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="http://ecsource.jove.com/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="http://ecsource.jove.com/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 border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Nikon Plan Fluorite Physiology Objectives 40X</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">MRF07420</td>
			<td style="width:393px;">Objective lens (Obj2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus Plan Fluorite Objective, 0.75 NA, 0.51 mm WD, 40X</td>
			<td style="width:71px;">Olympus</td>
			<td>RMS40X-PF</td>
			<td>Objective lens (Obj1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">N-BK7 Plano-Convex Lenses</td>
			<td style="width:71px;">Thorlabs</td>
			<td>LA1145-C</td>
			<td>For relaying spectral shower</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">FC/APC Fiber Collimation Package</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">F220APC-1064</td>
			<td>For outputing and collecting laser pulses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Pellicle beamsplitter</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">BP145B3</td>
			<td>For making beam replica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Protected silver mirror</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">PF10-03-P01</td>
			<td>For reflecting light</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">800-1650nm 12GHz single mode DC-coupled NIR Photoreceiver</td>
			<td style="width:71px;">Newport</td>
			<td style="width:119px;">1544-B</td>
			<td>For converting light into electrical signal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Infiniium High-Performance Oscilloscope</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">DSOX91604A</td>
			<td>To save the light-converted electrical signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">HI1060 optical fiber</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">HI1060</td>
			<td>Optical fiber for time-stretch</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">YTTERBIUM DOPED FIBER AMPLIFIER</td>
			<td style="width:71px;">Keopsys</td>
			<td style="width:119px;">KPS-STD-BT-YFA-37-BO-SM-111-FA-FA</td>
			<td>Optical in-line amplifier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Holographic grating</td>
			<td style="width:71px;">Wasatch Photonics</td>
			<td style="width:119px;">020305-6</td>
			<td>Grating</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">Infuse/Withdraw Syringe Pumps</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td style="width:119px;">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 ...

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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

Fabricating Metamaterials Using the Fiber Drawing Method

Metamaterials are man-made composite materials, fabricated by assembling components much smaller than the wavelength at which they operate 1. They owe their electromagnetic properties to the structure of their constituents, instead of the atoms that compose them. For example, sub-wavelength metal wires can be arranged to possess an effective electric permittivity that is either positive or negative at a given frequency, in contrast to the metals themselves 2. This unprecedented control over the behaviour of light can potentially lead to a number of novel devices, such as invisibility cloaks 3, negative refractive index materials 4, and lenses that resolve objects below the diffraction limit 5. However, metamaterials operating at optical, mid-infrared and terahertz frequencies are conventionally made using nano- and micro-fabrication techniques that are expensive and produce samples that are at most a few centimetres in size 6-7. Here we present a fabrication method to produce hundreds of meters of metal wire metamaterials in fiber form, which exhibit a terahertz plasmonic response 8. We combine the stack-and-draw technique used to produce microstructured polymer optical fiber 9 with the Taylor-wire process 10, using indium wires inside polymethylmethacrylate (PMMA) tubes. PMMA is chosen because it is an easy to handle, drawable dielectric with suitable optical properties in the terahertz region; indium because it has a melting temperature of 156.6 &deg;C which is appropriate for codrawing with PMMA. We include an indium wire of 1 mm diameter and 99.99% purity in a PMMA tube with 1 mm inner diameter (ID) and 12 mm outside diameter (OD) which is sealed at one end. The tube is evacuated and drawn down to an outer diameter of 1.2 mm. The resulting fiber is then cut into smaller pieces, and stacked into a larger PMMA tube. This stack is sealed at one end and fed into a furnace while being rapidly drawn, reducing the diameter of the structure by a factor of 10, and increasing the length by a factor of 100. Such fibers possess features on the micro- and nano- scale, are inherently flexible, mass-producible, and can be woven to exhibit electromagnetic properties that are not found in nature. They represent a promising platform for a number of novel devices from terahertz to optical frequencies, such as invisible fibers, woven negative refractive index cloths, and super-resolving lenses.

Metamaterials at terahertz frequencies offer unique opportunities, but are challenging to fabricate in bulk. We adapt the fabrication procedure for microstructured polymer optical fibers to inexpensively fabricate metamaterials potentially on an industrial scale. We produce polymethylmethacrylate fibers containing ~10 &mu;m diameter indium wires separated by ~100 &mu;m, which exhibit a terahertz plasmonic response.

制造超材料使用光纤拉丝法

Metamaterials are man-made composite materials, fabricated by assembling components much smaller than the wavelength at which they operate 1. They owe ...

科研

工程学

Determining 3D Flow Fields via Multi-camera Light Field Imaging

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and observed phenomena in fluid flows. Thus, a need exists for an accessible method capable of resolving three-dimensional (3D) data sets for a range of problems. We present a novel technique for performing quantitative 3D imaging of many types of flow fields. The 3D technique enables investigation of complicated velocity fields and bubbly flows. Measurements of these types present a variety of challenges to the instrument. For instance, optically dense bubbly multiphase flows cannot be readily imaged by traditional, non-invasive flow measurement techniques due to the bubbles occluding optical access to the interior regions of the volume of interest. By using Light Field Imaging we are able to reparameterize images captured by an array of cameras to reconstruct a 3D volumetric map for every time instance, despite partial occlusions in the volume. The technique makes use of an algorithm known as synthetic aperture (SA) refocusing, whereby a 3D focal stack is generated by combining images from several cameras post-capture 1. Light Field Imaging allows for the capture of angular as well as spatial information about the light rays, and hence enables 3D scene reconstruction. Quantitative information can then be extracted from the 3D reconstructions using a variety of processing algorithms. In particular, we have developed measurement methods based on Light Field Imaging for performing 3D particle image velocimetry (PIV), extracting bubbles in a 3D field and tracking the boundary of a flickering flame. We present the fundamentals of the Light Field Imaging methodology in the context of our setup for performing 3DPIV of the airflow passing over a set of synthetic vocal folds, and show representative results from application of the technique to a bubble-entraining plunging jet.

A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.

通过多摄像头的光场成像的三维流场测定

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and ...

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

纳米粒子的光学捕获

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Atom Probe Tomography Studies on the Cu(In,Ga)Se2 Grain Boundaries

Compared with the existent techniques, atom probe tomography is a unique technique able to chemically characterize the internal interfaces at the nanoscale and in three dimensions. Indeed, APT possesses high sensitivity (in the order of ppm) and high spatial resolution (sub nm).
Considerable efforts were done here to prepare an APT tip which contains the desired grain boundary with a known structure. Indeed, site-specific sample preparation using combined focused-ion-beam, electron backscatter diffraction, and transmission electron microscopy is presented in this work. This method allows selected grain boundaries with a known structure and location in Cu(In,Ga)Se2 thin-films to be studied by atom probe tomography.
Finally, we discuss the advantages and drawbacks of using the atom probe tomography technique to study the grain boundaries in Cu(In,Ga)Se2 thin-film solar cells.

Atom Probe Tomography Studies on the CuIn,GaSe2 Grain Boundaries

In this work, we describe the use of the atom-probe tomography technique for studying the grain boundaries of the absorber layer in a CIGS solar cell. A novel approach to prepare the atom probe tips containing the desired grain boundary with a known structure is also presented here.

原子探针层析成像研究在Cu（铟，镓）硒<sub&gt; 2</sub&gt;晶界

Compared with the existent techniques, atom probe tomography is a unique technique able to chemically characterize the internal interfaces at the ...

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

分析无节幼体的运动"<em&gt;卤虫</em&gt;&#39;由电浆纳米粒子的光学跟踪

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

相干反斯托克斯拉曼散射（CARS）显微可视化药片剂溶出过程中

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

Writing Bragg Gratings in Multicore Fibers

Fiber Bragg gratings in multicore fibers can be used as compact and robust filters in astronomical and other research and commercial applications. Strong suppression at a single wavelength requires that all cores have matching transmission profiles. These gratings cannot be inscribed using the same method as for single-core fibers because the curved surface of the cladding acts as a lens, focusing the incoming UV laser beam and causing variations in exposure between cores. Therefore we use an additional optical element to ensure that the beam shape does not change while passing through the cross-section of the multicore fiber. This consists of a glass capillary tube which has been polished flat on one side, which is then placed over the section of the fiber to be inscribed. The laser beam enters the fiber through the flat surface of the capillary tube and hence maintains its original dimensions. This paper demonstrates the improvements in core-to-core uniformity for a 7-core fiber using this method. The technique can be generalized to larger multicore fibers.

We describe a technique for inscribing identical fiber Bragg gratings into each core of a multicore fiber. This is achieved by introducing an additional surface into the optical path to mitigate lensing by the curved surface of the fiber cladding.

写布拉格光栅在多核纤维

Fiber Bragg gratings in multicore fibers can be used as compact and robust filters in astronomical and other research and commercial applications. Strong ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Semi-3-Dimensional 流聚焦微流控装置产生尺寸控制的聚乙二醇二丙烯酸酯液滴

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

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）

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