感谢杨先生为我们准备MCF-7。这项工作得到了中国香港特别行政区研究资助委员会（项目编号17259316,17207715,17207714,17205215和HKD 720112E），创新与技术支持计划（ITS / 090/14 ）和香港大学发展基金。

样品制备<ol><li> 样品制备（贴壁细胞; MCF-7细胞） <ol><li>从培养箱中取出细胞培养皿，排干培养基。 </li><li>用1x磷酸缓冲盐水（PBS）将培养皿上的细胞冲洗以除去过量的培养基。 </li><li>加入3 mL 0.25％胰蛋白酶溶液至培养皿（直径100 mm），并置于37℃，5％CO 2培养箱中4分钟。 注意:胰蛋白酶溶解细胞的粘合蛋白，使其从培养皿中分离。 </li><li>检查所有的细胞是否使用光学显微镜（10X物镜）从培养皿中分离出来。如果没有，轻轻摇动细胞培养皿。 </li><li>加入4mL标准培养基（用89％Dulbecco&#39;s Modified Eagle培养基（DMEM），10％胎牛血清（FBS）和1％青霉素 - 链霉素（PS））配制）以停止胰蛋白酶的作用。 </li><li> Ť将整个混合物（胰蛋白酶溶液和培养基中的细胞）转移到离心管中，并以200×g离心5分钟。 </li><li>取出所有液体，并将细胞重新悬浮在1 mL 1x PBS（pH值:7.4，预热至37°C）中。 </li></ol></li><li> 样品制备（培养的微藻） <ol><li>将培养的微藻和培养基（ 例如海水，琼脂或淡水）以1:5的体积比（微藻培养基）转移到新的玻璃培养管（〜15mL）中以建立一种新的亚文化培养基。 </li><li>在实验前72 - 120小时，根据光/暗（LD）循环（通常为LD 16:8），通过荧光灯的人工照明，将玻璃培养管放置在防尘室内。 </li><li>将分培养的样品转移到离心管中并充分混合。 </li></ol></li></ol> 2. ATOM系统设置 图1:ATOM系统示意图。使用宽带脉冲激光器将超快脉冲传送到（ a ）时间延伸模块和（ b ）直列光放大器模块。时间延伸模块产生一串时间波形，每个时间波形是激光源的波长谱的复制品（ 即波长到时间的映射）。放大器模块用于脉冲拉伸（色散）损耗补偿。然后（ c ）通过衍射光栅空间分散拉伸的脉冲，形成1D光谱淋浴照明，其中各个波长分量由中继透镜对中继并由物镜聚焦到（ d）内的流动单元上的不同位置）微流控芯片。这是频谱编码的过程。该光谱编码的光将再次通过另一个物镜和反射镜，返回到衍射光栅并重新组合为空间未分散的脉冲光束。然后，该图像编码的脉冲光束 （ e ）分成两个路径，使得两个梁在被耦合到纤维中之前被部分地被（ f ）刀刃而是从相反的方向阻挡。这两个光束代表最终图像的两个（相反）编码的相位梯度对比度。为了同时检测两个信号对比度，信号之一经历（ g ）时间延迟线，使得两个信号在时间上被多路复用（交错）。高速光电探测器和实时示波器用于数据采集。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55840/55840fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 时间st<html>保留模块<ol><li>采用宽带飞秒（fs）或皮秒（ps）脉冲激光源，推荐的中心波长在近红外（NIR）范围内，800-1,500 nm。 注意:典型的所需脉冲宽度范围可以从100 fs到几ps。脉冲激光源的详细要求见参考文献4和5。重要指标如表1所示 。 <ol><li>确保激光源具有高重复率，其应该在兆赫兹（ 例如，几十MHz）范围内，以确保ATOM中的超快成像。此外，将峰值输出激光功率设置得远低于光纤腔的损耗功率阈值，大约为1 kW。 </li><li>确保脉冲激光源的拍摄时间和光谱稳定性。 注意:频谱幅度波动的典型公差应保持在1.2％ 19,20 ，ss ="xref"&gt; 21，这通过该设置中的光纤锁模激光器实现。 注意:激光源的光学带宽预计为10-100nm，这对于在单细胞成像21中保证足够的成像视场（FOV）至关重要。 </li></ol></li><li>通过光纤准直器将激光脉冲光束耦合到单模色散光纤，其中脉冲通过群速度色散（GVD）进行拉伸（ 图1a ）。 注意:这是一个过程，每个脉冲的频谱作为波长扫描波形（ 即波长到时间映射）映射到时间上。 注意:总需要的GVD应足以确保整个ATOM图像分辨率不受波长到时间映射过程的影响（参见讨论）。通常，在NIR范围内，GVD应远远超出0.1ns / nm。例如，在thi中使用的单模光纤当前设置提供了波长1,060 nm（总光纤长度为10公里）的0.38 ns / nm的总GVD。 </li></ol></li><li> 光谱编码模块 <ol><li>构建光学显微镜系统以对沿着微流体通道流动的细胞进行光谱编码成像， 如图1所示 。 注意:该光学显微镜的关键部件包括:（1）衍射光栅，伸缩式中继透镜模块（ 图1中的 RL1和RL2）和两个物镜（ 图1中的 Obj1和Obj2）。 <ol><li>首先，将时间延伸和准直光束照射到衍射光栅上（在该设置中采用透射型光栅）以产生光谱淋浴（ 图1c ）。 注意:通过调整t可以使衍射光束的功率（ 即衍射效率）最大化他的光栅方向靠近Littrow配置。 </li><li>在4-f成像系统中配置两个中继透镜（RL1和RL2）（ 即，将衍射光栅放在RL1的焦平面上，并将RL1和RL2分开距离等于其焦距的总和。光谱淋浴然后将被成像到物镜Obj1）的后焦平面上。 </li><li>仔细对准光谱淋浴以填充Obj1的后孔，使得光谱淋浴可以投影并聚焦到显微镜的图像平面上。 注意:这里，物镜的NA（ 图1中的 Obj1）为0.75。 </li><li>在该物镜（Obj2）的后孔处放置另一个具有类似NA的物镜（Obj2）和平面镜，使得光谱淋浴器束可以对准以使图像平面双倍并返回到衍射光栅。 </li><li>调整所述一对物镜（Obj1和Obj2）使得它们的焦平面彼此重叠。检查光谱淋浴是否在相同位置双重通过图像平面，并按照相同的路径返回光栅。如果没有，请进行光学系统的进一步对准和调谐。 注意:返回的光线应通过一个附加的光束分离器，使得光线可以传输到非对称检测模块。 </li><li>将光放大器增益调整到合适的电平，使得所得到的信号可以由光电探测器以良好的SNR（通常在&gt; 10dB）来检测。 </li><li>放置并调整样品平台上的微流控芯片的位置，并确保光谱淋浴，从而将成像区域放置在微流体通道之间（ 图1d ）。 注意:光谱淋浴器与m内流体的流动方向正交地照亮微流控芯片，使得流动运动自动执行二维（2D）扫描。 </li></ol></li></ol></li><li> 不对称检测模块 <ol><li>放置一个附加的光束分离器将成像编码的光束分成两个（ 图1e ）。 注意:每个光束副本部分被刀刃阻挡。 </li><li>测量并记录束阻挡前光束的光功率。然后，手动放置刀刃（安装在线性平台上），使其大致阻挡梁的一半（通过目视检查）。之后，使用光功率计来监视光束的功率，同时通过将光束平移在光束上来微调刀刃的位置。 注意:建议最佳位置是功率下降约原始值的50％（ 即未阻塞的情况）。这是提供图像信号最佳组合的条件强度和图像对比度增强。 </li><li>对另一个光束副本重复步骤2.3.2。注意，一个光束的部分光束块的取向应该相对于另一个光束相反（ 图1f ）。 </li><li>将两个部分阻挡的光束通过双光纤准直器耦合到两个单独的单模光纤臂中。 注意:其中一个臂具有额外长度的纤维，用作基于光纤的延迟线，以引入相对于其他副本的延时（无延迟线）（ 图1g ）。在光电检测之前，两者均通过光纤耦合器引导到单个光纤。时间延迟应该足够长以便在时间上分离两个副本并足​​够短以避免与下一个波形的时间重叠（ 即，两个副本在检测之前总是在单个光纤中进行时间交织和时间复用（ 图2a ））。 </li><li>帽用实时示波器对检测到的光信号进行数字化处理。请注意，示波器的带宽和采样率应足够高以确保它们不影响最终的图像分辨率（参见讨论）。 注意:这里，GVD为0.38 ns / nm，需要后端采集带宽和采样率分别为&gt; 20 GHz和&gt; 40 GSa / s。 </li></ol></li></ol>实验程序<ol><li> 标本加载 <ol><li>在常规的相差显微镜下，在标准血细胞计数器中进行细胞计数。 </li><li>通过用1×PBS（用于微藻的弹簧水）稀释并用移液管充分混合来调节细胞密度。 注意:建议浓度为10 5至10 6个细胞/ mL。 </li><li>将微流体芯片安装到光学成像系统的样品平台上。 注意:微流体芯片主要由聚二甲基硅氧烷（PDMS）制成，并使用标准复制成型法制造。微流体通道被设计成具有不对称弯曲通道以产生惯性流聚焦效应，使得细胞可以在成像部分（尺寸为80μm×80μm（高×宽））的单个文件中高速流动。 </li><li>将密度调节的细胞溶液转移到10mL注射器。 </li><li>将注射器连接到微流体芯片的入口，将离心管连接到微流体芯片的出口以进行处理。 </li><li>将注射器安装在注射器泵上，并设置合适的流速以提供理想的产量，并避免在细胞和通道之间施加过大的剪切力。 注意:样品的线速度应在0.5和10 m / s之间。注意，在实际的图像记录之前，通常需要进一步微调系统，从而最大化图像信号强度，并通过重复步骤2.2.1.4-2.2.1.6优化图像聚焦。 </li></ol></li></ol>数据采集<ol><li>为每个实验设置一个合适数量的要保存的数据点。要保存的数据点的数量取决于样品的大小和流速。 注意:通常，在80 GSa / s的采样率下，将其设置为8-16万个采样点。 </li><li>使用示波器以批处理方式获取并保存数据。 </li></ol>后端处理<img alt="图2" class="xfigimg" src="/files/ftp_upload/55840/55840fig2.jpg" /> 图2:从行扫描时间跟踪重建ATOM图像。为了用吸收对比度和差分（增强）相位梯度对比来重构ATOM图像，从两个时间延伸的脉冲（见紫色和绿色脉冲）中提取两组（ a ）时间多路复用的时间波形迹线，然后被数字分段和堆叠以形成（ b ）两个2D图像，示出两个相反的相位梯度对比度。注意，需要剪切操作以通过补偿由于激光重复率估计中的舍入误差而导致的子索引移位来形成（ c ）无失真图像。通过从激光源的光谱强度包络中减去（ d ）原始线扫描，可以去除图像的背景。 （ g ）可以通过图像（ e ）和（ f ）的逐像素强度求和获得吸收对比图像，而（ h ）可以从图像的减法（e）获得差分相位梯度对比度图像）和（f）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55840/55840fig2large.jpg" target="_blank">请点击这里查看大图</a>的这个数字。 <ol><li> 将数据从示波器传输到计算机以进行离线图像重建。 注意:这里，数据存储在便携式硬盘中，并在示波器和处理计算机之间手动传送。每个实验的硬盘容量应该超过500 GB。 <ol><li>使用关键图像重建程序数字堆叠线扫描以形成2D图像（ 图2 ）。 注意:自定义程序（在MATLAB中）然后通过分离两个时间复用数据集（ 图2中的紫色和绿色波形）来产生两个不对称检测图像。 <ol><li>通过减去两个不对称检测图像的强度来获得差分相位梯度对比图像;可以通过添加两个不对称检测图像来获得吸收对比图像。查看MCF-7和微藻的选定图像图3 。注意，在图像求和和减法操作之前，首先应该消除单个图像的背景特征，并且将它们的强度归一化。 </li></ol></li><li>生成从图像派生的参数库，如单元格体积，圆形度和吸收密度等，以进一步分析。 </li></ol></li><li> 将数据库输入数据可视化平台（ 图4 ）。 <ol><li>将参数库和相应的重建图像加载到可视化界面平台。 </li><li>将轴设置为感兴趣的参数。 注意:参数是特定于问题的，由用户定义。它们是由MATLAB程序从ATOM图像派生出来的。这里，可以选择细胞的光吸收密度，细胞面积，细胞体积和细胞圆形度。 </l我&gt; <li>选择要显示的数据集，使得每个单元格图像都可以在散点图上显示为不同的数据点。 </li><li>将鼠标光标移动到每个点，使相应的图像和其他参数显示在浮动子窗口中。 注意:可以以交互方式在线性和对数刻度之间更改轴。 </li><li>通过在散点图上手动选通，进一步分析数据集的任何子集。 注意:门控子集的每个参数中的直方图可以对整个库的图形进行绘制。单独的直方图显示在另一个浮动子窗口中。 </li></ol></li></ol>

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R., Rabizadeh, S., Jalali, B. <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+high-throughput+cell+screening+in+flow.">Label-free high-throughput cell screening in flow.</a> Biomed Opt Express. 4 (9), 1618-1625 (2013).</li><li>Wong, T. T., 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=Asymmetric-detection+time-stretch+optical+microscopy+(ATOM)+for+ultrafast+high-contrast+cellular+imaging+in+flow.">Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow.</a> Sci Rep. 4, 3656 (2014).</li><li>Mata, T. M., Martins, A. A., Caetano, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microalgae+for+biodiesel+production+and+other+applications:+A+review.">Microalgae for biodiesel production and other applications: A review.</a> Renew Sustainable Energy Rev. 14 (1), 217-232 (2010).</li><li>Irigoien, X., Huisman, J., Harris, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+biodiversity+patterns+of+marine+phytoplankton+and+zooplankton.">Global biodiversity patterns of marine phytoplankton and zooplankton.</a> Nature. 429 (6994), 863-867 (2004).</li><li>Wei, X., 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=Coherent+Laser+Source+for+High+Frame-Rate+Optical+Time-Stretch+Microscopy+at+1.0+&#181;m.">Coherent Laser Source for High Frame-Rate Optical Time-Stretch Microscopy at 1.0 &#181;m.</a> IEEE J. Sel. Top. Quantum Electron. 20 (5), 384-389 (2014).</li><li>Wei, X., 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=Breathing+laser+as+an+inertia-free+swept+source+for+high-quality+ultrafast+optical+bioimaging.">Breathing laser as an inertia-free swept source for high-quality ultrafast optical bioimaging.</a> Opt. Lett. 39 (23), 6593-6596 (2014).</li><li>Xu, Y., Wei, X., Ren, Z., Wong, K. K. Y., Tsia, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+measurements+of+optical+spectral+coherence+by+single-shot+time-stretch+interferometry.">Ultrafast measurements of optical spectral coherence by single-shot time-stretch interferometry.</a> Sci Rep. 6, 27937 (2016).</li><li>Tsia, K. K., Goda, K., Capewell, D., Jalali, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+serial+time-encoded+amplified+microscope.">Performance of serial time-encoded amplified microscope.</a> Opt. Express. 18 (10), 10016-10028 (2010).</li><li>Alix-Panabi&#232;res, C., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+circulating+tumour+cell+research.">Challenges in circulating tumour cell research.</a> Nat Rev Cancer. 14 (9), 623-631 (2014).</li><li>Kling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+counting+tumor+cells.">Beyond counting tumor cells.</a> Nat Biotech. 30 (7), 578-580 (2012).</li><li>Hodgkinson, C. L., 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=Tumorigenicity+and+genetic+profiling+of+circulating+tumor+cells+in+small-cell+lung+cancer.">Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer.</a> Nat Med. 20 (8), 897-903 (2014).</li><li>Ozkumur, 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=Inertial+focusing+for+tumor+antigen-dependent+and+-independent+sorting+of+rare+circulating+tumor+cells.">Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells.</a> Sci Transl Med. 5 (179), (2013).</li><li>Hou, H. 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=Isolation+and+retrieval+of+circulating+tumor+cells+using+centrifugal+forces.">Isolation and retrieval of circulating tumor cells using centrifugal forces.</a> Scientific Reports. 3, 1259 (2013).</li><li>Karabacak, N. 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=marker-free+isolation+of+circulating+tumor+cells+from+blood+samples.">marker-free isolation of circulating tumor cells from blood samples.</a> Nat. Protocols. 9 (3), 694-710 (2014).</li><li>Krebs, M. G., 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=Molecular+analysis+of+circulating+tumour+cells-biology+and+biomarkers.">Molecular analysis of circulating tumour cells-biology and biomarkers.</a> Nat Rev Clin Oncol. 11 (3), 129-144 (2014).</li><li>Wu, 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=Ultrafast+Laser-Scanning+Time-Stretch+Imaging+at+Visible+Wavelengths.">Ultrafast Laser-Scanning Time-Stretch Imaging at Visible Wavelengths.</a> Light Sci Appl. , (2016).</li></ol>

作者没有什么可以披露的。

在ATOM系统设置过程中需要特别注意的几个技术细节。首先，必须注意的是，不对称/倾斜光谱编码的照明可以在吸收对比度中引入残余相位梯度分量（ 即阴影效应），并影响ATOM中相位梯度对比度的增强。因此，倾斜照明的这种效果应该最小化。第二，应该强调的是，涉及两个副本的时间复用或时间交织方案对成像速度没有显着的影响（ 即，仍然可以维持&gt; MHz的行扫描速率），假定...

光学成像提供了一个强大的工具和基于细胞的测定（几乎）非侵入性地显示许多细胞/亚细胞组分的详细空间分布，从而揭示了细胞的许多形态学，生物物理学和生物分子特征。然而，当必须调查巨大和异质的细胞群体时，从单细胞提取高含量信息的这种能力通常受到损害。这标志着测量吞吐量和内容之间基于细胞的测定中的共同权衡。一个显着的例子是，与传统非成像流式细胞仪相比，添加流式细胞术的成像能力导致吞吐量下降至少1-2个数量级。虽然它可以提供复杂的形态单细胞分析，这是不可能的标准流式细胞仪1 ，成像流式细胞仪通常缺乏足够的吞吐量id以高统计学信度诱导细胞异质性。这对于生物学的新发现和对疾病发病机理的了解是必要的。关键的技术挑战在于通常的光学成像策略固有的速度限制:激光束扫描（ 例如，通过电流镜）和/或图像传感器（ 例如电荷耦合器件（CCD）和互补金属 -氧化物半导体（CMOS））。激光扫描速度本身受到扫描镜的机械惯性的限制，而CCD或CMOS的帧速率受成像速度和灵敏度之间的基本权衡的限制（ 即，增加帧速率导致降低的信号检测灵敏度，反之亦然）。 基于全光学，超快速图像编码机制，光时延成像已经被证明是高通量成像流量cyt的有吸引力的平台米，不需要传统的图像传感器或机械激光扫描2,3 。参考文献4,5,6,7中可以看到时间延伸成像工作原理的详细描述。简而言之，它包括两个可互换的映射步骤:（i）频谱编码（波长空间映射），其中成像的样本的空间坐标映射到光脉冲束8,9的光谱上的不同波长，和（ii）分散傅里叶变换（波长 - 时间映射） 9 ，其中各个激光脉冲的波长分量通过组速度色散（GVD）变换（拉伸）成时间（波长扫描）波形（ 图1 ）。一个重要的时间延伸成像的特征是光学放大，其在抵抗由于超快光检测和GVD损耗引起的灵敏度损失方面起关键作用，从而增强图像信噪比（SNR）而不被光电探测器噪声9污染。由于每个激光脉冲编码与单元的单向流正交的成像样本的线扫描，因此通常超过10MHz的激光重复率来确定有效的线扫描速率。这种超快速操作使得能够以10,000-100,000个细胞/秒的吞吐量（ 即，比传统成像流式细胞术高10-100倍）的无模糊单细胞图像捕获。因此，时间延伸成像可以在高通量，单细胞，基于图像的筛选中找到独特的应用，特别是当需要在相当大的人群中识别未知的异质性或罕见/异常细胞（数千到数百万CEL<html> ls），如罕见癌细胞筛选10或微藻分类11 。 时间延伸成像主要依赖于明场（BF）图像捕获，通过来自细胞2,3,9,10,11的光散射和吸收产生图像对比度。这种无标记的单细胞成像能力可以绕过与荧光标记相关的有害作用，例如细胞毒性和光漂白，并且基于细胞和亚细胞纹理​​和形态为单细胞分析提供有价值的信息。这些无标记参数被证明对于细胞的深层图像分类是有效的，特别是当巨大的细胞群可用时，"外汇公司"&gt; 12。然而，在许多情况下，BF成像不能提供足够的对比度以揭示无标记透明细胞的详细形态。已经开发了不同的无标签，相位对比，时间延伸成像模式，用于在超快帧速率13,14,15下增强成像对比度。在这些技术中，开发了不对称检测时间拉伸光学显微镜（ATOM），以基于类似于Schlieren摄影的概念揭示相位梯度（差分 - 干涉 -​​ 对比 - （DIC）样）的对比度，在超高的微流体速度（高达10 m / s）下，单细胞的免费，高对比度成像16 。通过在光电检测之前部分地阻挡图像编码的光束路径或倾斜光束，可以容易地通过倾斜检测或照明来产生该效果。 ATOM的另一个优点是它的一个能够沿着相反的方向同时获取两个相位梯度对比度。两个对比度图像的强度减法和求和分别从同一行扫描得到差分相位梯度对比度和吸收对比度。这项工作提出了一个详细的协议描述ATOM的实现，包括建立光学设置，样品准备和数据采集和可视化。具体来说，这项工作通过对人类血液细胞，癌细胞和浮游植物（微藻）的单细胞成像证明了ATOM手术。这突出了ATOM对流式细胞术成像的适用性，不仅在生物医学领域，而且在海洋和生物燃料研究17,18 。

<table><tbody><tr><td>Nikon Plan Fluorite Physiology Objectives 40X</td><td>Nikon</td><td>MRF07420</td><td>Objective lens (Obj2)</td></tr><tr><td>Olympus Plan Fluorite Objective, 0.75 NA, 0.51 mm WD, 40X</td><td>Olympus</td><td>RMS40X-PF</td><td>Objective lens (Obj1)</td></tr><tr><td>N-BK7 Plano-Convex Lenses</td><td>Thorlabs</td><td>LA1145-C</td><td>For relaying spectral shower</td></tr><tr><td>FC/APC Fiber Collimation Package</td><td>Thorlabs</td><td>F220APC-1064</td><td>For outputing and collecting laser pulses</td></tr><tr><td>Pellicle beamsplitter</td><td>Thorlabs</td><td>BP145B3</td><td>For making beam replica</td></tr><tr><td>Protected silver mirror</td><td>Thorlabs</td><td>PF10-03-P01</td><td>For reflecting light</td></tr><tr><td>800-1650nm 12GHz single mode DC-coupled NIR Photoreceiver</td><td>Newport</td><td>1544-B</td><td>For converting light into electrical signal</td></tr><tr><td>Infiniium High-Performance Oscilloscope</td><td>Agilent</td><td>DSOX91604A</td><td>To save the light-converted electrical signals</td></tr><tr><td>HI1060 optical fiber</td><td>Corning</td><td>HI1060</td><td>Optical fiber for time-stretch</td></tr><tr><td>YTTERBIUM DOPED FIBER AMPLIFIER</td><td>Keopsys</td><td>KPS-STD-BT-YFA-37-BO-SM-111-FA-FA</td><td>Optical in-line amplifier</td></tr><tr><td>Holographic grating</td><td>Wasatch Photonics</td><td>020305-6</td><td>Grating</td></tr><tr><td>Infuse/Withdraw Syringe Pumps</td><td>Harvard Apparatus</td><td>PHD 2000</td><td>Syringe pump for sample loading in micro-fluidic channels</td></tr></tbody></table>

microfluidic imaging flow cytometry by asymmetric-detection time-stretch optical microscopy (atom)

缩放可测量参数的数量，这是允许进行多维数据分析和因此更高置信度的统计结果，一直是流式细胞术先进发展的主要趋势。值得注意的是，添加高分辨率成像功能允许细胞/亚细胞结构的复杂形态分析。标准流式细胞仪不可能。但是，提高我们的细胞功能知识是有价值的，有利于生命科学研究，临床诊断和环境监测。将成像能力纳入流式细胞术可以降低检测的吞吐量，这主要是由于相机技术中速度和灵敏度的限制。为了克服成像流式细胞术同时保持图像质量面临的这种速度或吞吐量挑战，已经证明了不对称检测时间延伸光学显微镜（ATOM）能够实现高对比度的单细胞图像具有亚细胞分辨率，成像吞吐量高达100,000个细胞/秒。基于通过使用超高速宽带激光脉冲依赖于全光图像编码和检索的常规时间拉伸成像的成像概念，ATOM通过增强未标记/未染色细胞的图像对比来进一步提高成像性能。这是通过访问细胞的相位梯度信息来实现的，其被频谱地编码成单次宽带脉冲。因此，ATOM在单细胞形态和纹理的高通量测量中特别有利 - 指示细胞类型，状态甚至功能的信息。最终，这可能成为用于细胞生物物理表型的强大的成像流式细胞仪平台，补充了当前最先进的基于生物化学标记的细胞测定法。本工作描述了建立ATOM系统关键模块的协议（从光学前端到数据p加工和可视化后端），以及基于ATOM的成像流式细胞术的工作流程，以人类细胞和微藻为例。

<html>这项工作说明了ATOM的两个单细胞成像试验:一个是哺乳动物细胞（人类外周血单个核细胞（PBMC）和乳腺癌细胞（MCF-7）），另一个是浮游植物（Scenedesmus和Chlamydomonas）。第一个实验是由于液体活检对血液中循环肿瘤细胞（CTC）的检测，计数和表征的兴趣日益增加23 。测量从原发肿瘤部位扩散到血液流的CTC的能力允许检测转移性癌症进展24...

Watch this Scientific Journal Video about Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM at JoVE.com

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

该协议描述了用于超快速微流体流中单细胞成像的不对称检测时间拉伸光学显微镜系统的实现及其在成像流式细胞术中的应用。

微流控成像流式细胞仪通过不对称检测时间延伸光学显微镜（ATOM）

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

microfluidic-imaging-flow-cytometry-asymmetric-detection-time-stretch

Universidad San Sebastian

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Unit 1

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Operational Amplifiers

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10.2K 次观看.  The University of Hong Kong. 该协议描述了用于超快速微流体流中单细胞成像的不对称检测时间拉伸光学显微镜系统的实现及其在成像流式细胞术中的应用。 

视频: Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

环境科学

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

免疫与感染

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

基于磁性纳米颗粒免疫分析的交错限制微流控亚克力装置的计算机数控微铣削

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

工程学

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

使用双光镊和微流体进行单分子研究

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

生物化学

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

微血管中场流的空间时间分析

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

<meta charset="utf8"></head><body>用于单细胞因子分泌分析的微流控液滴测定

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

<meta charset="utf8"></head><body>作者聚焦：揭示免疫反应中的多功能性和异质性

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌 

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...