A.奥兹坎衷心感谢支持总统早期职业科学家和工程师奖（PECASE），美国陆军研究办公室（ARO）青年研究者奖，美国国家科学基金会（NSF）CAREER奖，美国海军研究办公室（ONR）青年研究者奖美国国立卫生研究院（NIH）主任的新的的创新奖DP2OD006427从办公室的主任，美国国立卫生研究院。

在本节中，我们介绍了实验方案，为我们的手机为基础的宽视场荧光显微镜13日和光电流体成像仪平台14。我们将使用荧光微球和荧光标记的白血细胞，以测试这些影像平台。 A.制备手机的宽视场荧光显微镜及光电流体成像流式细胞仪手机为基础的宽视场荧光显微镜或流式细胞仪由两个主要部分组成：摄像头手机和紧凑的光流体添加附件。 1。在拍照手机虽然技术是适用于几乎所有的拍照手机，我们选择了索尼埃里克森艾诺作为这些设备的基础。这种手机有一个〜800万像素的RGB CMOS传感器，它安装在一个内置镜头的焦距（1）〜4.65毫米。 </p&gt; 2。宽视场荧光显微镜的光流体附件光学附件设计的Autodesk和打印的尺寸精英3-D打印机使用ABSplus热塑性材料。在该印刷过程中，模型和载体材料在打印机内的挤压头被加热，并建模基料层上的沉积层。当这一步完成后，辅助材料可被溶解，留下一个强大的3D模型所需的原型，我们的光学附件的设计包括发光二极体（中心波长在〜470 nm，DIGIKEY），塑料过滤器（NT54-46，埃德蒙光学元件），一个样品盘，和一个平凸的透镜 f 2 = 15毫米（＃NT45-302，爱特蒙特光学）。荧光光谱的基础上，所有的LED和塑料过滤器可以很容易地改变。该光电流体附件组装的步骤包括： <ol><li>到安装在其特定的透镜支架的位置，将镜头。 </li><li>将塑料过滤器的过滤器托盘上，将其滑入附件或磁带的塑料滤光器在手机相机镜头前的。 </li><li>将LED托盘附件。 </li><li>将样品置于载玻片到样品盘。将样品盘到附件。面对朝向样品的LED。 </li><li>剪辑附件到蜂窝电话，例如，额外的透镜是直接在触摸与手机相机透镜。 </li><li>使用附件上的开关打开的LED。 </li><li>图片样品的兴趣使用手机的摄像头单元的“夜间模式”。 </li></ol> 3。荧光成像流式细胞仪光电流体附件当有需要筛选大量罕见的事件的检测液体样品，光流体的流式细胞仪的移动设备可以被优选的。我们可以修改我们的宽视场荧光显微镜的设计，并把它转换成流式细胞仪，W基于的PDMS的微流体通道在这里是用于通过成像容积不断提供的液体试样。光学附件的设计也由Autodesk和精英的3-D打印机打印的尺寸。它还包括发光二极体（DIGIKEY在〜470 nm，中心波长），塑料过滤器（＃，爱特蒙特光学NT54-46），一个样品盘，和一个非球面透镜（= 4.5毫米）（产品＃C230TME-甲; Thorlab）。该光电流体附件组装的步骤包括： <ol><li>的非球面透镜放置到该附件。 </li><li>将塑料过滤器的过滤器托盘上，将其滑入附件或磁带的塑料滤光器的手机相机镜头前。 </li><li>将光电流体连接到同一个微流体通道。 </li><li>剪辑附件到蜂窝电话，使得额外的透镜是直接在触摸与手机相机透镜。 </li><li>使用附件上的开关打开的LED。 </li><li>将微流体集成电路通道的注射器泵，在以恒定的流速，并提供到微流体装置中的液体试样。 </li><li>捕获电影的荧光细胞/颗粒流经微流体通道，使用手机的摄像头的视频模式。 </li></ol> B.样品制备 4。的荧光微粒样品的制备<ol><li>荧光珠用10微米直径的（红色小珠：＃F8834激发/发射580nm/605nm产品;绿色小珠：产品＃F8836：激发/发射505nm/515nm）购自Invitrogen（卡尔斯巴德，加利福尼亚州）。 </li><li>混合绿色荧光珠，与40μlDI水的红色荧光珠的10微升10微升。 </li><li>此珠的混合物在载玻片上，用微量吸移管，并把它的顶部上，以使一个三明治结构的另一个载玻片place10微升。 </li><li>将这种三明治结构样品盘和滑动到手机附件。 </li&gt; </ol> 5。制备荧光标记的白血细胞<ol><li>就拿SYTO16核酸的荧光标记试剂盒（＃S7578，生命技术）和磷酸盐缓冲盐水（PBS），从冰箱，并为他们带来至室温。 </li><li>从EDTA血液收集管200微升的全血样品转移到1.5毫升聚苯乙烯试管中（＃05-408-129，Fisher Scientific）的。 </li><li>加入1毫升的红血细胞裂解缓冲液（＃R7757，Sigma-Aldrich公司），以200μL全血样品和调匀。 </li><li> 5分钟后，离心分离溶解的血液样品，并除去上清液的溶液。 </li><li>的白血细胞沉淀重悬到200μl的PBS缓冲液中，并轻轻混合。 </li><li>加入5μl1mM的SYTO16溶液的白血细胞样品。包裹样品与铝箔和孵育〜30分钟在黑暗的环境。 </li><li>再次离心样品。移去上层清液和标记的白血细胞沉淀重新悬浮到PBS缓冲液中。 </li><li>将5-10微升标记的白血细胞的液体试样的盖滑移，样品的顶部放置一个第二盖滑移。 </li><li>夹持的样品玻片插入到样品托盘和图像使用蜂窝电话的荧光显微镜。 </li></ol>另外<ol start="10"><li>不断提供的荧光标记的白血细胞通过微流体通道，使用一个自动注射器泵，同时也捕获在视频模式中使用的蜂窝电话的摄像头的流动细胞的荧光显微镜的电影。我们还应该强调的是，可利用的便携式电池供电的注射器泵甚至重力驱动流过微流体通道。 </li></ol>

<ol>
	<li>Sklar, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow Cytometry for BioTechnology. , (2005).</li><li>Nunez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow cytometry for research scientists: principle and applications. , (2001).</li><li>Mertz, 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> Introduction to optical microscopy. , (2010).</li><li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+deeper+than+microscopy:+the+optical+imaging+frontier+in+biology.">Going deeper than microscopy: the optical imaging frontier in biology.</a> Nature Methods. 7, 603-614 (2010).</li><li>Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+fluorescence+nanoscopy.">Toward fluorescence nanoscopy.</a> Nature Biotechnology. 21, 1347-1355 (2003).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proceedings of the National Academy of Science U.S.A. 102, 13081-13086 (2005).</li><li>Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., Hess, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM.">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM.</a> Nature Methods. 3, 793-796 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophysical Journal. 91, 4258-4272 (2006).</li><li>Ma, Z., Gerton, J. M., Wade, L. A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+near-field+microscopy+of+DNA+at+sub-10+nm+resolution.">Fluorescence near-field microscopy of DNA at sub-10 nm resolution.</a> Physical Review Letters. 97, 260801 (2006).</li><li>Chung, E., Kim, D., Cui, Y., Kim, Y., So, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+standing+wave+total+internal+reflection+fluorescence+microscopy:+superresolution+imaging+of+single+molecular+and+biological+specimens.">Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens.</a> Biophysical Journal. 93, 1747-1757 (2007).</li><li>Greenbaum, A., Luo, W., Su, T. -. W., G&#246;r&#246;cs, Z., Xue, L., Isikman, S. O., Coskun, A. F., Mudanyali, O., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+without+lenses:+achievements+and+remaining+challenges+of+wide-field+on-chip+microscopy.">Imaging without lenses: achievements and remaining challenges of wide-field on-chip microscopy.</a> Nature Methods. 9, 889-895 (2012).</li><li>Zhu, H., Yaglidere, O., Su, T. -. W., Tseng, D., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+and+compact+wide-field+fluorescent+imaging+on+a+cell-phone.">Cost-effective and compact wide-field fluorescent imaging on a cell-phone.</a> Lab on a Chip. 11 (2), 315-322 (2011).</li><li>Zhu, H., Mavandadi, S., Coskun, A. F., Yaglidere, O., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+fluorescent+imaging+cytometry+on+a+cell+phone.">Optofluidic fluorescent imaging cytometry on a cell phone.</a> Analytical Chemistry. 83, 6641-6647 (2011).</li><li>Suzuki, S., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+Visualand+Graphics.">Computer Visualand Graphics.</a> Image Processing. 30, 32-46 (1985).</li><li>Zhu, H., Sikora, U., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot+enabled+detection+of+Escherichia+coli+using+a+cell-phone.">Quantum dot enabled detection of Escherichia coli using a cell-phone.</a> Analyst. 137, 2541-2544 (2012).</li><li>Mudanyali, O., Dimitrov, S., Sikora, U., Padmanabhan, S., Navruz, I., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Rapid-Diagnostic-Test+Reader+Platform+on+a+Cellphone.">Integrated Rapid-Diagnostic-Test Reader Platform on a Cellphone.</a> Lab on a Chip. 12 (15), (2012).</li><li>Candes, E. J., Romberg, J. K., Tao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+signal+recovery+from+incomplete+and+inaccurate+measurements.">Stable signal recovery from incomplete and inaccurate measurements.</a> Communication of Pure and Applied Mathematics. 59, 1207-1223 (2006).</li></ol>

奥兹坎博士是一个初创公司的创始人，其目的是商业化计算和显微镜成像工具。

我们已经提出了我们的最近的结果基于宽视场荧光显微镜及光电流体成像流式细胞仪使用重量轻和紧凑的光电流体附件的手机相机手机。利用这个平台技术，包括微颗粒和全血样品中标记的白血细胞成像荧光物体。因此，此结构紧凑，成本效益的手机基于荧光成像工具包可能是有用的点 - 对 - 护理诊断，尤其是在资源有限的地区为打击各种全球性健康问题，如HIV +病人监测他们的CD4 +Ť淋巴细胞计?...

显微镜和流式细胞仪技术被广泛应用于1-12在生物医学和科研以及临床诊断为不同类型的细胞计数和表征。然而，常规的显微镜和流式细胞仪检测仪器是相对复杂和昂贵的，这限制了它们的使用，主要是行之有效的中央实验室。最近，我们已经开发出一款轻盈，小巧的荧光成像仪和显微镜设备集成在一个手机，13,14这表明承诺，符合成本效益的翻译荧光显微镜，流式细胞仪和相关的微分析技术，资源有限的环境各种远程医疗应用，影响了全球的健康。 在的光流体流式细胞仪的配置（ 例如，参见图1C和1D），定制设计的基于聚二甲基硅氧烷（PDMS）的微流体通道是positioned的前面的手机相机单元，其中发光二极管（LED）是对接耦合的通道的边缘。这种微流体芯片，与里面的液体试样一起，形成一个光电流体的平面波导（例如，PDMS-液体-PDMS组成的），使得激发光被引导，均匀地泵的微通道内的荧光标记标本。从这些标记的对象， 例如 ，细胞的荧光发射，进一步正确放置后，手机相机单元通过一个附加的透镜成像的，被映射到蜂窝电话的互补金属-氧化物-半导体（CMOS）图像传感器。由于收集荧光发射垂直于到激励光路径，廉价的塑料吸收滤波器是足以除去散射的激励光，并能提供一个体面的暗视场的背景荧光成像所需。使用类似的光电流体设计，我们也可以图像的荧光物体在stat集成电路模式（参见图1A和图1B），在该荧光体颗粒被夹在两片玻璃之间的滑动，而不是从这些荧光发色粒子的微流体通道和荧光发射流经由蜂窝电话的CMOS图像传感器，用于颗粒计数和捕获表征。根据不同的应用需求，流式细胞仪和宽视场荧光显微镜可以选择。例如，手机流式细胞仪的移动设备可以是用于筛选大量的稀有细胞或病原体的检测液体样品（ 例如几毫升）特别有用。 在这个手稿中，我们将回顾一些我们最近的研究结果荧光显微镜及成像流式细胞仪检测工具，一个手机上使用紧凑和具成本效益的光流体附件的整合。这些基于手机的微分析，成像仪和传感平台可以提供各种机会，远程医疗和护理点的诊断，尤其在资源有限的世界各地区对全球卫生挑战的斗争。

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

荧光显微镜和流式细胞仪被广泛应用于生物医学研究和临床诊断的工具。然而，这些设备一般比较笨重和昂贵，使得他们在资源有限的环境。为了解决这些潜在的限制，我们最近展示了集成的宽视场荧光显微镜及成像流式细胞仪检测工具，蜂窝电话用小型，轻重量和成本效益的光流体附件。在我们的流式细胞仪的设计中，荧光标记的细胞通过微流体通道被定位高于现有的手机相机单元被刷新。电池供电的发光二极管（LED）是对接耦合到该微流体芯片的一侧，它有效地作为一种多模式平板波导，其中的激发光被引导至均匀地激发荧光靶。手机的摄像头记录时间的推移电影的荧光细胞流经的微流体通道，在那里该数字这部电影的帧处理的标记的细胞的数量进行计数感兴趣目标溶液内。使用类似的光流体设计，我们也可以形象这些荧光标记的细胞在静态模式下， 例如夹在两片玻璃幻灯片的荧光颗粒和荧光图像捕捉他们使用手机的摄像头，可以实现空间分辨率，如 〜一个非常大的字段的图〜81毫米2的 10微米以上。该手机为基础的荧光成像流式细胞仪和显微镜平台可能是有用的，尤其是在资源有限的环境中， 例如点算的CD4 + T细胞向HIV（+）患者的监测或检测饮用水中的水传播的寄生虫。

我们的光流体泵/激励计划（ 图1C和1D），荧光标记的细胞可以连续使用注射器泵输送到微流体通道，同时手机的摄像头记录随时间推移的流动细胞的荧光显微镜电影。然后，可以快速分析这些荧光电影使用轮廓检测和跟踪算法14,15自动确定的绝对数量和通过微流体通道中流动的细胞密度，同时荧光摄像功能的流式细胞仪。基于上述描述的平台（ 图1C和...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

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

荧光显微镜及成像流式细胞仪检测工具，一个手机上使用紧凑和具成本效益的光流体附件的整合，我们回顾我们最近的研究结果。这些手机的微观分析设备可能是有益的，如流式细胞仪分析，以及高通量筛选，例如，水样在资源有限的环境中执行各种细胞计数任务。

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 ...

wide-field-fluorescent-microscopy-fluorescent-imaging-flow-cytometry

Research

JoVE Journal

Bioengineering

23.5K 次观看.  University of California, Los Angeles. 荧光显微镜及成像流式细胞仪检测工具，一个手机上使用紧凑和具成本效益的光流体附件的整合，我们回顾我们最近的研究结果。这些手机的微观分析设备可能是有益的，如流式细胞仪分析，以及高通量筛选，例如，水样在资源有限的环境中执行各种细胞计数任务。

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

片上的无透镜荧光显微镜

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

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

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

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

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

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

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

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

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

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

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

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...