This work was supported by DARPA grant number 84389.01.44908, an NSF CAREER award (DBI-1253293), an NIH exploratory/developmental research grant (CA195709), and NIH New Innovator Awards (HD080351, DP2-AR068129-01), and a New Directions grant from the UCSF resource allocation program.

1. SU8主制造<ol><li>设计使用设计软件三层制造微流体结构并通过在电路板胶片供应商与10微米的分辨率打印的设计。装置设计的细节都在所附的参考6给出的信道的几何形状在图1所示的层应包括对准标记，以帮助从每个制造层8并置的功能。 </li><li>放置在旋涂机的预清洗3英寸直径的硅晶片，并开启真空将其固定到卡盘。申请1毫升SU8-3050在晶片和自旋的中心为20秒在500rpm，然后在30秒在1750转，提供80μm的层厚度。 </li><li>取出晶片并在135℃加热板上烘烤30分钟。允许在移动到下一步骤之前，晶片冷却至室温。 </li><li>暴露涂覆的晶片到1层面具下的准直190米W，365纳米LED 3分钟。曝光后，放置在一个135℃热板上的晶片1分钟，然后冷却至RT在进行下一步骤之前。 </li><li>放置在旋涂机在晶片和打开真空将其固定到卡盘。申请1毫升SU8-3050在晶片和自旋的中心为20秒在500rpm，然后在30秒以5,000rpm，导致提供的40微米的附加厚度的层。 </li><li>移动到下一步骤之前，取出晶片并烘烤上的135℃热板上进行30分钟，然后冷却至室温。 </li><li>对准第二层掩模上在1.3图案化的几何形状和涂布的晶片暴露于准直的190毫瓦，365纳米LED 3分钟。曝光后，放置在135℃的热板4分30秒，然后冷却至RT在进行下一步骤之前。 </li><li>放置在旋涂机在晶片和打开真空将其固定到卡盘。申请1毫升SU8-3050在晶片和自旋的中心为20秒在500rpm，然后在30秒，以1,000rpm，导致提供100μm的附加厚度的层。 </li><li>移动到下一步骤之前，取出晶片并烘烤上的135℃热板上进行30分钟，然后冷却至室温。 </li><li>对准第三层掩模上在1.3图案化的几何形状和涂布的晶片暴露于准直的190毫瓦，365纳米LED 3分钟。曝光后，放置在135℃的热板9分钟，然后冷却至RT在进行下一步骤之前。 </li><li>通过在丙二醇单甲基醚乙酸酯的搅拌浴中浸渍30分钟，开发的掩模。洗在异丙醇和烘烤晶片上的135℃热板上进行1分钟。放置开发大师100毫米培养皿聚二甲基硅氧烷（PDMS）成型。 </li></ol> 2. PDMS器件制造<ol><li>加入50克聚硅氧烷基的与5g在塑料杯中的固化剂的组合1的PDMS:准备10。用安装有搅拌棒旋转工具混合内容物。度如30分钟干燥器内的混合物，或直到所有的气泡都被删除。 </li><li>倒入PDMS，得到厚度3mm以上的主站和放回干燥器进一步脱气。一旦所有的气泡除去，烘烤设备在80℃80分钟。 </li><li>削减清洁表面与图案化侧使用解剖刀和地点从模具装置上，并与一个0.75毫米活检穿孔冲流体的入口和出口。该设备必须被切割，以使120微米和220微米高的几何形状是从该装置的一侧触及。 </li><li>等离子体处理装置，具有特征面朝上，一个预净化2英寸3英寸载玻片沿着在1毫巴的 O 2等离子体在一个300瓦的等离子体清洁器20秒。通过将PDMS设备的图案化侧到玻璃载片的等离子体处理面上粘合PDMS设备。请将设备在80℃的烤箱烤组装设备40分钟。 </li><li>渲染通道通过使用注射器冲洗用氟化表面处理流体的装置疏水性的。在80℃立即烘烤设备10分钟以蒸发溶剂。 </li></ol> 3.光学元件的制备<ol><li>通过从最后5mm的105微米芯，125微米包层的去除绝缘制备激光激发纤维，NA = 0.22的光纤。 </li><li>通过重复3.1的第二相同纤维准备第二彩色激光激发光纤。 </li><li>制备纤维为通过重复3.1 200微米芯，225微米包层收集荧光信号，NA = 0.39的光纤。 </li><li>检查所有的光纤的尖端 - 如果提示不平坦的表面端，再切割用纤维划线的两端。 </li><li>附加一个激光光纤耦合到一个50毫瓦，405 nm激光和附加105微米芯纤维在激光中的一个。直接剥离端处激光功率的传感器计，并使用激光耦合器的微调最大化激光功率。 </li><li>重复3.5为50毫瓦，473纳米的激光。 </li><li>安装在使用透镜套管挡住激光并传输发射的荧光的PMT一个446/510/581/703纳米四带通滤波器。安装光纤耦合器，使光通过过滤器击中PMT前移动。 </li><li>从3.3光纤连接到3.7组装单元。 </li></ol> 4.离线混合乳化代<ol><li>获得具有60微米×60微米孔的流动聚焦微流体dropmaker设备。 </li><li>填充1ml注射器与HFE 7500油，用2％的离子氟表面活性剂9。 </li><li>填一系列1毫升注射器与（FITC）荧光素的以下组合和葡聚糖 - 缀合的蓝色染料（CB）的PBS:1nM的FITC / 10nM的CB，为10nM FITC / 10nM的CB，1nM的FITC / 100nM的CB，和10nM FITC / 100nM的CB。 </li><li>安装dropmaker设备上倒百万分之一的阶段应付和注射泵将水和油的注射器。夫妇注射器使用PE-2管道设备。 </li><li>在500微升/小时的油和250微升/小时的水溶液运行注射器泵，创建的从4.3含有溶液80μm的液滴的混合物。对于每种类型的水样中，使用新鲜的dropmaker装置，以消除交叉污染。收集约200微升每个染料组合乳液到一个空的注射器。 </li><li> 4液滴类型已经在单个收集注射器收集后，通过反复转动注射器混合该乳液。 </li><li>用含有0.1，1,10和100nM FITC标记的PBS水溶液重复步骤4.2-4.6。 </li></ol> 5.光纤插入<ol><li>放置在步骤2上加上能&lt;100微秒的快门速度的数字相机的倒置显微镜的阶段制造的微流体芯片。 </li><li>从侧面看，樱雪请小心操作室温联接到473纳米的激光光纤进入的最远上游120微米通道，小心不要通过穿刺到主流动通道。 </li><li>将连接到405 nm激光光纤到最下游120微米高端通道，提供300微米的纤维间隔。 </li><li>插入光电倍增管偶联纤维进入垂直于两个激光激发光纤220微米高的信道。 </li></ol> 6.混合乳液的荧光检测<ol><li>装入5毫升注射器填充有HFE 7500，用2％的离子氟表面活性剂的检测装置的隔离物进油口，用PE-2管。 </li><li>装入含有关于使用PE-2管垂直取向的注射器泵和联接到该设备的液滴回注入口混合的FITC / CB乳剂注射器。从装置出口到废物容器连接的PE-2管道的长度。 </li><li>灌注设备由1000微升/小时运行的每个泵，直至两根油和液滴看到在装置定期组合和下游流动。 </li><li>调节流速，使得所述间隔物油运行在6000微升/小时，并在100微升/小时的液滴，提供液滴通过检测区行进之间显著间距。 </li><li>开启激光器，启动数据采集程序，并调整PMT增益提供了比100倍基线噪声基底多个信号。调整激光功率以使所有双峰峰是清晰可见的单一，线性缩放timetrace。 </li><li>获取的PMT电压timetrace 60秒以至少20千赫​​。将数据导入计算机程序如Matlab和衡量使用自定义计算程序脚本记录的双峰峰的高度。 注意:这是作为补充信息。 </li><li>使用4.7创建的FITC只混合乳化，用405 nm激光关闭，重复步骤6.2-6.7。 </li></ol>

<ol>
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The authors have nothing to disclose.

光纤检测需要的光纤相对于流体通道的取向。因为我们的器件利用具有多层光刻制造导槽，以相对于彼此的掩模放置是非常重要的。如果光纤导槽太靠近流体通道，对于流体泄漏的可能性;如果导向通道位于太远或错位，由检测纤维收集的荧光信号可以显著减少。适当的对准可以通过设计对准标记，例如同心圆入口罩相片图案形成过程中共同定位来辅助。此外，光纤的手动插入装置是具有打破装置...

液滴的微流体通过在大量悬浮在载体油1水性液滴的compartmentalizing实验提供用于高通量生物学的平台。液滴已被用于应用程序作为不同的单细胞分析2中 ，数字聚合酶链反应（PCR）3，和酶进化4。荧光测定法检测的液滴的微流体的标准模式下，作为其亮信号和快速响应时间与在千赫速率检测子纳升液滴体积兼容。许多应用需要荧光检测用于同时至少两种颜色。例如，我们的实验室通常进行的PCR活化使用一个检测信道为一个化验结果液滴排序的实验中，并使用二次背景染料，使测定阴性液滴可数5。 液滴微流体典型检测站BAsed的上表面荧光显微镜，并需要复杂的光操作计划，以从自由空间激光引入激励光成显微镜被聚焦在样品上。荧光从液滴射出后，所发射的荧光的光进行滤波，使得每个检测信道利用中心波长频带设为一光电倍增管（PMT）。落射荧光显微镜基于光学检测系统，由于其费用，复杂性提供了一个进入门槛，并要求维修。光纤提供的装置构造的简化和健壮检测方案，由于纤维可以手动插入微流体装置，除去基于镜面光路由的需要，并且允许光路用光纤连接器相连接。 在本文中，我们描述了一个紧凑的模块化方案的组装和验证，利用光纤的阵列进行多色荧光检测DA单光电探测器6。光纤被耦合到单独的激光和被插入垂直于一个L形的流动通道以规则的空间偏移。的荧光收集纤维取向平行于激励区域，并连接到一个单独的PMT。因为液滴穿过在不同的时间的激光束，由光电倍增管记录的数据显示了一个时间偏移，允许用户向液滴由每个不同的激光束激发后发出的荧光区分。这个时间偏移消除了需要发射的光分离成使用一系列分色镜和带通滤波器的分离的光电倍增管。以验证检测器的效能，我们定量液滴种群包封不同颜色和浓度的染料的荧光。该系统的灵敏度进行了研究为单色荧光检测，并示出了以检测与浓度液滴向下至0.1nM，一个200×灵敏度曝光的能力ovement相比，在文献7日报道最近的基于光纤的方法为。

<table>
	<tbody>
		<tr>
			<td>Photomasks</td>
			<td>CadArt Servcies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; silicon wafers, P type, virgin test grade</td>
			<td>University Wafers</td>
			<td>447</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3035</td>
			<td>Microchem</td>
			<td>Y311074</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>Microchem</td>
			<td>Y111072</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Krayden</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringes</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>27 gaugue needles</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>PE 2 polyethylene tubing</td>
			<td>Scientific Commodities, Inc.</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec 7500</td>
			<td>Fisher Scientific</td>
			<td>98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr>
			<td>Ionic Krytox Surfactant</td>
			<td></td>
			<td></td>
			<td>Synthesis instructions in ref #10</td>
		</tr>
		<tr>
			<td>Dextran- conjugated cascade blue dye</td>
			<td>Life Technologies</td>
			<td>D-1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma</td>
			<td>28803</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/581/703-25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Thorlabs</td>
			<td>PMM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber port</td>
			<td>Thorlabs</td>
			<td>PAFA-X-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>lens tube</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 200 um core / 225 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 105 um core / 125 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>125 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T08S13</td>
			<td></td>
		</tr>
		<tr>
			<td>225 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T10S13</td>
			<td></td>
		</tr>
		<tr>
			<td>laser fiber adapter</td>
			<td>OptoEngine</td>
			<td>FC/PC Adapter</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MDL-III-405</td>
			<td>Distributor for CNI lasers</td>
		</tr>
		<tr>
			<td>473 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MLL-FN-473-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

multicolor fluorescence detection for droplet microfluidics using optical fibers

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

一个PDMS设备，其允许光纤的插入的制造需要多步光刻过程，以创建不同的高度（ 图1）的信道。首先，SU-8的80微米高的层被旋涂到硅晶片并使用掩模来创建流体处理几何图案。接着，附加40微米层的SU-8的旋涂在晶片上，并使用第二掩模来创建功能，将形成120微米高的激光光纤插入通道图案化。最后，100微米以上的SU-8旋涂在晶片上，并形成图案，得到220微米高?...

Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

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

使用多色荧光检测液滴微流控光纤

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

multicolor-fluorescence-detection-for-droplet-microfluidics-using

Research

JoVE Journal

Bioengineering

10.4K 次观看.  University of California, San Francisco. Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

视频: Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence detection methods for microfluidic droplet platforms

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

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

荧光检测方法，为微流体液滴平台

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

科研

生物工程

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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 Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

使用多色荧光检测液滴微流控光纤

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