The authors would like to acknowledge Dr. Matthew Heeney of Boston Children's Hospital/Dana-Farber Cancer Institute and Dr. Farzana Pashankar of Yale-New Haven Hospital for providing sickle cell patient samples. The authors would like to thank Chu H. Yu and Ashwini Joshi for their assistance in testing these samples and compiling the data.
S.T. acknowledges the American Heart Association Scientist Development Grant (15SDG25080056) and the University of Connecticut Research Excellence Program award for financial support of this research. S.K. acknowledges that this material is based upon work supported by the National Science Foundation Graduate Research Fellowship (DGE-1247393).

伦理声明:所有涉及人的血液样本程序是按照制度规定执行。所有协议进行审查和机构审查委员会批准。知情同意书是由所有参与者给出。 1.样品制备镰状细胞病诊断5,8 <ol><li>制备的Hank氏平衡盐溶液（HBSS）一个钆布醇的50mM的溶液。 </li><li>溶解于钆溶液的10mM焦亚硫酸钠。 注意:焦亚硫酸钠是吸入有毒，强烈刺激皮肤和组织。它是与水混合时的腐蚀性酸。它可能会分解时加热至高温以发射硫和钠的有毒氧化物烟雾。 </li><li> 通过获取无论指尖或静脉穿刺血液样本。 <ol><li>用采血装置用干净的一次性采血针抽血。美联社穿孔位点附近帘布层压力和擦拭使用移液管收集血液之前的三至四倍形成血液的液滴;注意不要挤压组织，因为这会导致与组织液样品的污染，"毒奶粉"的手指。 </li><li>另外，使用标准的静脉穿刺14抽血。 注意:如果样本将被储存超过几个小时，收集血液与抗凝剂如EDTA的真空采血管。 </li></ol></li><li>血少于1微升加入100微升钆 - 焦亚硫酸钠溶液中。 </li></ol> 2.样品制备WBC 7术<ol><li>无论是通过指尖或静脉穿刺获得血液样本，如在第1节。 </li><li>可选:红细胞裂解与RBC裂解液。吸管5微升的血液进入RBC裂解缓冲液500微升，在室温下进行3孵育 - 5分钟。 注意:这可以这样做，以确认白细胞的分离群体的悬浮范围。然而，这是没有必要执行此步骤，因为此处呈现的结果表明，红细胞悬浮在比白细胞明显较低位置。 </li><li>稀释全血1:1000在25mM钆在HBSS 注意:如果进行细胞裂解，稀释裂解样品9:1裂解的样品:250mM的钆以获得含有25mM的Gd的试样。 </li></ol> 3.样本使用磁悬浮平台4，5，6，7，8分析<ol><li> 启动磁悬浮装置: <ol><li>对于独立设备，插入该设备，使其电，并将其放置在平坦的水平面上。 </li><li>对于智能手机兼容的版本，推出智能手机应用，并向左滑动输入图像捕捉模式，并放置在平坦的水平面上。 </li></ol></li><li> 对于静态磁悬浮: <ol><li>通过浸渍结束到溶液和使样品通过毛细管作用填充毛细管加载制备的样品成方形玻璃微毛细管。 </li><li>慢慢推一端毛细管到材料与密封管密封结束。 </li><li>插入磁悬浮装置的磁铁之间的毛细管，使得只有1厘米的管保持可见（请参阅图3的说明，样品制备）。 注意:此步骤仍然是既智能电话兼容的和自包含设备相同。 </li><li>等待10分钟而不干扰设备或毛细管。 </li></ol></li><li> 对于流辅助磁悬浮: <ol><li>加载样品到样品管中。 </li><li>连接样品容器和英里之间的进口管crocapillary并连接微细和废物容器之间的出口管道。 </li><li>设置LED亮度和流量参数。 </li></ol></li><li>确保细胞在视野中是可见的，然后按拍摄按钮来拍摄的图像（按钮3的自包含设备和屏幕在智能手机应用底部的照相机按钮上）。 注:如果将USB用来存储图像，创建一个名为"图像"文件夹，并打开装置上之前插入USB。如果不存在驱动器，该设备将图像存储在其内部存储器和后来转移。 注:若干图像可以被捕获并分析了或缩小移动毛细管大约图像捕获之间半厘米，以减少异常的风险。 注意:如果单元中的视场范围内的数太高或太低（如检测和报告在用户界面上），移位毛细管在装置或出或准备另一山mple。 </li><li>取出样品，并根据机构或当地法规丢弃样品。毛细管应设置为尖锐。 </li></ol><img alt="图3" src="/files/ftp_upload/55012/55012fig3.jpg" /> 图3:样品制备和用户界面。 （ 一 ）将样品制备过程涉及穿刺被检者的手指，形成血液的液滴，转印血滴到样品测试溶液，搅拌该样品，并通过毛细作用装载到毛细管，样品插入到磁悬浮设备。 （b）中也显示在设备的屏幕上，这些样品制备步骤，以引导样品制备。 （ 三 ）该装置包括四个按键:一键放大到样本图像，以使用调节旋钮适当调整的重点;一个按钮，以获得一个单一的米easurement（5秒的延迟实施时间让用户插入样品）;时间推移测量（6图像以5秒的间隔拍摄）;和一个按钮，使用后关闭设备。再现时，经许可，从Yenilmez， 等。 8 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 4.图像分析4，5，6，7，8 <ol><li>运行包括为合适的样品（细胞分布或细胞类型分离）在设备上的图像分析软件。 注:对于自包含设备，自动进行分析并在图形用户界面上显示。对于智能手机兼容设备，进行分析，进入画廊ND选择所需的视频文件进行分析。 </li><li>观察并记录显示在屏幕上的分析的输出。 注:对于细胞分布分析（如镰状细胞病的诊断），输出将是细胞群体的禁闭的宽度。 注:对于细胞类型的分离（如白细胞识别），输出将与所确定的WBC种群的图像。为了计算每微升的细胞数，由2,000倍乘每图像白细胞的平均数目。例如，如果观察到5的白细胞，这将表明万白细胞/μL。在正常范围内被普遍认为是3500 - 11000白细胞/μL。 </li><li>通过或缩小约半厘米的设备的移动的样品管和捕捉另一图像如上所述待分析重复分析。 注:建议5重复分析 - 6次，每次采样，以避免错误。 </li></ol>

<ol>
	<li>Tasoglu, S., Khoory, J., Tekin, H., Thomas, C., Karnoub, A., Ghiran, I., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levitational+Image+Cytometry+with+Temporal+Resolution.">Levitational Image Cytometry with Temporal Resolution.</a> Advanced Materials. 27 (26), 3901-3908 (2015).</li><li>Tasoglu, S., Yu, C. H., Liadudanskaya, V., Guven, S., Migliaresi, C., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Levitational+Assembly+for+Living+Material+Fabrication.">Magnetic Levitational Assembly for Living Material Fabrication.</a> Advanced Healthcare Materials. 4 (10), 1469-1476 (2015).</li><li>Tasoglu, S., Yu, C. H., Gungordu, H. I., Guven, S., Vural, T., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+and+magnetic+self-assembly+of+magnetoceptive+gels.">Guided and magnetic self-assembly of magnetoceptive gels.</a> Nature Communications. 5, 4702 (2014).</li><li>Amin, R., Knowlton, S., Yenilmez, B., Hart, A., Joshi, A., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-phone+Attachable,+Flow-Assisted+Magnetic+Focusing+Device.">Smart-phone Attachable, Flow-Assisted Magnetic Focusing Device.</a> RSC Advances. 6, 93922-93931 (2016).</li><li>Knowlton, S. M., Sencan, I., Aytar, Y., Khoory, J., Heeney, M. M., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+Cell+Detection+Using+a+Smartphone.">Sickle Cell Detection Using a Smartphone.</a> Sci Rep. 5, 15022 (2015).</li><li>Knowlton, S., Yu, C. H., Jain, N., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-Phone+Based+Magnetic+Levitation+for+Measuring+Densities.">Smart-Phone Based Magnetic Levitation for Measuring Densities.</a> PLoS One. 10 (8), e0134400 (2015).</li><li>Yenilmez, B., Knowlton, S., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Contained+Handheld+Magnetic+Platform+for+Point+of+Care+Cytometry+in+Biological+Samples+.">Self-Contained Handheld Magnetic Platform for Point of Care Cytometry in Biological Samples .</a> Advanced Materials Technologies. 1, 1600144 (2016).</li><li>Yenilmez, B., Knowlton, S., Yu, C. H., Heeney, M., Tasoglu, S. <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+Sickle+Cell+Disease+Diagnosis+Using+a+Low-Cost,+Handheld+Platform.">Label-Free Sickle Cell Disease Diagnosis Using a Low-Cost, Handheld Platform.</a> Adv Mat Tech. 1 (5), 1600100 (2016).</li><li>Bender, M. A., Douthitt Seibel, G., Pagon, R. A., 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=.">.</a> GeneReviews. , (1993).</li><li>Kaul, D. K., Fabry, M. E., Windisch, P., Baez, S., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytes+in+sickle+cell+anemia+are+heterogeneous+in+their+rheological+and+hemodynamic+characteristics.">Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics.</a> J Clin Invest. 72 (1), 22-31 (1983).</li><li>Joiner, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardos+pathway+to+sickle+cell+therapies?.">Gardos pathway to sickle cell therapies?.</a> Blood. 111 (8), 3918-3919 (2008).</li><li>Finch, J. T., Perutz, M. F., Bertles, J. F., D&#246;bler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Sickled+Erythrocytes+and+of+Sickle-Cell+Hemoglobin+Fibers.">Structure of Sickled Erythrocytes and of Sickle-Cell Hemoglobin Fibers.</a> Proc Natl Acad Sci. 70 (3), 718-722 (1973).</li><li>Lew, V. L., Etzion, Z., Bookchin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehydration+response+of+sickle+cells+to+sickling-induced+Ca(++)+permeabilization.">Dehydration response of sickle cells to sickling-induced Ca(++) permeabilization.</a> Blood. 99 (7), 2578-2585 (2002).</li><li>Ernst, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NCCLS Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture: Approved Standard-Sixth Addition. 27 (26), (2007).</li></ol>

Authors Stephanie Knowlton and Savas Tasoglu are founders of and have an equity interest in mBiotics, a startup working to commercialize the magnetic levitation platform described herein for point of care diagnostic solutions. A provisional patent entitled "Magnetic Levitation with On-Board Optical Imaging and Image Analysis for Density-Based Separation, Identification, And Measurement" has been filed with UCONN Case No. 16-027 on the technology described here.

该议定书中的关键步骤 在此过程中的关键因素包括磁体的正确对准。如果磁体成为脱落或在装置内分离超过正常值，这可能会影响结果。为了控制这种故障或其他人在这个过程中，一个密度控制的颗粒，如聚苯乙烯微球，可周期性用于控制随时间的变化。另外，悬浮时间是很重要的，以使细胞以达到平衡。对于红细胞，10分钟是足以允许所有的细胞以达到平衡。然而，要注意的是更小的颗粒或细胞，可能需要较长的时间以达到平衡是很重要的。这可以通过在一个5秒的间隔拍摄时间推移图像和绘图随时间的限制宽度进行评估;平衡可以被确定为在其中禁闭宽度的变化是可以忽略的地步。 在协议中其他关键步骤包括预在精确的浓度，因为这钆液paration极大地影响样品的悬浮高度。这可以提前完成，从原液使用，但必须适当地密封，以避免该溶液中，在浓度不期望的增加的蒸发。此外，必须小心，以保持所用的细胞的健康状态。对于通过手指针刺抽取的人血液，应该抽血的一小时内被使用，不允许通过在密封容器中储存干燥。通过静脉穿刺抽取的人血液，样品应储存在4℃下不超过一个星期抗凝剂（乙二胺四乙酸（EDTA），一个共同的抗凝血剂，在这里被使用）。对于贴壁细胞系，死细胞应彻底从以胰蛋白酶和细胞应培养，直到使用前文化清洗。因为细胞健康已知会影响密度，因此悬浮唤起注意的细胞在悬浮的时间的健康至关重要吨。 修改和故障排除 我们已经证明两种不同密度的细胞在磁场可预测的位置的分离和禁闭。为了该技术扩展到其它的应用中，顺磁介质的不同制剂可被用于获得检测所需的范围替代应用3。钆的浓度支配检测的分辨率以及范围（请参考图6a）。因为钆更高浓度增加施加到细胞的磁力的强度，在悬浮液钆的浓度越大，越小的悬浮高度之差将是在细胞密度的任何差异。而这限制了分辨率，定义为在细胞密度小的差异来区分的能力，它增加了可分析的密度的范围ð。同样地，减小钆的浓度将增加分辨率而降低检测的范围内。此外，介质的密度可以改变，以向上或向下移动的检测的极限。控制悬浮高度的第二力是浮力，这取决于相比，悬浮介质的细胞的相对密度。这里，水性悬浮液时，这意味着细胞与密度是相同，在两个磁体具有较少致密的或致密的细胞分别漂浮高于或低于中心线之间的中线的平台漂浮的（与如先前所描述的范围内通过磁力控制）。因为浮力是依赖于相对密度，增加了介质的密度将检测的范围转移到一个较高范围的细胞密度。同样地，使用较低的密度介质将转向检测朝向较低范围的细胞密度的范围。&lt;<html> / P&gt; 我们还通过量化穿过毛细管宽度归一化的颗粒数（ 即颗粒的概率在整个毛细管宽度的距离，以流）研究流动辅助磁聚焦装置的性能。较低的流速有颗粒的更多限制，但是导致较低的体积可以通过，它可以是对于高体积样品在罕见物体检测的缺点。然而，这可以通过多遍在磁场中或通过较长的磁铁处理。多种细胞类型之间的分离，也可以在今后的研究利用通过在磁场的端部利用微流体分离器以不同密度的颗粒分离。 当分离多种细胞类型，它可能是有帮助的，以便建立的平均高度和之前漂浮的同质种群中每个细胞类型的范围单独地悬浮每个种群。 e_content"&gt;技术的局限性 这里介绍的方法被限制为不同密度的颗粒的分离。为了实现多种细胞类型的可靠识别，但重要的是它们具有不重叠的离散密度范围。此外，它可以被检测的颗粒在尺寸上受到限制。他们必须能够在微毛细管内自由移动 - 200微米是粒径推荐上限。另外，粒子必须足够大，也可以清楚地成像 - 5微米是在直径推荐下限。 关于到现有/替代方法的技术意义 这种方法的细胞分析很简单，让现场用户友好的分析。许多医疗诊断程序必须在临床检测实验室中进行，需要专门的检测设备和由受过训练的实验室的特别执行程序吨。然而，该协议需要一个简单的装置是对医疗诊所更容易获得。样品制备是简单和分析是自动的，最大程度地减少用户错误的风险。 此装置将实现快速，现场测试用于各种医疗条件。该设备是用户友好的，无标签，便携，使其成为理想的点保健疾病诊断。相比于远程临床实验室检测的现行标准，这种做法将使医生能够迅速做出关于病人护理明智的决策，从而防止因延误护理并发症。该平台的设计具有方便和廉价的元件，从而在临床上广泛设置使用这种方法，让发展中国家和改善全球无障碍的医疗服务。 掌握这一技术后，未来的应用方向或 需要注意的是它是重要的此处所描述的测试尚未验证上的大规模的患者群。迄今为止，镰状细胞疾病的诊断已证实在一个小的患者队列2,5和WBC术已被证明是一个概念证明。与这些应用程序和那些在未来开发的临床试验中，必须以验证临床使用之前这种方法来进行，但在这里给出的结果表明，对于最终使用这种技术和技术在点 - - 关心临床诊断的承诺。 使用这种方法对于基于密度的术分析可能最终被扩展到其他疾病的诊断应用程序。单细胞如这里所描述的疾病的诊断的磁悬浮的这种方法需要使用的患者的细胞的单细胞悬液，并可以使用当前系统以其限制地成像在分辨率由于使用细胞的智能手机摄像头或低成本光学。此外，这种技术可应用于其中满足下列条件之一的疾病:当他们携带的疾病与健康对照相比的兴趣（ⅰ）细胞必须达到改变的密度，（ii）细胞密度的变化必须通过加入诱导的试剂（或任何可用的替代治疗），或（iii）诊断必须涉及单个样品它或者固有地或通过一些治疗具有独特的密度在识别不同的细胞类型。使用同一平台设备对其他疾病的未来应用可能对全球健康产生了巨大影响。这些可以包括生物组分，这是由密度区分的检测。某些细胞类型，细胞正在经历细胞死亡，和患病细胞都被证明具有独特密度签名，因此不同的磁性图案，因此可以量化并使用此平台分离。在非常低的数字的单元格也可以be。通过利用在流动辅助磁悬浮装置的流体流动检测。

这里，我们提出了一个技术平台，它采用加上自动成像和分析磁悬浮来分析患者的细胞的密度分布作为疾病指示器的技术。对于基于密度的术分析这种通用方法最终可应用于一系列疾病的诊断。但是，为了能与床边血糖检测兼容，而在发展中国家使用，该技术必须满足低成本，便携，和可用性要求。该设备和消耗品必须以低成本容易地获得。样品制备必须简单，分析应与用户输入或解释的最低要求是自动的，并且结果应该可以快速恢复。此外，该装置必须是紧凑和便携是在临床设置，以及发展中国家是有用的。因此，我们已开发了一种装置和方法，在点的护理兼容TECHNOL使用磁悬浮术通过耦合自动成像和图像分析来对病人的细胞群的密度分布返回结果。 点护理技术提供了显着的优势超过目前临床实验室测试程序。目前可用的技术是太昂贵的由临床医生所拥有或过于复杂，由医务人员来进行。许多这些步骤需要必须由受过训练的技术人员才能进行劳动密集型的协议。由于这些原因，患者样品如血液或尿液中的医生的办公室，然后转移到一个远程的，集中的测试实验室进行的临床试验，这可能需要几天的时间医师收到测试的结果通常收集。这可能会导致在治疗在某些情况下的过程的延误或并发症，使该试验很昂贵和低效率的（导致保险付款人财政负担），并进一步使许多诊断在低资源设置和发展中国家无法访问。 这里，我们提出加上两者具有嵌入摄像处理（ 图1）和智能电话兼容设备（ 图2）的设备自动成像和分析一个磁悬浮技术。这些磁性基于悬浮装置表示具有潜在一个广泛适用的平台技术被应用到一个范围的不同的医疗诊断应用。磁力的浮力1，2，3:基于两种力量之间的平衡磁悬浮方法的功能。当颗粒悬浮在顺磁介质，并插入到由两个磁体彼此面对，磁力作用在朝向两MAGN之间的中心线的方向上的粒子像磁极产生的磁场<html> ETS。浮力是由比悬浮介质的粒子的相对密度引起的是向上的颗粒比中等密集和向下中的颗粒比周围介质致密的情况下少的情况下。基于这两种力量，颗粒将达到它平衡这两股力量领域的磁悬浮平衡位置;该位置是直接关系到颗粒的密度，以更密集的颗粒小于致密颗粒漂浮在外地较低。成像模块，无论是一个内置的智能手机摄像装置4，5，6或配有放大镜7,8独立的光学元件，被用于可视化粒子的位置。图像处理，或者通过智能手机应用4,5="外部参照"&gt; 6或嵌入处理部7，8，然后处理所捕获的图像，以量化的空间分布，因此，人口密度分布。为了分析大样品（如那些具有每毫升兴趣只有几个颗粒，流动可以直接集成到这种颗粒悬浮在设备和当它们通过所述成像区域（ 图2）进行分析。 <img alt="图1" src="/files/ftp_upload/55012/55012fig1.jpg" /> 图1:自含磁悬浮平台。 （ 一 ）紧凑磁悬浮装置包括磁聚焦组件，成像组件（发光二极管（LED），光学透镜，和一个照相机检测器），和一个处理单元与显示屏幕。 （b）该CRO磁场强度其中，被插入所述样本磁铁之间的区域的β-部分。场强最大的在磁体的表面上，并在它们之间的中心线接近零。 （ 三 ）的粒子，如细胞，磁场经验几个部队内:一个磁力（F M）朝向磁性之间的中心线，与大小不同而不同基于所述粒子的位置;一重力（F G&#39;），它依赖于相对于该悬浮介质的粒子的密度，和一拖力（F D）抵抗粒子运动。再现时，经许可，从Yenilmez， 等。 8 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55012/55012fig2.jpg" /> 图URE 2:智能手机兼容的流辅助磁悬浮平台。 （ 一 - 三 ）前面（a）中 ，一侧（b）和背面（ 三 ）次磁悬浮装置（d）该装置的组成部分包括:1）磁悬浮模块，包括永久磁铁，放大透镜，和LED和光扩散，2）智能手机的情况下，3）电子产品，包括微控制器，泵驱动器和蓝牙接收器，4）微泵座，5）调节孔口，6）废液管夹，7）电池座，8 ）样品架，9）两用底座和盖子。 （ 五 ）流动示意图，示出了通过磁场的样品的泵送。 （ 六 ）磁悬浮模块，显示如何不同密度的颗粒将对齐，因为它们是通过现场泵的横截面;密度较小的颗粒，如颗粒1，将平衡在较高的悬浮高度t汉致密颗粒，如颗粒2转载，经许可，从阿明， 等。 1 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig2large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 使用任何样品的在该系统中的最低要求的密度分布分析包括以获得悬浮细胞或颗粒大于约5微米，小于约250微米的尺寸（用于成像和图像处理）及其与相容性的能力在一个顺磁性溶液中的溶液混合，如这里使用的钆布醇。用于疾病诊断，兼容的应用包括那些，其中（i）所关注的细胞固有地具有改变的密度相比健康对照，当他们携带的疾病，（ii）一种密度变化所用的细胞通过加入试剂或部分被诱导替代治疗一个短cubation时间，或（iii）不同类型的细胞在单个样品中的被识别和固有（或通过某些治疗）具有独特特征的密度。 镰状细胞病是一种遗传性疾病引起的血红蛋白，HbS的，在一个人的红血细胞（红细胞）要生产的突变形式，其可以导致间歇性血管闭塞事件和慢性溶血性贫血9。它是使用任一血红蛋白等电聚焦，高效液相色谱（HPLC）分离，或血红蛋白电泳是高度准确的，但必须在临床测试实验室，因为它们是与点 - - 关心设置不兼容进行诊断。溶解性和纸基测试镰状细胞病已经被提出，但一般需要用户的主观解释和确证试验。在这里，我们使用了基于密度的方式来确定镰刀红细胞，从而达到比RB更高的密度CS由人没有镰状细胞病。该机制涉及血红蛋白，HBS的突变形式，这将导致红细胞脱水的镰状细胞病的RBCs脱氧条件10，11，12，13下的聚合。 这种基于密度的方法还可以应用到密度的基础上分开的不同类型的细胞:白血细胞（白细胞）和红细胞7。白细胞通常负责在体内抵抗感染。白细胞术可用于量化这些细胞在血液中的数量，并作为一个有用的诊断工具。白细胞计数高于正常（一般认为每微升更大的超过11,000细胞）可能表明感染，免疫系统疾病，或白血病。白细胞计数低于在正常范围（约3500每μL细胞）可能由自身免疫性疾病或康迪特引起离子的损害骨髓。不像替代技术，这里提出的方法不以鉴定白细胞依赖于红细胞或污渍的裂解。这种基于细胞的试验利用了两种细胞类型的独特固有密度进行分离，作为WBC种群密度已经报道为比如先前使用密度梯度离心2,3计算出的RBC群体的低。 相比在远程位置变质测试，这个测试是快速，简单的样品制备（ 图3），在该装置的分离单元10内- 15分钟，自动成像和分析需要不到1分钟。以这种方式，该装置可迅速地返回结果以更好地告知医疗决定，允许处理被立即施用，以减轻身体和心理痛苦，并减少并发症associ的风险ated与医疗保健的延迟。可以在现场或在临床环境，由于简单的样品制备和自动成像和分析返回以最小的用户输入或解释的结果来执行该技术。因为使用的使用试样分析永久磁铁和使用任一智慧型手机或用于成像和图像处理简单的电子部件的一个简单的方法，相对于一些复杂的测试程序的装置，以及每次测试成本是最小的。

<table border="0" cellpadding="0" cellspacing="0" width="938">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Gadavist (Bayer)</td>
			<td style="width:257px;">Jefferson Medical and Imaging</td>
			<td style="width:119px;">2068062</td>
			<td style="width:241px;">Gadavist contains 1M gadobutrol, a chelate of gadolinium. We purchased 2 mL vials with 15/ca.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Square glass microcapillary tubes</td>
			<td style="width:257px;">Vitrocom</td>
			<td style="width:119px;">8270</td>
			<td>50 mm length is sufficient</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Sodium metabisulfite</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9000</td>
			<td>Chemical formula: Na2S2O5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Leica Microsystems Critoseal tube sealant</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9269&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Trypsin-EDTA</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td>Or other reagent as recommended for the cell type used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">MICROLET 2 Adjustable Lancing Device</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">246567</td>
			<td>Any lancing device is acceptable when used according to biosafety protocols</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microlet Lancets</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">667474</td>
			<td>Must be dispoable and not reused</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hausser Bright-Line Phase Hemacytometer</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td>Or any preferred method for cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">ACK Lysing Buffer</td>
			<td style="width:257px;">ThermoFisher</td>
			<td style="width:119px;">A1049201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic levitation coupled with portable imaging and analysis for disease diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

为细胞密度分布分析，这是用于镰状细胞疾病的诊断的技术中，其目的是确定细胞群的分布的宽度。患者血细胞无镰状细胞病将在可预见的宽度内被限制。从患者的镰状细胞疾病的细胞将在整个较宽的区域分布，与小区分配一个向下偏斜（参见图4）对于任何特定的应用中，一个阈值可以控制样品的分布宽度与该健康之间设定样品为"健康的"和"积极的疾病"5,8之间的截止。 <img alt="图4" src="/files/ftp_upload/55012/55012fig4.jpg" /> 图4: 磁悬浮到DENSIT分析实例Ÿ分布在血液样本镰状细胞病的指标。在左边，红血细胞是公一个狭窄的区域内密闭。在右边，红血细胞的子集实现更大的密度，因此，较低的悬浮高度，向下偏斜的分布和增加限制的宽度。比例尺= 200微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 分析样品的密度分布，计算算法在设备内实现。首先，沿垂直和水平轴的像素亮度梯度计算。磁铁边缘和毛细管边缘被检测为在垂直像素梯度轮廓的峰。的内毛细管边缘之间的距离，以像素为单位，已知为0.7毫米，因此被用作斯卡利纳克因素的距离从像素转换成毫米。沿着水平轴的像素亮度梯度是最大的，其中细胞的位置。此梯度分布进行了分析和拟合高斯曲线。该曲线的值的4倍的标准偏差被报告为约束宽度。 结果在图5显示了控制和镰状细胞病样坐月子宽度。这里，具有更大的限制宽度（超过50微米）的样品将被认为是镰状细胞病阳性和那些低于阈将被认为是负面的疾病。应当指出的是，镰状细胞分布分析的其它方法进行了调查和由Yenilmez报道， 等。 8 <img alt="图5" src="/files/ftp_upload/55012/55012fig5.jpg" /> 数字5:坐月子宽度镰状细胞病诊断的量化。实验结果为对照组（n = 48的图像超过400例）和镰状细胞病（N = 93的图像超过10例）红细胞禁闭宽度。结果根据曼-惠特尼秩双侧检验（正常近似值，N 1 = 3，N 2 = 10，Z = -2.6764，P = 0.0074），有统计学显著。晶须表示从样品中的最小和最大限制宽度测试和星号代表异常值。再现时，经许可，从Yenilmez， 等。 8 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig5large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 用于颗粒分离，其可用于识别血液样品中的白细胞，其目的是确定两个不同波波尔ations。如果种群具有不同的密度，它们将被在不同的区域中的视场观察。因此，具有不同密度的两种或更多种颗粒的均一群体可悬浮，一经分离，所述多个群可以在图像中可以观察到，并使用图像分析算法4检测（ 见图6）。 分析两种不同类型的细胞的分离，一个算法被实现其中在平衡状态下区分两个分开的人口。以类似的方法，使对镰状细胞疾病的分析说明，两个高斯分布是配合到样本，而不是单一的曲线。在像素的亮度梯度的每个峰代表不同的细胞群。高斯曲线拟合到这些数据得到两个平均悬浮高度（相对于底磁铁的位置），为高斯曲线的平均值和禁闭宽度为曲线7的标准偏差。 <img alt="图6" src="/files/ftp_upload/55012/55012fig6.jpg" /> 图6:微粒与密度鲜明混居的磁悬浮的例子。 （ 一 ）的校正曲线，在五个不同的Gd浓度悬浮高度范围从12.5至200mM微球体密度相关。斜率是最大的钆的浓度最低，从而提供更大的分辨率（ 即小的密度差异的敏感性）。斜率对于较高浓度的Gd，表明检测的增加范围，但以较低的分辨率是最低的。 （b）与在两分钟的过程中两种不同密度的均匀样本微球体的时间相关的分离。在平衡时（右），两个不同的频带被图像肛门检测ysis算法。再现时，经许可，从Yenilmez， 等。 7 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig6large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 为了识别各个细胞类型的任何给定的应用程序不同的密度，最好是在一个时间进行量化的预期悬浮高度的第一悬浮一种细胞类型。 图7a示出了从其中红细胞已被溶解的血样的白细胞的悬浮高度。这定义作进一步的分析白细胞约束的区域。结果表明，红细胞悬浮比白细胞，因此，白细胞可以基于悬浮位置血样来区分低。视任何领域内的体积为0.5μL。在其中稀释1样品:1000，白细胞的数量/μl可以通过计数来计算在视和乘法场白细胞由2,000 7倍的数量。 <img alt="图7" src="/files/ftp_upload/55012/55012fig7.jpg" /> 图7:WBC 流式细胞术全血。 （ 一 ）以下RBC裂解从血液白细胞悬浮。这定义了白细胞在在25mM钆磁场悬浮的范围内。 （ 二 ）白细胞计数的示例（白细胞标记为蓝色箭头）。顶框镶嵌显示白细胞和底框和底框镶嵌显示RBC群体。再现时，经许可，从Yenilmez， 等。 7 <a href="http://ecsource.jove.com/files/ftp_upload/55012/55012fig7large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics at JoVE.com

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

磁悬浮加上便携式成像与分析诊断疾病

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

magnetic-levitation-coupled-with-portable-imaging-analysis-for

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Mechanical Engineering

Bioengineering

Unit 1

Equilibrium of a Particle

SCIENTISTS IN ACTION

8.7K 次观看.  University of Connecticut. We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

视频: Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

科研

生物工程

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

用原位滞后测量进行磁畴成像的优化设置和协议

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

纳米等离子体增强散射和低放大显微镜成像对肿瘤衍生外质体的定量

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

测量超极化 [1-13C]硫化的旋转-莱迪兹松弛磁场依赖性

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

磁悬浮加上便携式成像与分析诊断疾病

摘要

探索更多视频

此视频中的章节