Name: multicolor fluorescence detection for droplet microfluidics using optical fibers
Uploaded: 2016-05-05T14:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

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>

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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&quot; 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 &amp;#956;m core / 225 &amp;#956;m 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 &amp;#956;m core / 125 &amp;#956;m 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 &amp;#956;m fiber stripping tool</td><td>Thorlabs</td><td>T08S13</td><td></td></tr><tr><td>225 &amp;#956;m 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&amp;nbsp;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&amp;nbsp;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

10.5K 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

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

科研

遗传学

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

折光传感荧光芯微腔的合成和操作

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

工程学

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 ...

生物工程

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 ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

生物化学

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

微流控成像流式细胞仪通过不对称检测时间延伸光学显微镜（ATOM）

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

使用双光镊和微流体进行单分子研究

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

生物学

<meta charset="utf8"></head><body>用于单细胞因子分泌分析的微流控液滴测定

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

<meta charset="utf8"></head><body>作者聚焦：揭示免疫反应中的多功能性和异质性

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌 

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...

使用多色荧光检测液滴微流控光纤

摘要

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此视频中的章节

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

折光传感荧光芯微腔的合成和操作

片上的无透镜荧光显微镜

荧光检测方法，为微流体液滴平台

应用三色单分子焦虑研究蛋白质相互作用的相关性

微流控成像流式细胞仪通过不对称检测时间延伸光学显微镜（ATOM）

使用双光镊和微流体进行单分子研究

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌