This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1.实验装置和样品制备<ol><li>使用光刻，蚀刻和如先前6所述熔化过程制造microtoroids。制造上的硅晶片（芯片）的顶部microtoroids通常具有80-100微米的大直径，以及2微米的小直径。 </li><li>放松大致从它的纤维卷轴的米单模光纤（125微米包层，4.3微米的模场直径）。 </li><li>在光纤的开卷部分的中间，剥去光纤使用剥线围绕聚合物涂层的一小部分（2.5厘米）。注意：这是光纤，其将被用来渐逝地将光耦合到所述微型环的部分。 </li><li>清洁剥离的纤维用异丙醇酒精和无尘擦拭。 </li><li>持使用由磁性压块的一个光纤夹持该部分的纤维就位。 </li><li>薄的剥离纤维〜500nm下meltin克，并用氢气火炬和两个步进电机在相反的方向移动，在60微米/分钟牵引。光纤定位火焰的顶端部，它应该是约10毫米高的内部。停止拉光纤时通过光纤传输将停止脉动，它可以通过视觉监视（通过观察即从纤维眨眼侧向散射和关闭光），或通过光纤连接到其连接到示波器的光电二极管。 </li><li>切割光纤的一端，并把它插入到裸光纤适配器。放置光纤的这一端进入光接收器的输入端。 </li><li>纤维耦合光纤线轴到激光的通过使用一个光纤耦合器的另一端。 </li><li>放置使用任一环氧树脂或双面胶带的微型环芯片上的样品架的顶部（不锈钢，37.8毫米×6.4毫米×3.2毫米）。 </li><li>装入样品支架上的定位台，其包括3轴纳米定位的顶部（正anocube）阶段（参照设备列表）上的3轴千分尺的顶部。执行一个气动隔离光表中的所有实验，以减少震动。 </li><li>粗略位置使用3轴千分尺的样品芯片。 </li><li>对准芯片平行于光纤使用纳米定位器的含微型环-。注意：输入光（〜633纳米）中的一个波长的距离内对齐微型环。为这一过程的可视化使用两个成像列（管与物镜和相机，见设备清单），其定位在顶部和在芯片的一侧。 </li><li>优化通过光纤使用一种在线偏振控制器引导所述激光的偏振（见设备列表）与旋钮调节极化。注意：当在光纤的传输所测量的倾角出现最窄最佳偏振的实现。注意这浸在示波器（详情参见步骤2.2）。 </li><li>构造通过环氧树脂将玻璃盖玻片上使用玻璃显微镜载玻片作为间隔物样品台上的样品室。注：有机玻璃外壳覆盖了整个安装程序可能最大限度地减少气流非常有用。一个小的开放应该留给允许的解决方案中使用管道进行抽水。 </li><li>热平衡粒子混悬液或单分子水溶液为≥1小时在室温水浴（〜500毫升）。注：样品使用从制造商， 例如 ，PBS或HEPES中指定的相关联的缓冲器稀释至所需浓度在微量离心管中。如果没有检测到结合事件，增加缓冲液的盐浓度。 </li><li>含有溶液（1毫升）中简要地为〜2秒涡旋粒子。 </li><li>注入含粒子溶液到样本室以1ml / min，使用1ml注射器泵。 </li><li>后样品室填补，关闭注射器泵。 </li><li>等待30秒前记录数据，以减少从流体流动会影响测量所得的机械振动的影响。 </li></ol> 2.频率锁定<ol><li>重新耦合圆环到光纤通过移动样品支架与纳米定位，因为耦合将因受到干扰到注射液。 </li><li>由通过各种波长的扫描计算机控制的输入激光定位微型环的谐振波长。执行此步骤通过发送一个三角波电压信号，以调整所述激光的波长的激光控制器内的压电元件。执行使用可见光（635纳米±2.5纳米），因为是光在水在此波长吸收少的实验。 </li><li>通过插入的光纤的输出入一个自动均衡光器测量的光透射通过光纤。插入受光的输出转换成使用BNC电缆的示波器。观察Øn中的示波器，在该微型环的谐振波长，通过光纤传输下降。 </li><li>附加光接收器的输出对所述频率通过电缆锁定反馈控制器（见设备清单）的主输入端。 </li><li>采用顶级的高峰期为2 kHz的抖动频率和19个FM波长振荡的幅度锁定在运行模式下自动锁定的频率锁定反馈控制器。经验设定在使用齐格勒- Nichols整定规则14的软件窗口的比例-积分-导数设置。注意：这些值只需要在所有实验的开始设置一次。 </li><li>自动锁定激光器的波长到微型环的谐振波长。执行填充样品室之后此步骤。注意：如果波长偏移过大，则反馈控制器将失去锁定并自动切换到扫描模式以找到谐振波长位置。这OC小人的波长偏移超过约一线宽（至少600 FM这里考察的所有系统）放大。 </li><li>使用24位数据采集卡上记录所述反馈控制器的输出在20千赫。导出的数据，经由数据采集软件的文本文件。 </li></ol> 3.数据处理和分析<ol><li>傅立叶变换在MATLAB中的数据。 </li><li>低通使用“砖墙”滤波器的截止在1 kHz，除去2千赫强加抖动频率的数据（见补充代码文件截图1）。 </li><li>采用16赫兹的窗口大小计算陷波滤波器的数据。注意：这样做是为了除去已知噪声源，在这种情况下，60赫兹电子线路噪声和谐波，以及100赫兹（从激光驱动未来）和它的谐波（见补充编码文件截图1）。 </li><li>逆傅立叶变换的数据回时域。 </li><li>值滤波器使用窗口中的数据1,001样品尺寸（见补充代码文件截图2）。 </li><li>定位使用Kerssemakers 等人 15的步骤寻找算法步骤中的变化谐振波长。 </li><li>生成每个结合步骤的幅度的直方图。 </li><li>使用公式计算粒径。 （1）（见讨论）。 </li></ol>

<ol>
	<li>Lu, T., 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=High+sensitivity+nanoparticle+detection+using+optical+microcavities.">High sensitivity nanoparticle detection using optical microcavities.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 5976-5979 (2011).</li><li>Su, J., Goldberg, A. F. G., Stoltz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+single+nanoparticles+and+biological+molecules+using+microtoroid+optical+resonators.">Label-free detection of single nanoparticles and biological molecules using microtoroid optical resonators.</a> Light: Science and Applications. , (2016).</li><li>Knight, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single molecule biology. , (2009).</li><li>Jonsson, U., 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=Real-time+biospecific+interaction+analysis+using+surface+plasmon+resonance+and+a+sensor+chip+technology.">Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.</a> BioTechniques. 11, 620-627 (1991).</li><li>Vollmer, F., Arnold, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whispering-gallery-mode+biosensing:+label-free+detection+down+to+single+molecules.">Whispering-gallery-mode biosensing: label-free detection down to single molecules.</a> Nat. Methods. 5, 591-596 (2008).</li><li>Armani, D. K., Kippenberg, T. J., Spillane, S. M., Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-Q+toroid+microcavity+on+a+chip.">Ultra-high-Q toroid microcavity on a chip.</a> Nature. 421, 925-928 (2003).</li><li>Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+microcavities.">Optical microcavities.</a> Nature. 424, 839-846 (2003).</li><li>Arnold, S., Khoshsima, M., Teraoka, I., Holler, S., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shift+of+whispering-gallery+modes+in+microspheres+by+protein+adsorption.">Shift of whispering-gallery modes in microspheres by protein adsorption.</a> Opt. Lett. 28, 272-274 (2003).</li><li>Vollmer, F., Arnold, S., Keng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+virus+detection+from+the+reactive+shift+of+a+whispering-gallery+mode.">Single virus detection from the reactive shift of a whispering-gallery mode.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 20701-20704 (2008).</li><li>He, L., Ozdemir, S. K., Zhu, J., Kim, W., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+single+viruses+and+nanoparticles+using+whispering+gallery+microlasers.">Detecting single viruses and nanoparticles using whispering gallery microlasers.</a> Nat Nano. 6, 428-432 (2011).</li><li>Dantham, V. R., 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=Label-free+detection+of+single+protein+using+a+nanoplasmonic-photonic+hybrid+microcavity.">Label-free detection of single protein using a nanoplasmonic-photonic hybrid microcavity.</a> Nano Lett. 13, 3347-3351 (2013).</li><li>Baaske, M. D., Foreman, M. R., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+nucleic+acid+interactions+monitored+on+a+label-free+microcavity+biosensor+platform.">Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform.</a> Nat Nanotechnol. 9, 933-939 (2014).</li><li>Su, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Single+Exosome+Detection+Using+Frequency-Locked+Microtoroid+Optical+Resonators.">Label-Free Single Exosome Detection Using Frequency-Locked Microtoroid Optical Resonators.</a> ACS Photonics. (9), 1241-1245 (2015).</li><li>&#197;str&#246;m, K. J., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Feedback systems : an introduction for scientists and engineers. , (2008).</li><li>Kerssemakers, J. W., 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=Assembly+dynamics+of+microtubules+at+molecular+resolution.">Assembly dynamics of microtubules at molecular resolution.</a> Nature. 442, 709-712 (2006).</li><li>Su, T. -. T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Label-free detection of single biological molecules using microtoroid optical resonators. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

作为粒子结合，的环形增大的谐振波长 （λ）。如果粒子解除绑定，谐振波长相应减少（降压事件）。粒径（d）可以通过各波长步骤的幅度的直方图来确定。每个波长台阶的高度而变化，由于结合的粒子的尺寸变化和由于对那里的粒子结合的微型环的位置。当颗粒粘合在微型环其中电场（E 0，max）为最高的赤道在共振波长（步骤高度）的最大变化发生。这个最?...

单分子检测的实验是用于降低用于疾病的早期检测的分析物的生物传感器的使用量，是有用的，并且用于检查分子3的基本性质。这种实验是使用标签，但是，标签不总是能够获得对特定蛋白质，增加了成本，可能扰乱正在研究中的事件，并且可以是不方便，尤其是即时现场实验或点OF-典型地护理诊断。 目前金标准的无标记生物传感器是表面等离子体共振4，但是商业表面等离子体共振系统通常具有检测纳米的量级的典型下限。近日，光学谐振器已成为一个有前途的技术，无标签的单分子生物检测5。基于长期（NS）光学谐振器的工作光6,7限制。光是渐逝典型地通过光纤耦合到这些设备。当经过光纤的光的波长的谐振器的谐振波长相匹配时，点亮有效地耦合到谐振器。此耦合光谐振器的空腔中的谐振器的周附近产生的渐逝场中的全内反射。作为颗粒进入渐逝场和绑定到谐振器中，谐振器的变化成比例地粒子8的容积的谐振波长。 在检测能力方面，微谐振器前面已用于检测单个A型流感病毒颗粒（100纳米）9,10。最近，plasmonically增强微球的光学谐振器已经被用于检测的单牛血清白蛋白分子 11和8-mer的寡核苷酸12，但是这种方法限制了粒子捕捉区域至0.3μm2每日副。面积较大的捕捉生物传感器是理想的最大化粒子探测的机会。当前的解决方案为基础的无标记生物传感技术与大（&gt; 100微米2）捕获的区域被限制于检测到聚苯乙烯颗粒≥25纳米。 我们已开发了一种基于光谐振器技术被称为频率锁定光学耳语倏逝谐振器（花）13（图1）一个无标记生物传感系统，其能够时间分辨单分子检测在溶液中的。花使用微型环光谐振器结合频率锁定反馈控制，平衡检测，并计算滤波以向下检测小颗粒以单个蛋白质分子的长光子寿命。使用频率锁定的允许系统始终跟踪微型环的移位共振作为颗粒结合，而无需扫或扫描激光波长过大范围。可以使用花的原理来增强的其他技术的检测能力包括电浆增强。在下文中，该程序用于执行FLOWER进行说明。

<table border="0" cellpadding="0" cellspacing="0" width="396">
	<tbody>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Tunable diode laser</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Laser controller</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300-LN</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Frequency locking feedback controller</td>
			<td class="xl63" style="width:99pt" width="132">Toptica Photonics</td>
			<td class="xl63" style="width:99pt" width="132">Digilock 110</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Auto-balanced photoreceiver</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">Model 2007</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">In-line polarization controller</td>
			<td class="xl63" style="width:99pt" width="132">General Photonics</td>
			<td class="xl63" style="width:99pt" width="132">PLC-003-S-90</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">24-bit data acquisition card</td>
			<td class="xl63" style="width:99pt" width="132">National Instruments</td>
			<td class="xl63" style="width:99pt" width="132">NI-PCI-4461</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">Recombinant human interleukin-2</td>
			<td class="xl63" style="width:99pt" width="132">Pierce Biotechnology</td>
			<td class="xl63" style="width:99pt" width="132">R201520</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">20 nm polystyrene beads</td>
			<td class="xl63" style="width:99pt" width="132">Thermo Scientific</td>
			<td class="xl63" style="width:99pt" width="132">3020A</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">NanoCube XYZ Piezo Stage</td>
			<td class="xl63" style="width:99pt" width="132">Physik Instrumente</td>
			<td class="xl63" style="width:99pt" width="132">P-611.3</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Optical table</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">VH3660W-OPT</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Objective lens for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl64">1-60228</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Imaging column (adaptor tube)</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl65">1-60228</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">High-Res CCD camera for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Edmund Industrial Optics</td>
			<td class="xl64">NT39244</td>
		</tr>
	</tbody>
</table>

label-free single molecule detection using microtoroid optical resonators

检测低浓度的分子到单分子限制对如能早期发现疾病，并在分子的行为基础研究领域的影响。单分子检测技术通常利用诸如荧光标签或量子点，标签然而，标签不总是可用的，增加了成本和复杂性，并可能扰乱所研究的事件。光学谐振器已成为一个有前途的手段来检测单分子，而不使用标签。目前在溶液通过非plasmonically增强裸光谐振器系统检测到的最小的颗粒是25纳米的聚苯乙烯球1。我们已经开发了一种称为频率锁定光学花落逝谐振器（花），可以在水溶液中2超过这个限制，实现无标记单分子检测的技术。由于信号强度与尺度颗粒体积，我们的工作表示&gt; 100X improveme核苷酸中的信号，以对现有技术的当前状态的噪声比（SNR）。这里后面FLOWER的程序都在努力提高在该领域的使用情况。

粒子结合事件清楚地视为阶梯状变化微型环的谐振波长随时间（图2A）。这些步骤的高度被示为在图2B中的直方图。 图2-4示出从外来体的结合代表迹线（纳米囊泡），5纳米二氧化硅珠，和单人白细胞介素2分子，分别。该阶梯状的事件比例与粒径的事实表明，该技术已被正确地执行。这可以通过产生的台阶高度（图2B）的直方图和比较观察到的理?...

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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 ...

label-free-single-molecule-detection-using-microtoroid-optical

Research

JoVE Journal

Bioengineering

9.1K 次观看.  California Institute of Technology. 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. 

视频: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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 ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

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.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

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 ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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.

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 ...