我们非常感谢，组装振动探测系统的帮助下，普渡大学，瘫痪研究中心教授理查德Borgens。这项研究是由内授予美国国立卫生研究院1R01EY019101支持MZ和BR，和部分的加州再生医学RB1 - 01417研究所，美国国家科学基金会MCB - 0951199，赠款和不受限制从研究津贴，防止盲目性，加州大学戴维斯分校的眼科。

1。探头制造空白探头均购自世界精密仪器（elgiloy /不锈钢聚对二甲苯涂层的微电极）（见下文"特定的试剂和仪器表"）。探头切背后的提示25-30毫米，二甲苯绝缘约5毫米刮切端用手术刀（＃11刀片），以确保良好的连接。探头安装在一个黄金R30的连接器使用载银导电环氧树脂（如祭乐SL65）[*见下文注]。探头是在室温下储存在一夜之间让环氧树脂硬化。接下来，探头尖端镀有黄金和铂金，使用纳安的电源。探头尖端在丙酮冲洗，并连接到一个纳安电源负输出。探头尖端被认为在解剖显微镜下（X40），镀金溶液（钾dicyanoaurate（W / V蒸馏水九（CN）2（DH 2 O）的0.2％）。一个参考线连接到正摆在首位输出和在溶液中完成的电路。电流5娜是5分钟，然后上升到20 nA的，直到提示是大约一半所需的最终大小（约10-15微米）应用。卫生署冲洗探头尖端2，然后platinizing解决方案（氯铂酸水合物; 1％W / V H2PtCl6 * 6H 2 O）加铅（II）醋酸（0.1％W / V PB（CH3CO 2）2 * 3H 2 O在卫生署2 O），适用于电流250 nA的是5分钟，然后直到提示是理想的最终大小约80％提高到500 nA的电流增加至1μA和应用阵阵，直到最后的尖端直径在1秒（约30-35微米），最后提示是在卫生署2 O探针冲洗，可以在室温下保存下去。如果探头损坏，黄金R30的连接器可重新使用。 [*注:一些探测系统的探头安装直接连接到放大器进行信号。其他系统有一个单独的安装点和信号连接器。在后一种情况下，短期（2-3厘米）与R30的一端连接器的导线焊接到第一R30的连接器前安装探头（见图2A）。] 2。探针系统探头连接到一个压电式电动弯管机安装在一个3维微定位器（图1）。探头振动控制，振动探头电源，这也使得振动幅度和频率调整。探头电源发送一个参考信号锁定放大器，这也显示了振动频率和相位角。连接示波器，以便有一个快速视觉参考探头振动，它也是有用的。从探头的信号锁定放大器。要测量探针和样品可以被看作在解剖显微镜（放大倍数5233至X40）光纤照明。在校准和样品测量，参考和地面（接地线），必须在解决方案（见图2b）。放大器是通过模拟 - 数字（I / O）接口连接到电脑上。数据记录使用斯特拉斯克莱德电软件的全细胞程序（WinWCP）。 锁定放大器设置:灵敏度200μV]，动态分辨率[正常]，[]的偏移，展开[X1]，时间常数:前[10秒]，后[0.1秒]。如果需要更快的反应是，预时间常数可以减少到3秒偏移量的控制是用来将探头跟踪，靠近屏幕中心的。如果大的反应，预计，跟踪可移动或向下。 WCP的软件设置:记录时间[204.8小号]，每个声道的样本[1024]，采样间隔[0.2秒]，电压范围+ / - 0.2 V]。如果预计大电流，电压范围可提高到1 V或5 V。 3。探头设置新探针进行测试，并确定其独特的频率和相位角。是摆在新的探头校准室含生理盐水（图2B）。电源开启时，出现的频率，直到最大的震动是观察。这是探头的谐振频率。使用在这个频率的探头，可以导致不稳定和在录音产生的噪声，使探头是"调整"减去10 Hz到探头的工作频率（一般为150-200赫兹）。振幅调整，使探针振动的距离是尖端直径相同，从而使振动探头时探头尖端的"双重形象"是看到（见图2b）。要确定的相位角，探头放在生理盐水中，在校准室和电流1.5μA/ 厘米 2反复应用。相位角放大器锁进行调整，直到没有任何反应。加上或减去90 °角度给出了最大的反应，这个角度是探头的工作相角。每个探头的频率和相位角指出以供将来使用。在一项实验中，重要的是，这些设置不会改变频率，振幅和相位角，因为这将改变探头的响应。为方便起见，当电流流经南到北"，这应该产生在录音跟踪向上偏转（这里被称为"峰"），电流流过"北 - 南"应该表现出向下偏转（参见图3A）。如果这是走错了路，再加入180 °相位角将修复翻转反应一轮。探测函数背后的理论，校准等详细信息， 请参见雷德等。 校准:一个"标准化"目前正是1.5μA/厘米2，适用于在校准室的探头，探头的响应是用于计算样品中的电流（见图2B，3A） 。样品测​​量之前，将探头校准在适当的解决方案，如BSS +人工泪液对角膜解决方案。当前应用:南北和南北两个方向产生一个向上和向下偏转，分别相当于外向和内向电流，根据样品的方向。探头跟踪应该有一个稳定的基线，噪音低（比较图3A和3B）。在实验中使用的解决方案，以补偿由于蒸发的渗透压改变的探头校准。在分析数据时，从上半年的实验测量，计算结束校准下半年开始校准值，测量，可以计算出。 样品测​​定:分庭可以进行设计，以保持和固定样本。例如，图3C显示培养皿用钢丝环举行的眼睛角膜测量。含生理盐水的培养皿置于显微镜下解剖和重点放在视野样本的兴趣领域。探针，然后放置在溶液中和导向平行样品的表面和重点也的，所以要测量的样品点同一水平上。探头移动方便（例如1-2厘米）远离样品，振动开启（和记录的计算机软件），以建立一个稳定的（水平）基线（见图4A）。然后探头移动到测量位置，从表面约50微米。当新的（"峰"）的价值是稳定的，探头是搬回参考位置和跟踪返回到基线。这可以在固定的时间点重复生产timelapse数据，或样品移动/旋转略有反复在不同的位置，产生空间的当前映射数据（见图4B，5B）。 数据分析:数据分析使用WinWCP（见图5a）。水平的红色"零"线是向上或向下移动，所以它是平行跟踪基线测量峰值前。垂直绿化测量线北移，以跟踪基准。输出绿色阅读在绿线的底部应接近零（如0.00012）。这个数字显示的红色和绿色线的交叉点，的，和蓝线之间的differnce。红线，然后向上移动，直到它与高峰的顶端平行。绿色输出读数mV峰的大小。的数据，所有测量的山峰，和校准数据放入一个Microsoft Excel电子表格模板（见表1）。如日期，探头的数量，电压范围（VR），解决方案中使用，测量位置，时间点等相关信息，也可以在电子表格中。电流的方向（或样品的那样:"I / O"）指出，和内向电流也给予了在"高峰期"一栏中负值。在右栏的电流计算公式:电流=峰*（1.5/calibration）1.5μA/ 厘米 2应用目前在校准。因此:"unw1&#39;= 12.45 *（1.5/56.12）= 0.33276907μA/厘米2。 4。成功的秘诀在所有的电，重要设备的接地（接地），有助于消除噪音。因此，至少微定位器和显微镜的机箱应接地，也许还有光源（见图1）。用户也可以将静电的来源，因此，通过手腕带接地可以防止在某些情况下，探针不稳定，但并不总是必要的。一个法拉第笼是没有必要的，在锁定了所有的频率（如60Hz的电网），除了频率放大器，过滤器的探头振动。一个振动隔离表是有用的，但不是必需的。坚实，稳定的工作台或桌子作品一样好。除了基本的在Excel试算表的信息（见上文），它是有用的记录在自己的实验室书额外的有用信息，如温度的变化，药物补充，等，如果照片是显微镜，注意放大倍率。如果相关，它也是有用的绘制草图显示位置和/或探针测量的方向（见图6）（S）的样品。 挑战性的步骤<ol class="lalpha"><li>使探头:探头和R30的连接器之间必须有一个良好的电气连接。如果没有在电镀阶段的情况，那么这可能是原因。 </li><li>校准:使用适当生理盐水或培养基中，你将测量样品。不要过高或过低，填补校准室，因为这可以改变的响应。液体的表面应平整，跨室顶部。 </li><li>样品测​​量:提前计划，如你需要作出特别室举行/装入样品（见图3C）的吗？从样品测量时，探头应为导向的长期轴平行样品表面，使探头的振动方向（并因此目前正在测量方向）是垂直的样品表面（例如（图4B） ）。样本可移动和/或在不同的位置测量旋转。重要的是保持一致的探针和样品表面之间的距离，测量时。测量电流是从样品表面的距离成正比;探头移动距离样品表面，由平方反比定律的电流下降。也就是说，当测量样品表面产生了电流，电流检测是从表面的距离的平方成反比。目镜刻度可以用来判断探针和样品表面之间的距离。 </li></ol>故障排除<ul><li>问题:没有回应，在校准。解决方案:检查生理盐水接触电极。在恒定电流校准器，检查电池。 </li><li>问题:小的反响。解决方案:在DH 2 O和/或丙酮的清洁探头。检查相位角。 </li><li>问题:噪音或不稳定的基准（参见图3B）。解决方案:检查接地线。 </li><li>问题:关闭屏幕跟踪跳跃。解决方案:不要让探头尖端触摸样品。 </li></ul> 5。代表性的成果图3a显示校准跟踪的一个很好的例子。注意稳定（水平）基准，低噪声和大的反响。作为比较，图3b显示嘈杂的跟踪与不稳定的基线。在小鼠角膜伤口的不同位置的电流测量，如图4B所示。顶部面板显示探头的位置，中间的面板显示的探头在电脑上记录的痕迹，下部面板是在不同位置的电流的的图形，呈现出伤口目前的个人资料。图5b给出了在小鼠的皮肤伤口，在固定的时间间隔生产在伤口上的当前时间，当然的数据的测量。 <img src="/files/ftp_upload/2358/2358fig1.jpg" alt="图1" /> 图1。振动探测系统。见详细的说明文字。蓝色文字描述的连接线的功能。绿色符号显示接地点。 <img src="/files/ftp_upload/2358/2358fig2.jpg" alt="图2" /> 图2:A。探头安装。探头粘成黄金R30的载银环氧树脂，以电穿孔前连接器。在某些系统中，第二个连接器进行信号的短丝焊接。比例尺3毫米。白金球是不是规模。 B.探头校准。恒定电流校准器（左）适用于目前的1.5μA/厘米2的探头校准室（右）。左下:近距离的探测。当探头（右下），振捣振幅调整，所以被认为是一个尖端的双图像。比例尺100微米。 <img src="/files/ftp_upload/2358/2358fig3.jpg" alt="图3" /> 图3。A。探头校准跟踪。一个良好的探头校准跟踪，基线平稳，低噪声和大的反响，示例。电流南到北给出了一个向上的偏转，北到南的电流产生一个向下偏转。 B.一个不稳定的，嘈杂的探测跟踪。 C.钱伯斯提出安装鼠标（左）或鼠（右）眼睛角膜测量。比例尺5毫米。 <img src="/files/ftp_upload/2358/2358fig4.jpg" alt="图4" /> 图4证明测量的例子。答:建立一个稳定的基线是1-2从样品中的参考位置的探头。当探头移动到附近的样品的测量位置，它检测到一个当前和跟踪偏转向上（向外目前）。 B.在整个鼠标角膜的不同位置测量。探头导向平行于表面，使振动方向垂直。向上峰显示外向电流。最大电流在伤口边缘（职位B＆F）。探头的原理是测量位置F（右伤口边缘）所示。比例尺300微米。 <img src="/files/ftp_upload/2358/2358fig5.jpg" alt="图5" /> 图5。A。分析探头的痕迹。上游面板:红色零线是向上或向下移动，因此它是平行与跟踪基准，那么绿色的测线跨越基线移动，因此，在绿色的输出读数接近零（0.00012）。下半部分:红线是当时的感动，了，直到它与跟踪峰顶部平行，输出读数显示mV峰值（12.45）的大小。电流计算使用校正数据（见表1）。 B.老鼠的皮肤伤口timelapse数据。前伤人的电流在零时刻（红色符号）。测量是在相同的位置在伤人后定期的时间点的小鼠皮肤伤口。经过初步的瞬态内向电流（低于零），目前扭转和外向电流（正）缓慢上升，并趋于稳定。 <img src="/files/ftp_upload/2358/2358fig6.jpg" alt="图6" /> 图6显示探针测量位置的实验室书草图。 A.大鼠脑红点显示，测量位置和探头符号显示探头的方向。 B.大鼠角膜伤口;红点显示测量位置和箭头显示当前测量的方向。 <img src="/files/ftp_upload/2358/2358tab1.jpg" alt="表1" /> 表1。Excel中用于存储和量化探针数据的电子表格的例子。 pN2_cal1 =开始校准; CAL +和CAL - = mV校准值; VR =电压范围，峰值在MV =样品测量，I / O =电流（或样品）。

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我们描述了一种成本低，基本的，但高度敏感的振动探头在各种生物系统的非侵入性电流测量系统。 可能的修改<ol><li>如果铂/铱电极（世界精密仪器;猫＃PTM23B20）是用于代替不锈钢，镀金阶段被淘汰。 </li></ol>应用我们用振动探头测量电流:大鼠角膜2;大鼠晶状体3,4;小鼠皮肤5; 6 爪蟾蝌蚪;人体皮肤的7...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eligoy-Stainless Electrode</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SSM33A70</td>
<td>76 mm, 7 M&Omega;, 1-2&mu;m tip</td>
</tr>
<tr>
<td>Gold R30 connector</td>
<td>&nbsp;</td>
<td>www.vectorelect.com</td>
<td>R30</td>
<td>Re-usable</td>
</tr>
<tr>
<td>Silver-loaded epoxy</td>
<td>&nbsp;</td>
<td>3M</td>
<td>SL65</td>
<td>Mix 1-part Resin with 1-part Hardener</td>
</tr>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZ40</td>
<td>Magnification x6 to x40</td>
</tr>
<tr>
<td>Potassium dicyanoaurate (KAu(CN)2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>379867</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Chloroplatinic acid hydrate (H2PtCl6 x 6H2O)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>520896</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Lead(II) acetate trihydrate (Pb(CH3CO2)2 x 3H2O)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>185191</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Nano-Amp power source</td>
<td>&nbsp;</td>
<td>Made in-house</td>
<td>-</td>
<td>Powered by six 1.5 V (AAA) batteries</td>
</tr>
<tr>
<td>3-dimensional micro-positioner</td>
<td>&nbsp;</td>
<td>Line Tool Co.</td>
<td>Model H</td>
<td>&nbsp;</td>
<tr>
<td>Lock-in amplifier</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR530</td>
<td>&nbsp;</td>
<tr>
<td>Digital I/O interface</td>
<td>&nbsp;</td>
<td>National instruments</td>
<td>PCI-6220</td>
<td>&nbsp;</td>
<tr>
<td>Shielded Connector Block with BNC connections</td>
<td>&nbsp;</td>
<td>National instruments</td>
<td>BNC-2110</td>
<td>&nbsp;</td>
<tr>
<td>Strathclyde Electrophysiology Software</td>
<td>&nbsp;</td>
<td>University of Strathclyde Institute of Pharmacy and Biomedical Sciences, UK</td>
<td>WinWCP V4.1.5</td>
<td>Free download from: <a href="http://spider.science.strath.ac.uk/sipbs/software_ses.htm">http://spider.science.strath.ac.uk/sipbs/software_ses.htm</a></td>
</tr>
<tr>
<td>Calibration Chamber</td>
<td>&nbsp;</td>
<td>Made in-house</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Constant Current Calibrator</td>
<td>&nbsp;</td>
<td>Vibrating Probe Company</td>
<td>&nbsp;</td>
<td>Powered by one 9 V (PP3) battery</td>
</tr>		</tbody>
		</table>
		</div>

measurement of bioelectric current with a vibrating probe

离子的主动运输所产生的电场，在许多生物系统中，往往成为重要的组织和器官的功能。例如，他们发挥了重要作用，直接在伤口愈合细胞迁移。在这里，我们描述了超灵敏的振动测量细胞外的电流探头的制造和使用。该探测器是一个绝缘的，削尖的一个小的铂黑尖（30-35微米），它可以检测在μA/厘米2的范围在生理盐水中的离子电流的金属丝。该探测器是由压电德尔振捣在200赫兹左右。离子电流的存在，探测器检测到其运动的极端之间的电压差。一个锁在了外来噪声放大器，过滤器，锁定探头的振动频率。数据记录到计算机中。探头校准的开始和结束在适当的盐水实验，用一腔适用于电流完全相同1.5μA/厘米2。我们描述了如何使探针，将系统设置为和校准。我们也证明了角膜测量技术，并表现出一些有代表性的成果从不同标本（角膜，皮肤，脑）。

Watch this Scientific Journal Video about Measurement of Bioelectric Current with a Vibrating Probe at JoVE.com

Measurement of Bioelectric Current with a Vibrating Probe

制造，校准和使用非侵入性的振动探头来测量各种生物系统中的生物电的电流的描述。

生物电电流测量振动探头

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

measurement-of-bioelectric-current-with-a-vibrating-probe

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Unit 1

Current and Resistance

SCIENTISTS IN ACTION

14.1K 次观看.  University of California, Davis. 制造，校准和使用非侵入性的振动探头来测量各种生物系统中的生物电的电流的描述。

视频: Measurement of Bioelectric Current with a Vibrating Probe

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

科研

生物工程

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

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

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

在生物系统中的离子浓度测量荧光纳米粒子

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

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜:测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

水样中偶极辅助固相萃取Microchip的痕量金属分析的制作

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

用原位滞后测量进行磁畴成像的优化设置和协议

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...