Name: optical control of living cells electrical activity by conjugated polymers
Uploaded: 2016-01-28T14:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA, 
Telethon - Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).

1.准备光活性基质<ol><li>准备一个P3HT:PCBM溶液（1:1重量/重量）的氯苯以20克/升的P3HT浓度。混合用磁力搅拌器将溶液至少4小时，在60℃。考虑150微升溶液的体积为每个衬底被制备。 </li><li>清洁的ITO涂覆的玻璃载片（R S = 10Ω/□，18×18毫米2，厚度170微米）的去离子水，丙酮和异丙醇中超声波仪连续浴（10分钟为每个工序）。干燥用氮气枪的盖玻片。把样品中的等离子体清洁器在100瓦的功率进行10分钟。 </li><li>沉积活性层的薄膜上经由旋转涂布清洁过的基材上。 <ol><li>用金刚石尖，切显微镜载玻片，（厚度≈1毫米），以在ITO涂覆的基材的相同的横向尺寸。卸下P3HT:从加热板PCBM溶液，让其冷却至环境温度。 </li><li>设置一个两圣EPS旋转涂布机程序:ⅰ）30秒以80​​0rpm; ⅱ）30秒1600转。放置在旋涂机的卡盘滑动，并开始真空进行修复。放丙酮在滑动下降，然后将清洗衬底，以在ITO涂覆侧朝上。启动旋转涂布机旋转甩掉多余的丙酮。 </li><li>把150微升的P3HT:PCBM溶液在基片上并再次开始旋转涂布机（使用在1.3.2中描述的相同的协议）。 </li><li>后旋涂机已完成，取出样品，小心地分离从载玻片上涂布的基材。清洁衬底的同一个洁净室拭子浸入丙酮背面​​。 注意:光活性基材可以直到用于存放几天在黑暗的框。对于更长的时间，让他们在与惰性气体的手套箱。 </li></ol></li></ol> 2.准备细胞培养基和电解决方案<ol><li>制备完全生长培养基（CGM），用于HEK-293细胞:杜尔贝科改良伊格尔培养基（DMEM），补充有10％胎牛血清（FBS）的未热灭活的，100U / ml青霉素和100μg/ ml链霉素。消毒用0.2微米的过滤系统和存储介质在4℃下。 </li><li>准备在超纯水（以mM浓度）的胞外溶液:氯化钠135，氯化钾5.4，5 HEPES，10葡萄糖，1.8 氯化钙 2，1 MgCl 2的。调节pH至7.4，用NaOH。消毒在4℃的溶液用0.2微米的过滤系统和存储。 </li><li>准备在超纯水中的细胞内溶液（浓度以mM计）:12的KCl，125 K-葡萄糖，1 MgCl 2的，0.1 氯化钙 2，10 EGTA，10 HEPES，10的ATP 的 Na 2。需要注意的是三磷酸腺苷的 Na 2是热敏感和最后添加。调节pH至7.4，用NaOH。消毒在-20℃在1毫升管中的溶液用0.2微米的过滤系统，等分和存储。 </li></ol> 3.电镀的HEK-293细胞的在活性基底<ol><li>放置在覆盖有铝或封闭的陪替氏培养皿，并把在烘箱中于120℃进行2小时的灭菌玻璃烧杯光敏衬底。从这一刻起，在无菌条件下进行的所有操作。把样品在无菌罩，让他们冷静下来。 </li><li>涂层光活性基片的粘合层，以减少表面疏水性，并促进细胞粘附。 <ol><li>放置在一个P35培养皿的活性基片，或将6多孔板。给出的共轭聚合物层的疏水性，使聚二甲基硅氧烷（PDMS）以及固定样品和限制的解决方案。 <ol><li>混合PDMS和催化剂在10:1的比例用玻璃棒，得到粘稠浆液。 </li><li>倒在一个6多孔板浆料，分配的量足以覆盖一半的每个孔。 </li><li>等待PDMS聚合反应在室温。 </li><li>切固化硅橡胶，中间为obta寄存器中聚合物样品的相同尺寸的正方形。 </li><li>为至少30分钟灭菌的PDMS孔浸在70％的乙醇 - 水混合物中。 </li></ol></li><li>如果使用的是PDMS好，刮沿着井用尖头镊子的整个边界的光活性层，为了做好后细胞培养取出PDMS时避免整个层的分离。 </li><li>覆盖纤连蛋白溶液（2毫克/升的PBS）各样品的整个表面上。每个样品500-750微升之间的体积通常是足够的，如果一个PDMS以及使用（井明面上:15×15 平方毫米）。如果没有PDMS好，约2毫升纤维连接蛋白的解决方案是必要的，完全沉浸样品;注意，虽然在溶液的基材不浮动。孵育样品在37℃下至少1小时。 </li><li>取出用玻璃吸管纤连蛋白溶液，并用2毫升的PBS洗涤一次。 </li></ol></li><li> PL吃HEK-293细胞上的纤连蛋白处理的样品以15,000细胞/ cm 2，在完全生长介质的密度。使用750-1,000微升CGM的体积为每个样品如果一个PDMS井的情况下，否则CGM 2-3毫升是必要的。孵育细胞，在37℃和5％CO 2的24-48小时。 </li></ol> 4.光刺激方案和电生理学测量<ol><li>放于水浴中的胞外溶液，在37℃至热能化;解冻细胞内的解决方案，把它放在1毫升注射器用34号针头，并保持与冰的接触，以避免ATP的降解。 </li><li>采取从孵化器细胞的基板，仔细去除PDMS以及如果存在的话。取出用玻璃吸管生长培养基和冲洗用胞外溶液的样品。缓慢进行，以避免细胞脱落。 </li><li>把设备插入到电车站与细胞外搜索解决方案的样品架n和参考电极。准备新鲜的玻璃微的膜片钳与拉马（理想的电阻范围为2-4MΩ）。填写一半的吸管与细胞内的解决方案，并将其安装在显微操作。 </li><li>寻找一个健康细胞来修补设备上。为了避免光活性基片的不需要的激励，则使用红外线照明进行成像，将长波长通滤波器（例如，一个切波长λ= 750纳米，可用于聚合物吸收在可见）中的显微镜照明器的前方。 </li><li>修补所选择的小区和通过经由显微镜（图1）的荧光激发路径传送光到样品申请光学刺激所需的协议。膜片钳协议的详细描述可以在参考20中找到。 <ol><li>膜片吸管靠近定位于细胞膜用测微操纵器。在定位，适用overpressur以e为吸移管，以避免在刀尖上的污垢粘附。移液器应该是几微米的单元格上方。 </li><li>开始降低吸移管向细胞膜，同时控制上的补丁控制软件移液器阻力。当吸液管电阻已增加大约1兆欧，从吸管同时施加轻柔的吸力，以形成所述吸液管和细胞膜之间的千兆欧密封移除超压（即，所测量的电阻应增加在大于1的值GΩ） 。 </li><li>施加负电位加到吸管接近预期细胞静息电位（对HEK293细胞的-40电位毫伏可以使用），以帮助稳定密封。补偿电容瞬变由于吸管电容与贴片上的放大器和/或补丁控制软件的相对的命令。 </li><li>打破隔膜，使电进入细胞质通过施加短暂而强烈的吸力脉冲到吸管。设置补丁放大器电流钳模式（I = 0，即，没有电流注入细胞）来跟踪细胞膜电位。等待几分钟，看细胞电位是稳定的。 </li><li>应用所需的照明协议向小区和记录在膜电位的影响。 注意:照明可以被传递到样品或者从底部或从顶部，视显微镜架构。 <ol><li>选择的激发波长是深受活性材料吸收;对于P3HT:PCBM共混，在450-600纳米范围内的波长都可以使用。合适的光激发密度的范围是10-100毫瓦/毫米2。 注意:光的短脉冲（以下:10-20毫秒）结果在小区的一过去极化，而与长期照明（几百毫秒或更长的脉冲）的持续超极化观察直到灯被关闭。 </li></ol></li&gt; </ol></li></ol>

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The authors have nothing to disclose.

关键步骤所报告的协议为体外细胞光学刺激主要涉及光敏聚合物的选择，热灭菌参数，强度和光刺激的持续时间。一个P3HT:PCBM薄膜在这里被选中，因为它保证了良好的时间和电化学稳定性。然而，人们应该注意到，并非所有的感光聚合物可提供模拟表演，22更具体在照明。此外，在这种情况下，选定的加热杀菌参数不导致的聚合物光电特性相当大的退化;通知然而，在情况下，灭菌法或热处理参数被改变，关于聚合物性质可能作用，应仔细评估。它是已知的，例如，该UV曝光迅速产生不可逆的聚合物漂白和降解，由于缀合损失。此外，不同的退火温度和持续时间可以导致聚合物表面的不同形态顺序，与电荷传输性的负面影响。光激发协议的参数应仔细评估，以及:激发密度大于100毫瓦/厘米2，和/或延长的刺激，可能容易导致聚合物降解和光刺激协议失败。在协议中的另一个关键步骤是使用一种蛋白质粘附层，由于P3HT的高疏水度:PCBM表面。如果没有粘附层采用在实现生物聚合物的界面，细胞接种和增殖的可能受到严重损害。 内上述关键步骤和约束，但是，所报告的协议可以被直接地修改，根据具体的实验需要和目标。其它稳定的，光敏感聚合物，可以发现，从7几个COM商用供应商或通过适当合成新化合物。这允许使用不同的激发波长的，从蓝色跨越到红色和NIR范围。此外，该聚合物沉积技术并不限于旋涂使用。事实上，其它方法确保良好的均匀性和合适的膜厚度可以设想，如喷墨印刷，喷涂，或刮除的灭菌方法的细胞生长介质和延长的接触的作用，应为每个新的材料进行评估考虑。尽管使用蛋白粘附层是必要的，以获得良好的细胞培养物上的聚合物表面，具体使用的纤连蛋白的顶部，如这里报告，不是强制性的。不同粘附蛋白层可以使用，也按照该细胞系生长21，例如，神经元细胞为通常在聚L-赖氨酸层进行培养。最后，照明协议可以改变并适于用上ecific需要，在波长，光斑尺寸，功率密度激励，时间的刺激持续时间和频率，光入射方向（从基板或从电解液浴）。如上面的简要说明，但是，协议参数的定义应考虑到该聚合物的光吸收范围和可能的降解作用，其可以有很大的差异，从个别情况。 一些光敏感的聚合物的有限时间和电化学稳定性实际上代表了该技术的主要局限性。另外，使用商品聚合物无需任何进一步纯化可导致在某些情况下，以不同批次的材料的，由于不同的纯度程度之间的结果的限制重复性。 我们也注意到，在在CSPP协议的光转导作用的基的确切机制仍然需要进一步阐明和特征。热学，电学和化学pH值enomena有可能参与其中，在不同的生物模型不同程度的冲击。一个完整的理解将是关键，充分利用技术。特别是，热介导的现象的发生，应审查，作为该技术的限制，因为它可以几乎不控制，并可能导致局部过热的影响，特别是在体内应用的情况下。具体实施该装置的，其目的是通过热传导路径的适当的工程控制在细胞外环境中的热扩散，可能会在下一将来揭示所必需的技术的充分开发。 在文献中，使用光脉冲对细胞和组织的温度介导的刺激不同的技术，无论是在体外 和体内都可以找到。在这些研究中，红外激光束通过水的吸收，通常用作转导机制。23-25 ​​对于红外神经刺激，所述CSPP机构具有明显的优点，特别是对在体外实验。 CSPP是基于光在中等强度的可见范围内，并且使刺激可通过标准荧光显微镜的激发路径来提供。与此相反，水的吸收需要的波长在红外（主要是围绕1.45微米和1.93微米），其具有被传递到通过外部源，如micromanipulated在制备的附近的光纤的样品中，由于一个标准的光学列车显微镜不能使用在这样的波长。在CSPP的情况下，用激光扫描系统以及空间光调制器所获得的光图案可以直接耦合到大多数显微镜的光学系统。以这种方式，衬底的基于有机半导体的光刺激可以使有可能得到几个细胞的刺激在视场独立地，既有高的时间一<html>第二空间分辨率。 光学刺激另一个非常有价值的替代方案是由遗传方法提供，基于在细胞基因光活化探针的靶向表达。光遗传学现今提供了前所未有的时间和空间分辨率，具有典型的光照强度在几十毫瓦/毫米2的范围内 ，可以既激发和抑制电活动，并且可以遗传靶向特定亚群的感兴趣的特定脑区。然而，它仍存在有需要解决的一些问题。特别是，关于使用的病毒的基因表达的安全问题，特别是在人类中，以及实现稳定和控制长期异源蛋白质的表达仍然代表了重大挑战。 CSPP技术允许以规避需要的基因转移，和所有相关的安全问题，这是特别相关的体内应用。 jove_content"&gt;所有的一切，CSPP方法提供了几个优点相对于内源性光刺激技术。由于它不是侵入性的，它可以方便地耦合到任何现有的电生理工作站，它不需要复杂的激光源或遗传转染，而保持较高的空间和时间的选择性。由于这些原因，CSPP有希望成为神经科学体外调查补充工具，并在体内的应用开辟新的前景。然而，从材料科学，物理学和工程学大的力气社区预计在未来几年，改进，优化和充分利用技术需要。

能够操作与精确的空间和时间分辨率的细胞活性的可能性表示在神经科学研究和在神经和精神障碍的治疗的关键策略1的传统方法是基于使用电极细胞电刺激定位成接近或接触目标系统，2可以是不同的复杂性（单电池，蜂窝网络，脑切片， 在体内脑组织）。在过去的一个世纪中，利用膜片钳，金属基板集成电极所提供的生理学和神经网络的运作机制的单个神经元和病理生理学的详细图片。然而，电刺激患有重要的限制。第一个涉及一种普遍较差空间分辨率由于电极和它们的固定几何形状的的物理尺寸，这是不能容易地适应像生物组织复杂的组织系统。另外，有关的刺激和记录系统之间的串扰的电极阻抗和问题可能恶化的测量值的最终信噪比3另一方面，利用光的刺激可能有助于克服许多局限性电方式。首先，它提供了前所未有的空间（&lt;1微米）和时间分辨率（&lt;1毫秒），使得能够针对特定的细胞类型或甚至亚细胞区室。此外，它是高度非侵入性，因为它避免了与感兴趣的组织的任何物理接触和disentangles刺激从记录。而且，无论光的强度和波长，可以精确地调节，从而多样化刺激方案都可以应用。3,4- 然而，动物细胞中的绝大多数不呈现给光任何特定的灵敏度。几种策略用于光学stimulation的因而被提出，或者利用附近或在细胞内的感光分子介质，或使用光敏器件外部设置的，靠近小区。前一类是指内生机制，例如通过可见光或红外光（IR）的刺激，以及利用或者光异构化/光解的化合物或光敏分子致动器（光遗传学）基因的表达。后一类包括技术实现了与使用无机纳米/微米颗粒或光电的硅衬底的外源性刺激。5然而，所有这些系统都具有光明的两侧和缺点。具体地，细胞在可见光范围内源性的吸收弱，不可靠，以及伴随的产生活性氧的物质可以是有害的细胞。在一般情况下，红外用于诱导局部热加热因吸水，但水的消光系数小，因此要求圣荣红外光（从几十到几百瓦/毫米2），它是难以通过标准显微镜光学器件以提供并可能造成安全问题为在体内的应用程序。另一方面，光笼蔽切换化合物具有时间限制的动作，往往需要UV光，是很难传递由于有限的组织穿透。此外，他们还从激活的化合物后，光解照射范围外扩散问题的困扰。最后，光遗传学工具使得科学家能够针对特定的细胞亚群和副舱室，并迅速成为的关键技术在神经科学研究之一。然而，通过病毒载体中插入的外源DNA区段提出了重要的安全问题，特别是考虑通过对人类患者。5,6-出于这些原因，新材料和设备能够细胞的光学操纵研究是一个非常热门的话题。 最近，一种新型基于使用的光敏共轭聚合物，能够有效地转导的光刺激成细胞电活动的调制手段，已经提出了。由聚合物光激发的细胞刺激（CSPP）技术利用许多键支持特性的典型的有机半导体:它们在本质上是敏感的光在可见光范围内; 7它们是生物相容的，柔软和适形和它们的机械灵活性允许与组织亲密的接口无论是在体外 和体内8-10。除此之外，他们可以很容易地官能化，以更好地适应与活细胞的界面，并启用特定激励，探测和感测能力。11,12而且，它们支持电子以及离子迁移，从而使它们适合的组合电子广告生物学13,14有趣的是，他们可以在光伏模式下工作，避免了需要应用外部偏置˚F或有效的细胞光刺激。15 CSPP技术的可靠性已在几个系统先前已经证实，包括原代神经元，星形胶质细胞15,16，17二次电池线 18和取出的视网膜组织16在这项工作中，所有必要的步骤，以制造光敏感的生物聚合物的接口 19 在体外系统的光学刺激进行详细说明。作为研究的情况下，区域规则性聚（3-己基噻吩）的一个典型的有机光生伏打共混物（RR-P3HT），用作电子给体，和苯基C61-丁酸 - 甲基酯（PCBM），充当电子受体采用。作为生物系统，人胚肾（HEK-293）细胞中被使用。设置有细胞的活性通过电生理学测量的相对记录一个光刺激协议的一个例子。 所描述的平台然而一般有效性，并且它可以很容易地扩展到使用其它共轭聚合物（通过适当地调节溶液的制备方法和淀积参数），不同类型的细胞（通过适当改变细胞培养协议，电镀过程和时间请求细胞接种和增殖）和不同的刺激方案（光的波长，刺激频率和持续时间，光激发密度）。

<table><tbody><tr><td>rr-P3HT</td><td>Sigma Aldrich</td><td>698989-5G</td><td></td></tr><tr><td>ITO-coated substrates</td><td>Nano-CS</td><td>IT10300100</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>chlorobenzene</td><td>Sigma Aldrich</td><td>319996</td><td></td></tr><tr><td>PCBM</td><td>Nano-C</td><td>Nano-CPCBM-BF</td><td></td></tr><tr><td>acetone&amp;nbsp;</td><td>Sigma Aldrich</td><td>270725</td><td></td></tr><tr><td>isopropyl alcohol</td><td>Sigma Aldrich</td><td>563935</td><td></td></tr><tr><td>HEK cells</td><td>LGC standards srl</td><td>ATCC-CRL-1573</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5244</td><td></td></tr><tr><td>E-MEM</td><td>LGC standards srl</td><td>ATCC-30-2003</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E8008-</td><td></td></tr><tr><td>FBS</td><td>LGC standards srl</td><td>ATCC-30-2020</td><td></td></tr></tbody></table>

optical control of living cells electrical activity by conjugated polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

细胞可以在P3HT容易培养:PCBM基板，提供一个合适的粘附层沉积（如在所描述的协议的步骤3.2中使用的纤连蛋白）。 P3HT:PCBM光学吸收峰在可见光谱的绿色部分;然而其他光敏感共轭聚合物可以被选择，根据优选光刺激的波长范围（ 图2）。这些基板的生物相容性已经证明不仅与细胞系18,21一样的HEK-293，也可与神经元15和星形胶质细胞的原代培养17名健康的HEK-293细胞上的培养的P3HT的典型显微照片:PCBM膜示在图3中。细胞由光敏衬底介导的光学刺激可导致细胞膜上的不同的效果，这取决于光刺激的持续时间。18后的光脉冲的开始，一快速尖峰（具有约3毫伏的强度和大约1毫秒的持续时间）所用的细胞膜电位记录（图4）中观察到。这个信号是由于在活性材料上的电荷产生的聚合物/电解质界面的电容充电。 在此之后初始的快速尖峰，一过去极化（具有约1毫伏的强度和几十毫秒量级的持续时间）中的细胞（图4）观察到。为光的短脉冲，如光被关闭时，相对的行为是观察。这些信号已被归因于在薄膜电容的变化，由于局部加热下的光吸收。 对于延长的照明，但是，在光脉冲的初始去极化变成细胞超极化（图5）。这种现象有获得热原点，但在这种情况下，它是与所述膜平衡电位的变化，由于在诱导由增加的温度的离子通道的电导率的变化。 <img alt="图1" src="/files/ftp_upload/53494/53494fig1.jpg" /> 图1.草图光活性基片的。用于蜂窝光刺激的光活性接口是由沉积在ITO膜的玻璃基板的有机半导体薄膜的。细胞可以直接在设备表面上生长后，它已被用适当的粘接层（ 例如，纤连蛋白）。 <a href="https://www.jove.com/files/ftp_upload/53494/53494fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53494/53494fig2.jpg" /> 图2. Absorp<html>不同的共轭聚合物的灰谱。使用薄膜的有机半导体可以允许在激发波长可调谐性极大，同时保留在衬底的局部透明。举个例子，不同的共轭聚合物通常用在光伏吸收光谱的报道。 <a href="https://www.jove.com/files/ftp_upload/53494/53494fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53494/53494fig3.jpg" /> 图3. HEK-293细胞培养上的光活性基片上。HEK-293细胞的光敏衬底的温育24小时后的表面上培养的典型显微照片。比例尺，20微米。 <a href="https://www.jove.com/files/ftp_upload/53494/53494fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> e_content"FO:保持together.within页="1"&gt; <img alt="图4" src="/files/ftp_upload/53494/53494fig4.jpg" /> HEK-293细胞的图4.用20毫秒光脉冲的光学刺激。一个的HEK-293细胞通过的光的20毫秒的脉冲引起的膜电位的变化。时的光的发病进行初始的快速尖峰之后是细胞的脉冲期间去极化。由于光线被关闭，相反的行为是观察。 <a href="https://www.jove.com/files/ftp_upload/53494/53494fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53494/53494fig5.jpg" /> HEK-293细胞用200毫秒的光脉冲的图5的光学刺激。变异的HEK-293细胞通过光的一个200毫秒的脉冲引起的膜电位。观察短ILLUM最初的去极化萌发的只是一个短暂的现象，这几十延长光照毫秒后自动进入细胞的超极化。 <a href="https://www.jove.com/files/ftp_upload/53494/53494fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

由共轭聚合物活细胞电活动的光控制

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

optical-control-living-cells-electrical-activity-conjugated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Istituto Italiano di Tecnologia. A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

视频: Optical Control of Living Cells Electrical Activity by Conjugated Polymers

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

科研

遗传学

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

生物工程

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

金纳米棒辅助神经细胞的刺激光

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

神经科学

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

一种通过非遗传光刺激研究 Ca2+ 传播的心脏微生理系统

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

用于同步光遗传学调制和电神经记录的光驱阵列

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.