with+single-shot+quantitative+phase+microscopy.">Optical detection and measurement of living cell morphometric features with single-shot quantitative phase microscopy.</a> J Biomed Opt. 17 (7), (2012).</li><li>Aknoun, S., 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=Living+cell+dry+mass+measurement+usinq+quantitative+phase+imaging+with+quadriwave+lateral+shearing+interferometry:+an+accuracy+and+sensitivity+discussion.">Living cell dry mass measurement usinq quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.</a> J Biomed Opt. 20 (1), 1-4 (2015).</li><li>Cotte, Y., 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=Marker-free+phase+nanoscopy.">Marker-free phase nanoscopy.</a> Nature Photonics. 7 (2), 113-117 (2013).</li><li>Choi, W., 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=Tomographic+phase+microscopy.">Tomographic phase microscopy.</a> Nat Methods. 4 (9), 717-719 (2007).</li><li>Kesavan, S. V., 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=High-throughput+monitoring+of+major+cell+functions+by+means+of+lensfree+video+microscopy.">High-throughput monitoring of major cell functions by means of lensfree video microscopy.</a> Sci Rep. 4, 1-11 (2014).</li><li>Zheng, G., Lee, S. A., Antebi, Y., Elowitz, M. B., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ePetri+dish,+an+on-chip+cell+imaging+platform+based+on+subpixel+perspective+sweeping+microscopy+(SPSM).">The ePetri dish, an on-chip cell imaging platform based on subpixel perspective sweeping microscopy (SPSM).</a> Proc Natl Acad Sci USA. 108 (41), 16889-16894 (2011).</li><li>Pushkarsky, I., 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=Automated+single-cell+motility+analysis+on+a+chip+using+lensfree+microscopy.">Automated single-cell motility analysis on a chip using lensfree microscopy.</a> Sci Rep. 4, 4717 (2014).</li><li>Allier, C., 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=Imaging+of+dense+cell+cultures+by+multiwavelength+lens-free+video+microscopy.">Imaging of dense cell cultures by multiwavelength lens-free video microscopy.</a> Cytom Part A. 91 (5), 1-10 (2017).</li><li>Su, T. -. W., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+lensfree+imaging+and+characterization+of+a+heterogeneous+cell+solution+on+a+chip.">High-throughput lensfree imaging and characterization of a heterogeneous cell solution on a chip.</a> Biotechnol Bioeng. 102 (3), 856-868 (2009).</li><li>Delacroix, R., 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=Cerebrospinal+fluid+lens-free+microscopy:+a+new+tool+for+the+laboratory+diagnosis+of+meningitis.">Cerebrospinal fluid lens-free microscopy: a new tool for the laboratory diagnosis of meningitis.</a> Sci Rep. 7, 39893 (2017).</li><li>Tinevez, J. -. Y., 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=TrackMate:+an+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: an open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2016).</li><li>Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+mass+and+growth+dynamics.">Optical imaging of cell mass and growth dynamics.</a> Am J Physiol Physiol. 295 (2), 538-544 (2008).</li><li>Liu, P. Y., 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=Cell+refractive+index+for+cell+biology+and+disease+diagnosis:+past,+present+and+future.">Cell refractive index for cell biology and disease diagnosis: past, present and future.</a> Lab Chip. 16, 634-644 (2016).</li><li>Rapoport, D. H., Becker, T., Mamlouk, A. M., Schicktanz, S., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+validation+algorithm+allows+for+automated+cell+tracking+and+the+extraction+of+biologically+meaningful+parameters.">A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.</a> PLoS One. 6 (11), e27315 (2011).</li><li>Al-Kofahi, O., 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=Automated+cell+lineage+construction:+A+rapid+method+to+analyze+clonal+development+established+with+murine+neural+progenitor+cells.">Automated cell lineage construction: A rapid method to analyze clonal development established with murine neural progenitor cells.</a> Cell Cycle. 5 (3), 327-335 (2006).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Semin Cell Dev Biol. 20 (8), 894-902 (2009).</li><li>Posakony, J. W., England, J. M., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+growth+and+division+during+the+cell+cycle+in+HeLa+cells.">Mitochondrial growth and division during the cell cycle in HeLa cells.</a> J Cell Biol. 74 (2), 468-491 (1977).</li><li>Zocchi, E., 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=Expression+of+CD38+Increases+Intracellular+Calcium+Concentration+and+Reduces+Doubling+Time+in+HeLa+and+3T3+Cells.">Expression of CD38 Increases Intracellular Calcium Concentration and Reduces Doubling Time in HeLa and 3T3 Cells.</a> J Biol Chem. 273 (14), 8017-8024 (1979).</li><li>Reitzer, L. J., Wice, B. M., Kennell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+glutamine,+not+sugar,+is+the+major+energy+source+for+cultured+HeLa+cells.">Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells.</a> J Biol Chem. 254 (8), 2669-2676 (1979).</li><li>Benedetti, A. D. E., Joshi-barve, S., Rinker-Schaeffer, C., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Antisense+RNA+against+Initiation+Factor+eIF-4E+mRNA+in+HeLa+Cells+Results+in+Lengthened+Cell+Division+Times,+Diminished+Translation+Rates,+and+Reduced+Levels+of+Both+eIF-4E+and+the+p220+Component+of+eIF-4F.">Expression of Antisense RNA against Initiation Factor eIF-4E mRNA in HeLa Cells Results in Lengthened Cell Division Times, Diminished Translation Rates, and Reduced Levels of Both eIF-4E and the p220 Component of eIF-4F.</a> Mol Cell Biol. 11 (11), 5435-5445 (1991).</li><li>Kumei, Y., Nakajima, T., Sato, A., Kamata, N., Enomoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+G1+phase+duration+and+enhancement+of+c-myc+gene+expression+in+HeLa+cells+at+hypergravity.">Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity.</a> J Cell Sci. 93 (2), 221-226 (1989).</li><li>Ginzberg, M. B., Kafri, R., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+being+the+right+(cell)+size.">On being the right (cell) size.</a> Science. 348 (6236), 1245075 (2015).</li><li>Mathieu, E., 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=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li></ol>

作者没有什么可透露的。

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><tbody><tr><td>Cytonote lens-free video microscope&amp;nbsp;</td><td>Iprasense</td><td></td><td></td></tr><tr><td>Horus acquisition software</td><td>Iprasense</td><td></td><td></td></tr><tr><td>6-well glass bottom culture plates</td><td>MatTek corporation</td><td>Part No: P06G-0-14-F&amp;nbsp;</td><td></td></tr><tr><td>DMEM + GlutaMAX medium&amp;nbsp;</td><td>Gibco</td><td></td><td></td></tr><tr><td>heat-inactivated fetal calf serum&amp;nbsp;</td><td>Eurobio</td><td></td><td></td></tr><tr><td>penicillin and streptomycin&amp;nbsp;</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Matlab, image processing toolbox&amp;nbsp;</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

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

lens-free-video-microscopy-for-dynamic-quantitative-analysis-adherent

Research

JoVE Journal

Biology

9.4K 次观看.  Université Grenoble Alpes. 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。

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

科研

生物工程

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as ~18 cm2. Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of ~5 mm without the need for refocusing which corresponds to up to ~9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments.

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

无透镜片上的细胞成像提供了一种高通量细胞生物学和医疗诊断的新工具

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

生物学

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

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

摘要

探索更多视频

此视频中的章节

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

无透镜片上的细胞成像提供了一种高通量细胞生物学和医疗诊断的新工具

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

随机使用时移视频显微镜细胞迁移的定量分析

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

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

摘要

探索更多视频

此视频中的章节

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

无透镜片上的细胞成像提供了一种高通量细胞生物学和医疗诊断的新工具

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

随机使用时移视频显微镜细胞迁移的定量分析

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术