The authors have nothing to disclose.

1.纳米结构的制备<ol><li>选择直径25mm玻璃盖玻片上为170微米（第1.5）作为用于纳米加工基板的近似厚度。 </li><li>浸入食人鱼溶液盖玻片（3:1的比例的硫酸和过氧化氢的）至少6小时。用超纯水18.2MΩ去离子蒸馏水（DDW）的量丰富的洗净浸泡食人鱼盖玻片。 注意:食人鱼酸发生剧烈反应与有机材料的，必须格外小心。 </li><li>上的盖玻片通过电子束蒸发存款10nm的铬薄膜，以避免纳米结构的图案化和成像期间电荷效应。 </li><li>旋双层的第一层抗蚀剂选自乳酸乙酯甲基丙烯酸甲酯（MMA_EL6）共聚物以2000rpm进行45秒，然后烘烤，在150℃。以3000rpm进行45秒旋的聚甲基丙烯酸甲酯（950PMMA_A2）所述第二层，然后烘烤，在180℃。 </li><li>行话n中的双层抗蚀剂使用电子束光刻（EBL），在25千伏为300μC/ cm 2的区域的剂量。开发中的异丙醇（IPA）/甲基异丁基酮（MIBK）:2/1和冲洗在IPA。 </li><li>存款的Ti（5nm）的使用电子束蒸发器在衬底上/金（80纳米）的膜。 </li><li>继金沉积，升空浸泡在丙酮基板4小时的共聚物双层抗拒。 </li><li>使用扫描电子显微镜（SEM）确认纳米结构的形状和大小检查基材，使用的CR-7腐蚀剂通过湿蚀刻除去从基板剩余铬60秒在RT，然后冲洗在DDW。 </li><li>设计的33微米中心对中心阵列间距，以留出空间阵列间的细胞成像。图案在20×20模式为每个阵列的使用电子束写入300nm的间距的纳米结构。每个芯片包含300阵列的80±2.5纳米的高度典型的纳米结构尺寸，70±2.5纳米diame之三。 </li><li>检查使用原子力显微镜（AFM）对尺寸和均匀性的验证阵列的一个子集。 </li><li>附加的支撑环，通常是硅，以使用UV固化环氧树脂盖玻片的背面。 </li></ol> 2.芯片清洁和自组装膜中的应用<ol><li>进行清洗和再生芯片，等离子体灰在40瓦，在5％的氢，清洁腔室5分钟在相同条件下经过95％的氩气，持续45秒的300毫乇混合物的功率。 </li><li>通过浸入一种双组分的乙醇硫醇溶液中的芯片组成的3官能的金纳米结构等离子体灰化后，立即:1的比例的SH-（CH 2）8 -EG 3 -OH（SPO）和带胺的成分或羧基官能团，即，SH-（CH 2）11 -EG 3 -NH 2（SPN）或SH-（CH 2）11 -EG 3 -COOH（SPC）。 </li><li>离开芯片的In中的硫醇溶液O / N，以形成自组装单层（SAM）。 </li><li>冲洗用乙醇，干燥，用氮气芯片。 </li><li>如果需要的话，存储该官能化芯片进行长达2周，在4℃。 </li><li>当准备使用反应该SPN或者SPC组分与使用取决于所选择的配位体的化学配位体（见下文）。 注意:该芯片可以再生和再官能数十次。一个给定的芯片可以用于周期，范围从6个月到一年以上。一个给定的阵列上的所测量的光谱通过等离子体灰化反复再生，随后biofunctionalization之后可靠地再现。29 </li></ol> 3.表面功能化和动力学特性注意:使用在商业SPR仪器的官能化芯片与配体和分析物之间的动力学速率常数表征，以及研究的SAM非特异性b上电阻inding。有一个广泛的流量和微流体设计，以便有效的表面功能化。既然我们有市售SPR我们围绕标准化的推荐流量。我们注意到，这些流量是典型的所有SPR仪器等等都没有限制。 SPR仪器不是必须的，因为所有的官能化可以直接在LSPR芯片上进行，但它并减少我们的工作负荷，因为它是一个复用的仪器，而我们的微流体LSPR设置不。 <ol><li>功能化商用裸金片与SAM在第2节所述。 </li><li>如果使用SPC基于SAM中，激活羧基用1:133毫1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）和33 N-羟基的毫琥珀酰亚胺（NHS）的DDW 10 1混合物分钟。 </li><li>缀合的活化的羧基与感兴趣为使用30微升/分钟的流速300秒的抗体/配体。准备配体pH为6的磷酸缓冲液，典型地，但是这可以根据不同的分子变化。 </li><li>所述配体缀合后，流的0.1M乙醇胺在磷酸盐缓冲盐水（PBS），为300秒，在30微升/分的速率失活剂步骤。乙醇胺有助于减少非特异性结合。 </li><li>引入感兴趣的分析物在100微升/ min，使用浓度范围的流速和计算使用动力学分析软件的动力学速率常数。 </li><li>如果非特异性结合是有问题的，增加的SPO到SPC或SPN的比率。 </li></ol> 4. LSPR常规设置<ol><li>显微镜设置: <ol><li>使用100瓦的卤素灯克勒照射样品。使用在光路中的长通滤波器（一般593纳米的截止），以消除波长不向谐振移位（ 图2）。 </li><li>对于LSPRi数据的收集，使用倒置显微镜与40X石油观鱼ñ目标（1.4 NA）和热电冷却的16位CCD相机。 </li><li>放置一个光束分离器，在显微镜的输出端口，以同时获得图像和光谱。 </li><li>将温度控制的显微镜阶段至37 °C和平衡4小时。 </li><li>合并在显微镜额外孵育组件，以调节CO 2浓度，湿度为5％和95％，分别为。 </li></ol></li><li>芯片的制备和安装: <ol><li>官能LSPR的芯片作为第2部分所述用来自SPR实验确定最佳的双组分的SAM比率。 </li><li>一个定制的微夹持器内装入芯片如下。放置在铝底片的芯片。夹着该底部片和硅酮密封垫和一个透明的塑料顶部件之间的芯片上。用4个螺钉夹紧组件。 </li><li>对于一个典型的SPC基硫醇应用，降外套300微升1:1混合的133毫的EDC和33毫NHS在DDW TURE激活SPC硫醇组分的羧基。 </li><li>等待10分钟，并手动冲洗表面用10mM PBS中。 </li><li>缀合的活化的羧基与感兴趣的配体（通常是抗体或抗体片段）通过滴涂覆300微升的配体溶液。 </li><li>等待30分钟，用10mM的PBS手动冲洗。 </li><li>降外套300微升的0.1M乙醇胺的PBS在芯片上，以减少非特异性结合。等待10分钟。 </li><li>用含0.005％吐温20的PBS（PBS-T20）的PBS洗乙醇胺。 </li><li>将一个石英片芯片以上，以减少相关的变化半月板的数据波动。 </li><li>保持芯片用PBS-T20缓冲湿而安装在显微镜。 </li><li>放置LSPR芯片组件使其进入加热阶段，样品架，并附上微管。 </li><li>装上微流体管的组装和流动BUFFER（或无血清培养基进行细胞研究），直到达到稳定状态。 </li><li>允许该组件和显微镜下平衡至少2小时。 </li><li>使用操纵杆，使得中央阵列与光纤的光谱对准芯片对齐。光谱数据是使用光谱仪和频谱分析软件。 </li><li>请重点阵列整个实验过程由软件自动对焦，蔡司定聚焦或等效的自动对焦装置。 </li></ol></li></ol>抗c-myc的分泌物9E10杂交瘤细胞5 LSPR成像注意:用于此研究的杂交瘤细胞系表达抗c-myc的抗体组成，因此不需要化学触发<ol><li>功能化与c-myc基因肽纳米结构。这具有1.77纳米用于通过克隆9E10杂交瘤细胞分泌的抗c-myc的抗体A K D值 。 </li><li>文化杂交瘤细胞在COMPLETË生长培养基在37℃，5％CO 2条件下10％胎牛血清（FBS）和1％抗生素的T75烧瓶。保持4×10 5个细胞/ ml的细胞密度。 </li><li>为细胞分泌的研究中，从T75烧瓶通过离心沉淀细胞，用RPMI-1640无血清培养基（SFM）洗涤两次以除去分泌的抗体，并调整细胞密度4×10 6个细胞/ ml。 </li><li>他们介绍到LSPR芯片之前收获细胞并测试可行性。 </li><li>引入50微升的细胞溶液的手动到LSPR芯片用微量。几分钟后25到50个细胞附着在LSPR芯片的表面上。 </li><li>与溶液中的新鲜SFM利用微流体灌注系统洗去剩余的细胞。 </li><li>选择在10微米LSPR阵列成像这是接近，但不与细胞重叠。 </li><li>以确保该信号是特定于分泌的抗c-myc的antibodies引入细胞培养基具有和不存在的抗体以及与抗体，但与堵塞的c-myc基因的溶液肽饱和浓度的存在下它们的结合位​​点。 </li><li>校准传感器在每次运行与抗c-myc的抗体（250纳米）的饱和溶液的末端。这有助于在传感器的响应归一化，并确定基于每个游程的biofunctionalization轮廓分数占用。 </li><li>校正用图像比对软件的X和Y方向上的漂移。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Imaging+of+Protein+Secretions+from+Single+Cells+in+Real+Time.">Quantitative Imaging of Protein Secretions from Single Cells in Real Time.</a> Biophysical Journal. 105, 602-608 (2013).</li><li>Raphael, M. P., 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+New+Methodology+for+Quantitative+LSPR+Biosensing+and+Imaging.">A New Methodology for Quantitative LSPR Biosensing and Imaging.</a> Analytical Chemistry. 84, 1367-1373 (2011).</li><li>Raphael, M. P., 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=Quantitative+LSPR+imaging+for+biosensing+with+single+nanostructure+resolution.">Quantitative LSPR imaging for biosensing with single nanostructure resolution.</a> Biophysical Journal. 104, 30-36 (2013).</li><li>Raphael, M. P., 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+new+methodology+for+quantitative+LSPR+biosensing+and+imaging.">A new methodology for quantitative LSPR biosensing and imaging.</a> Analytical Chemistry. 84, 1367-1373 (2012).</li><li>Henn, A. D., 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=Modulation+of+single-cell+IgG+secretion+frequency+and+rates+in+human+memory+B+cells+by+CpG+DNA,+CD40L,+IL-21,+and+cell+division.">Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division.</a> Journal of Immunology. 183, 3177-3187 (2009).</li><li>Bramswig, K. H., 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=Soluble+Carcinoembryonic+Antigen+Activates+Endothelial+Cells+and+Tumor+Angiogenesis.">Soluble Carcinoembryonic Antigen Activates Endothelial Cells and Tumor Angiogenesis.</a> Cancer Research. 73, 6584-6596 (2013).</li></ol>

We thank George Anderson for helpful comments and discussions. This work was supported by the Naval Research Laboratory&#8217;s Institute for Nanoscience and the National Research Council Research Associateship Award.

The LSPR imaging technique described in this work has numerous advantages over more traditional methodologies for detecting cell secretions. First, the time resolution of our technique is on the order of seconds whereas the commercial alternative, an immunosandwhich assay known as EliSpot, has a typical time resolution of 2 to 3 days.7,32 As a result we were able to resolve sudden changes in the rate of protein secretion, such as that shown in Figure 6. Second, having arrays distributed over the chip allows for the secreted signal to be tracked in space and time which enables more rigorous comparisons to diffusion-based models of cell secretion. In addition, arrays like the control array shown in Figure 6 can be used to subtract out global changes in the image that typically arise from instrumental factors such as focus drift. Third, our technique requires no modification of the cells. If desired, the experiment can incorporate commonly used tags such as fluorescent proteins, but if there is concern that such tags may negatively affect cell viability or homeostasis the label-free nature of our approach does not require them. Fourth, using the spectroscopic data we have demonstrated that quantitative information regarding the fractional occupancy of surface bound ligands can be calculated.
There are numerous alternative methods to EBL for fabricating metallic nanoparticles. However, we have found that the EBL provides considerable flexibility for optimizing nanostructure and array dimensions to best suit the optics and the cells under investigation. Also critical is the fact that the chips can be readily regenerated by plasma ashing. In this way, a typical chip can be used dozens of times. Biofunctionalization details must be modified for the specific application. The protocol presented here conjugated the surface with relatively small c-myc peptide ligands. Larger ligands such as whole antibodies typically require more spacing and thus a higher SPO to SPN/SPC ratio. Regardless, a well formed SAM layer is essential for preventing non-specific binding in live-cell experiments. In general, larger molecular weight analytes are more readily detected by LSPR. Thus, in its single-cell manifestation, this technique may not be appropriate for detecting the secretion of small proteins, such as cytokines. 
The current setup has been used for studying individual non-adherent cells. There are significant number of secreted signaling proteins and vesicles to which the results reported in this work are directly applicable. For example carcinoembryonic antigen (CEA) which for decades now has been a diagnostic marker for cancer. Colon cancer cells are known to secrete CEA at the rates of thousands of molecules/cell/hr and the molecular weight is 180 kDa which exceeds that of IgG antibodies. CEA is believed to be involved in autocrine and paracrine signaling pathways but the spatio-temporal nature of these secretions have never been measured. Our technique can directly address these signaling questions. An extension of this work will be to measure the spatio-temporal nature of CEA secretion from single cells.33 Future work will also focus on integrating LSPRi with two and three dimensional cell cultures of adherent cells. By incorporating multiplexed arrays capable of detecting a number of secreted proteins in parallel, this technique has the potential to open a new window into cell secretions and how they influence neighboring cells.

间的蜂窝通信是对许多生理活动的内部和细胞外部的调节是至关重要的。各种蛋白质和囊泡的可分泌其随后引发复杂的细胞过程如分化，伤口愈合，免疫应答，迁移和增殖。1-5故障的细胞间信号传导途径已牵涉许多疾病，包括癌症，动脉粥样硬化和糖尿病，仅举几例。 最优细胞分泌测定应当能够在空间和时间映射感兴趣的分泌蛋白而不破坏相应信号通路。以这种方式，浓度分布和接收单元的响应之间的因果关系可以推断。不幸的是，许多的最常用的荧光为基础的技术不符合这些标准。荧光融合蛋白可以用于标记分析物瓦特ithin的细胞，而且可以破坏分泌途径，或者如果分泌，结果，其中是难以量化单元外部的扩散辉光。荧光immunosandwich基测定法是最常用的技术，用于检测蜂窝分泌物但通常需要单个细胞的分离。6-11此外，引入感测抗体的通常停止或结束实验和的抗体标签的大小， 150 kDa的对IgG，是妨碍下游信号。 这些路障，因为它是优选无标记技术被用于图像蛋白分泌物和之间现有无标记技术，表面等离子体共振（SPR）和局域型表面等离子体共振（LSPR）传感器是优良的候选12-17这些传感器已被广泛地用于蛋白，外来体和其他生物标志物的分析物结合研究。在18-24 LSPR，电浆nanostr的情况下uctures可以将光刻图案化到玻璃盖玻片，并通过标准的宽视场显微镜的配置利用可见光激发。由于它们的纳米级尺寸，大多数的玻璃基板的可用于诸如亮场和荧光显微镜使这些探针非常适用于与活细胞显微术集成常见的成像技术。25-28我们已经证明了实时测量从使用官能化金电浆纳米结构的225毫秒和10微米，分别时空分辨率杂交瘤细胞抗体的分泌物。基本芯片配置示于图1。28显微镜的输出光路被分离用于成像的CCD照相机和一个光纤光学地耦合分光计用于定量测定纳米结构的给定阵列的分数占用（图2的间）。 该protoc醇在本文提出描述了实验设计为单细胞的分泌物的实时测量，同时监测使用标准明场显微术的细胞的响应。多学科方法包括纳米结构的细胞株的制造，纳米结构为高亲合力的分析物结合的官能化，这两个最小化非特异性结合和使用商业表面等离子体共振表征动力学速率常数（SPR）仪器表面优化，集成到衬底上，并且图像和光谱数据的分析。我们预计这种技术是一种使能技术进行细胞分泌物和接收单元的因果关系的时空映射。

<table><tbody><tr><td>25&amp;#160;mm diameter glass coverslips</td><td>Bioscience Tools&amp;nbsp;</td><td>CSHP-No1.5-25</td><td>&amp;nbsp; 170&amp;plusmn;5 &amp;micro;m is optimal</td></tr><tr><td>Poly-methyl methacrylate</td><td>Microchem</td><td>PMMA 950 A4</td><td></td></tr><tr><td>Ethyl lactate methyl metacrylate</td><td>Microchem</td><td>MMA EL6</td><td></td></tr><tr><td>Electron beam evaporator</td><td>Temescal</td><td>FC-2000</td><td></td></tr><tr><td>Electron beam lithography</td><td>Raith</td><td>Series 150</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>320110</td><td></td></tr><tr><td>CR-7 chromium etchant</td><td>Cyantek</td><td>CR-7</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Zeiss</td><td>Ultra 55</td><td></td></tr><tr><td>Atomic force microscope</td><td>Veeco</td><td>Nanoscope III</td><td></td></tr><tr><td>Plasma ashing system</td><td>Technics</td><td>Series 85 RIE</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;8&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-OH (SPO)</td><td>Prochimia</td><td>TH 001-m8.n3-0.2</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;11&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-COOH (SPC)</td><td>Prochimia</td><td>TH 003m11n3-0.1</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;11&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-NH&lt;sub&gt;2&lt;/sub&gt; (SPN)</td><td>Prochimia</td><td>TH 002-m11.n3-0.2</td><td></td></tr><tr><td>Surface plasmon resonance system</td><td>Biorad</td><td>XPR36</td><td></td></tr><tr><td>Bare gold chip</td><td>Biorad</td><td>GLC chip</td><td>Plasma ashed to remove the monolayer</td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide</td><td>Thermo</td><td>22980</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-hydroxysuccinimide (NHS)</td><td>Thermo</td><td>24510</td><td></td></tr><tr><td>Pentylamine-Biotin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermo</td><td>21345</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>Neutraavidin</td><td>Thermo</td><td>31000</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Thermo</td><td>28374</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P2287</td><td></td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axio Observer</td><td>Microscope is equipped with 40X oil immersion objective; CO&lt;sub&gt;2&lt;/sub&gt; and humidity incubation from Pecon GmbH</td></tr><tr><td>CCD camera</td><td>Hamamatsu</td><td>Orca R2</td><td>Thermoelectrically cooled (16 bit)</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>QE65Pro</td><td></td></tr><tr><td>Spectrasuite</td><td>Ocean Optics</td><td>version1.4</td><td></td></tr><tr><td>c-myc peptide HyNic Tag</td><td>Solulink</td><td>SP-E003</td><td></td></tr><tr><td>monoclonal anti-c-myc antibody</td><td>Sigma-Aldrich</td><td>M4439</td><td></td></tr><tr><td>Hybridoma cell line</td><td>ATCC</td><td>CRL-1729</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100&amp;times;)</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Serum free media RPMI 1640&amp;nbsp;</td><td>Invitrogen</td><td>&amp;nbsp;11835-030&amp;nbsp;</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Rhodamine DHPE</td><td>Life Technologies</td><td>L-1392</td><td></td></tr></tbody></table>

a label-free technique for the spatio-temporal imaging of single cell secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

在典型的活细胞分泌研究有数据收集发生的多种模式。 图3示出了LSPRi图像，其中突出的正方形阵列的叠加，和一个透射光照明图像，突出的细胞在左下方。数据通常收集在3小时内，随后通过引入的分析物为后述的归一化计算的饱溶液。荧光图像，也可以通过一个过滤器立方体的自动切换集成到数据收集例程。 在图4染色荧光膜染料若丹明DHPE细胞呈现伪足状延伸（箭头）。如果这样的扩展是与阵列重叠，他们将得到的假阳性蛋白质分泌。有图像的多种模式可以帮助识别这种情况的发生。 图5示出前光谱数据和船尾器引入一个饱和溶液（400纳米）的商购抗c-myc的抗体与c-myc的官能化阵列。没有细胞存在于该实验。光谱显示两个红移和增加强度。两条曲线下的区域之间的差异导致在CCD摄像机上的增加，阵列图像强度LSPRi模式。甲非线性最小二乘数据分析方法已被开发来推断从光谱面结合的配体的分数而定。30,31 在实验结束时，饱和的强度值（即，分数占用≈1）被用来计算使用以下公式中的每个阵列的归一化的反应: 哪里的归一化强度在时间点t，在该实验中，最终的饱和强度的开始初始强度，并且阵列的被测强度在时间点t </eM&gt;分别。 从两个阵列标准化的值示于图6中。一个阵列是在10微米的细胞的受调查的同时，另外，作为对照，从一小区的距离为130微米。在最接近的阵列相对于所述控制阵列的平坦响应的单元的归一化响应的突然增加是表示一个局部突发分泌的抗体的。 <img alt="图1" src="/files/ftp_upload/53120/53120fig1.jpg" /> 图1.传感器的设计。甲绘图描绘了典型的活细胞分泌实验的几何形状的细胞（蓝色球体）沉积到LSPR芯片，其中包含的biofunctionalized金纳米结构阵列。在放大视图中，感兴趣的细胞分泌，在示出为Y形分子这种情况下的抗体，因为它们结合到表面，测量功能化纳米结构。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig1large.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/53120/53120fig2.jpg" /> 图2.光学设置。来自卤素灯的照明光首先由一个长通滤波器（LP）过滤。的光被线性偏振（P1）和照射经由40X / 1.4 NA物镜的样品。散射光被物镜通过交叉偏振器（P2）的收集和传递。 50/50分束器（BS）被插入到所收集的光路用于同时光谱分析和图像分析。右上:由300纳米的间距隔开9个人纳米结构的原子力显微镜图片<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig2large.jpg" target="_blank">请点击这里</a>查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53120/53120fig3.jpg" /> 图3.活细胞LSPRi研究。甲合并透射光和LSPRi的图象，显示了12阵列包围的单个杂交瘤细胞（左下）。这是一个对比度增强的图像。比例尺为10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53120/53120fig4.jpg" /> 图4.活细胞荧光分析。沾上罗丹明DHPE，这是一种膜染料单个杂交瘤细胞的荧光假彩色图像。在荧光成像模式下的阵列通常不可见的，然而，附近的数组这里可观察到作为从头缺少方在右下角。该细胞可以被看作是从虽然触手状延伸（可能丝状伪足或者伪足）向外从细胞（箭头）延伸的所述阵列分离。比例尺为10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53120/53120fig5.jpg" /> 图5.频谱形态。之前和引进抗c-myc基因抗体的400纳米的解决方案后，从c-myc基因功能化的阵列获得的光谱。无细胞存在于这项研究。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/文件/ ftp_upload / 53120 / 53120fig6.jpg"/&gt; 图6.单细胞分泌的影响。位于15μm的单细胞的和一个位于130微米远（控制）的阵列的响应。比例尺为10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53120/53120fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions at JoVE.com

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

细胞间的通信为内和细胞外的控制各种生理活性的关键。本文描述了测量的单细胞的分泌物的时空性质的协议。为了实现这一目标，多学科的方法是使用集成了无标记纳米等离子体激感测与活细胞成像。

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

a-label-free-technique-for-spatio-temporal-imaging-single-cell

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.5K 次观看.  Naval Research Laboratory. 细胞间的通信为内和细胞外的控制各种生理活性的关键。本文描述了测量的单细胞的分泌物的时空性质的协议。为了实现这一目标，多学科的方法是使用集成了无标记纳米等离子体激感测与活细胞成像。 

视频: A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

科研

生物化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

工程学

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

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

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

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

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

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

免疫与感染

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

生物学

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

定量微模式细胞细胞外细胞异细胞的时态参数

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

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

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

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

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

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

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

无标记技术的单细胞分泌物的时空成像

摘要

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此视频中的章节

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

受体动力学通过单分子荧光显微镜的高分辨率时空分析

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

成像本地CA

定量微模式细胞细胞外细胞异细胞的时态参数

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

无标记技术的单细胞分泌物的时空成像

摘要

探索更多视频

此视频中的章节

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

受体动力学通过单分子荧光显微镜的高分辨率时空分析

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

成像本地CA

定量微模式细胞细胞外细胞异细胞的时态参数

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术