这项工作得到奖项从HHMI 56006760 JDV，美国国立卫生研究院（NIH）（2 TL1 RR 24158-6）到DMAW，埃利森医学基金会（AG-SS-2465）和美国国立卫生研究院（GM45735 GM45735S1 GM096445）的LP。 GM45735S1从NIH发行根据美国复苏与再投资法案2009。该显微镜用于这些研究支持部分通过NIH / NCI资助（5 P30 CA13696）。

1。酵母细胞转化与生物传感器<ol><li>变换所需的酵母菌株质粒轴承的线粒体roGFP或mitGO ATeam2使用醋酸锂方法27。 </li><li>要确认改造质粒传播的生物传感器和防止质粒亏损，选择，并保持适当的选择性合成完全培养基（SC-市建局线粒体roGFP，或SC-列伊为mitGo ATeam2的）转化。如果已经比这里所描述的不同的质粒中亚克隆的荧光探针，使用适当的选择性培养基。通过荧光显微镜确认，他们表达的荧光生物传感器，并表现出正常的线粒体形态的可视化转化。 </li></ol> 2。细胞生长和准备成像细胞的功能和对药物治疗反应是高度依赖于细胞密度和代谢活性。得到最好的结果，当细胞活跃分裂的（数中期，〜0.5 - 1×10 7个细胞/ ml）。最可靠的方法来生成日志中期阶段文化一致的密度是从固定相的预培养接种。 <ol><li>准备一个固定相的预培养:挑一盘转化细胞的单个菌落接种选择性液体介质（5毫升50毫升锥形筒底部）。生长在30℃，以225 rpm振摇，直至在600nm（OD 600）的光密度培养达到平台期（24-48小时）。 </li><li>准备一个中等日志阶段文化观察:使用适当体积的预培养选择性培养基接种5毫升，50毫升锥形筒底部。 YPD介质的自体荧光和不应该被用于这些研究。使用合成完整的，基于葡萄糖，辍学（SC-市建局）媒体。细胞生长震动225转，在30℃，时间为4-16小时，直到他们到达日志中期阶段（〜0.5 - 1×10 7个细胞/ ml）。可确定细胞密度通过测量OD 600，以确定正确的OD 600读数，校准分光光度计。在我们的贝克曼DU530（Beckman Coulter公司，印第安纳波利斯，IN），日志中期阶段对应的OD 600 0.1-0.3。 </li></ol>美图roGFP1感官细胞器代谢变化波动。例如，在该测定线粒体氧化还原状态的变化时，酵母生长在发酵的碳源（ 如葡萄糖，SC媒体）对非发酵碳源（ 如甘油，如在媒体SGlyc），即使是在不同批次的同媒体上。因此，使用同一批次的媒体的所有实验。 <ol start="3"><li>细胞是正在执行的处理，如果没有准备浓度（见步骤2.4）和成像。如果正在接受治疗的细胞，培养细胞与相应的处理和继续到下一个步骤。 </li><li>浓缩液通过离心分离1ml培养在6000×g离心15秒，并将细胞沉淀重新悬浮在20μl的介质。在这些条件下的视场中可区分的细胞数量最大化。 </li><li>将2微升悬浮细胞的幻灯片。盖上盖玻片（1.5号，最好是高性能170±5微米厚），密封盖玻片边缘透明指甲油或valap的（见试剂）。要密封与valap，少量融化金属刮刀超过本生灯火焰，然后蔓延少量沿盖玻片边缘。 </li><li>在成像过程中，细胞保持在30°C。一个客观的加热器100倍油镜用于成像的工作原理以及这种应用。在这些条件下，线粒体形态，氧化还原状态和ATP水平保持不变，在成像过程中10-15分钟。 </li></ol> 3。影像设定<ol><li>宽场荧光显微镜成像线粒体roGFP1的设置 步骤都针对AxioObserver.Z1显微镜配备一个Colibri的LED激发光源，宽领域的奥卡ER相机Axiovision采集软件。两个通道及经光氧化线粒体roGFP1漂白显着减少使用LED照明相比，汞弧灯照明（见下文）。 为了最大限度地提高信号的分辨率，使用最高的数值孔径在客观上可能，提供了足够的空间分辨率的最低倍率。此外，对于线粒体roGFP成像，验证目标发送以及在365 nm。 EC PlanNeofluar目标100x/1.3NA（蔡司）以及这种应用。 配置采集软件捕获的氧化和还原线粒体roGFP1的品种。我们用下面的条件。 <ol><li>配置氧化线粒体roGFP的使用在365海里（100％LED功率）和发射滤波器适用于GFP，如38他过滤集（蔡司）激发的通道，包括激励下N个滤波器从立方体中删除。激发滤光片去除，而不需要切换过滤器允许激发波长为365纳米和470纳米，从而增加实现的时间分辨率。 </li><li>配置的信道为减少线粒体roGFP的使用激发波长为470 nm（100％LED电源），如上文所述，使用相同的发射过滤器立方体为氧化的线粒体roGFP的。将相机设置为1x1的分级优化的空间分辨率。 </li><li>设置软件采集AZ栈由11片0.5微米的间距，收集每个z位置的两个通道。这种收购模式比获得每个z堆栈反过来慢，但它可以防止氧化和还原渠道收购线粒体之间的运动而产生的文物。 </li><li>图像的一些细胞，以确定适当的曝光时间，产生一个强有力的，但不饱和的信号。重要的是要保持氧化和还原线粒体roGFP1的所有实验的比率的曝光时间秒。例如，如果曝光时间为氧化和还原线粒体roGFP1是300和100毫秒，然后为氧化线粒体roGFP1的曝光时间应该是3倍，可降低线粒体roGFP的所有实验。 </li></ol></li><li>光谱共焦显微镜设置的成像mitGO ATeam2 为了量化，使用mitGO ATeam2，GFP的荧光（最大发射510纳米）的ATP水平必须区别于OFP（最大发射560纳米）。有解决从这些荧光团发出的荧光的荧光过滤器集。然而，我们发现，光谱检测仪，有许多激光扫描共聚焦显微镜，最适合这个应用程序。光谱探测器分隔成许多组件（通常为32个或更多），根据波长的荧光发射。几个相邻的波长带，可以组合成一个图像通道。凭经验选择要合并的波长，以尽量增加从G信号FP和OFP，同时避免交叉信号，将混淆FRET测量。使用最高的数值孔径镜头。我们使用一个100x/1.49，或60x/1.49 APO TIRF镜头。我们使用尼康A1R共聚焦显微镜运行NIS Elements软件。配置采集软件捕捉ATP绑定和的ATP未绑定mitGO种ATeam2。我们用下面的条件。 <ol><li>设置针孔1.0艾里单位（AU），在我们的系统上对应AZ分辨率为0.42微米。 </li><li>设置扫描变焦接近奈奎斯特采样的限制，这将最大限度地提高图像的空间信息。在我们的系统的像素大小为0.12微米。 </li><li>由于线粒体可能在成像过程中移动，也可以是通过裁剪字段512×256像素，有助于提高成像速度。在我们的系统中的图像的一帧所需的总时间为1.0秒，包括了2倍的扫描平均。根据所需的空间分辨率，可以进一步裁剪领域越来越e时代的分辨率。 </li><li>在488 nm激发，并收集排放从500 - 520 nm的绿色荧光蛋白，并从550 - 580纳米的OFP。实际的最佳值可能会发生变化的成像系统的特性。 </li><li>我们的系统是最佳​​的激光功率在6.0％和6.4％之间。每个显微镜系统的最佳的激光功率会有所不同，但最好是尽可能的低，同时仍然产生一个解释的图像。使用内部或外部功率计来监控激光功率的变化，这通常发生在任何光学系统中随着时间的推移。 </li><li>调整检测器的增益和照明光的强度，以最大限度地提高检测到的范围内的象素值，但避免饱和的信号，为尽可能多的细胞。不分析任何细胞中含有超过1％饱和像素。所有样品图片使用相同的目标，激光功率，扫描变焦，像素的大小，增益和偏移。 </li></ol></li></ol> 4。图像采集<ol><li>找到焦点解放军<html>还使用透射光，以尽量减少漂白的荧光探针的细胞之一。 </li><li>定位一个或多个感兴趣的细胞后，收集的z系列，通过使用步长为0.5μm的典型细胞（大约7微米）的整个深度。 </li><li>图片其他细胞，在幻灯片上的利益，但不超过15分钟，单个幻灯片图像。在幻灯片上15分钟后，细胞失去活力。 </li></ol> 5。分析通过测量在560 nm处的mitGO ATeam2排放的比例在510 nm处测定线粒体ATP水平27的氧化还原状态的细胞器是减少氧化比（R / O）线粒体roGFP的测量， 即排放除以排放在510 nm处后，在365 nm激发波长为470 nm激发后在510 nm处。在计算比率，减去背景，并确定一个阈值的像素属于荧光线粒体。 帐篷"公共领域（ 如 ImageJ的30），或市售的软件（ 如 Volocity，Perkin-Elmer公司）可用于分析的线粒体roGFP1或mitGO ATeam2。根据软件，用于图像采集和分析，图像可能需要先被转换为另一种格式，例如TIFF，打开它们之前在分析软件中，如果图像被转换时，它是必不可少的，以验证在转换过程中不改变像素值。作者:线粒体roGFP1数据分析，使用这两个程序如下所述。 粗体斜体突出显示每个菜单内选择程序菜单和选项。 5.1 ImageJ的分析<ol><li>打开图像和变化类型为32位: 图像→类型→32位。 </li><li>绘制在一个地方有没有细胞的权益回报率（ROI）的区域。在这个投资回报率计算的平均强度分析→测量。 </li><li>从堆栈中减去计算的平均值背景: 处理→数学→减去。 </li><li>减去Z-Stack的使用，找到中间片和线粒体门槛: 图像→调整→阈值和阈值窗口上单击" 应用 "。适用堆栈中的所有切片。检查设置背景像素为NaN。 </li><li>创建比Z图库: 处理→图像计算器堆栈将减少氧化Z图库线粒体roGFP1分析。除以560纳米的（ATP方向）510纳米（ATP绑定）图像为mitGO的ATeam2分析图像。 </li><li>感兴趣的区域周围绘制的投资回报率。选择分析→工具→ROI经理 ，并单击" 添加到记录的投资回报率。可被存储在管理器的多个区域。在投资回报率管理器中，选择所有的ROI，然后选择M矿石→多功能测量，测量所有的堆栈片。数据导出到电子表格分析。 </li></ol> 5.2 Volocity分析<ol><li>导入图像到Volocity库和2个通道创建一个图像序列。 </li><li>绘制在一个地方有没有细胞的权益回报率（ROI）的区域。选择: 工具→比率。 </li><li>使用Volocity计算背景: 从投资回报率。 </li><li>调整阈值，包括线粒体的结构。 </li><li>检查选择申请一个彩虹LUT的比例通道。的强度调制的信道，可能会产生一个减少噪音的图像进行表示，但它不应该被用于定量的平均比例。 </li><li>选择" 测量 "选项卡上，选择比例通道和感兴趣的区域周围绘制的投资回报率。测量比通道，但不包括零值。多个区域可以被选择和测量在同一时间。数据导出到电子表格分析。 </li></ol>

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没有利益冲突的声明。

在这里，我们介绍使用方法线粒体roGFP1 mitGO ATeam2的生物传感器在活酵母细胞线粒体的氧化还原状态和ATP水平评估。我们发现，表达的质粒量化目标线粒体，线粒体的形态和分布或细胞生长率没有任何明显的影响的的传染的的线粒体roGFP1或mitGO ATEAM结果。3美图roGFP1的检测线粒体氧化还原状态的变化，从高氧化，高度降低的状态。同样，mitGO ATEAM可以测量发生呼吸抑制剂治疗呼吸驱动增长和酵母在酵母线粒体ATP水平的变化发生。此外，由于两种生物传感器提供亚分辨率，他们可以检测单个细胞内线粒体的氧化还原状态或ATP水平的异质性。最后，由于这两种生物传感器比例式传感器，它们不影响样品的厚度或生病的变化在浓度或变化在可选最底照度。这些探头提供了一个学习分辨率在活细胞与亚细胞线粒体功能的微创方法。 要成功地应用这种方法，图像应以最大可能的信号噪声比收购。第一个重要步骤是从10-20转化菌落在显微镜下检查细胞，并选择那些具有强大的荧光和正常线粒体的形态和生长率。接着，成像条件进行优化，以产生高，但不饱和，强度，而不会造成荧光蛋白或细胞的光损伤。人口中的一些细胞（&lt;1％）可能有异常高或低的整体荧光由于质粒拷贝数变化可以忽略这些有利于可解释的图像捕捉多数细胞。 虽然好处线粒体roGFP的是明确的，它也是重要的是要意识到潜在的陷阱。我们检测线粒体的氧化还原电位的差异，不只是用在细胞代谢的碳源引起的差异，而且还取决于在不同批次的相同的培养基中的酵母传播。因此，必须小心选择时，线粒体的氧化还原测量细胞的生长介质。此外，，虽然线粒体roGFP提供了前所未有的空间分辨率的线粒体的氧化还原状态，时间分辨率roGFP是不足够检测阵阵ROS，都与ROS信号转导31。最后，必须注意，当选择的激发波长和强度的氧化形式roGFP。在400 nm处的照明优化激发的氧化的roGFP物种。然而，它也激发减少roGFP的，在更大的程度相比，在365nm处激发。这将导致线粒体氧化还原状态的测量灵敏度降低。此外，在400 nm处的高强度照明曝光线粒体roGFP1的蛋白质的光转化的规范指改变光谱特性。我们还没有看到光转化后在365纳米的高强度照明。因此，我们建议使用365纳米的激发，如果可能的话。 MitGO ATeam2也有其局限性。例如，，，自mitGO ATeam2是本身就是一种蛋白质，它可以被损坏以蜂窝辱骂，包括活性氧，这可能会损害其生物传感器活性。此外，必须解决GFP和OFP发射波长（510和560 nm）的荧光共振能量转移分析，这可能是困难的传统的荧光显微镜。 不像在体外酶试验，这些活细胞检测报告线粒体ATP或氧化还原状态的相对变化，不ATP浓度测量绝对或减少半胱氨酸。虽然在理论上可以通过测量传感器的蛋白质在溶液中的反应产生的标准曲线，这可能不适用于线粒体内的不同的分子环境。进一步<html>安伏，这些检测方法的比较，在不同的条件下的细胞提供了一种手段。此外，成像条件不尽相同，因此可能不会被复制在另一个收购的一个成像设置确切的比值。 这些比成像技术可以适用于其他比例的指标，包括的其他roGFP或GO-ATEAM的亚型和其他功能探头。宽视场或共聚焦显微镜的选择将取决于探头和所需的空间分辨率。宽视场的方法，用在这里线粒体roGFP的，因为它允许在365nm处激发，从而提高了传感器的灵敏度和稳定性。这里所用的为mitGO ATEAM，光谱检测仪，共聚焦方法提供了一个方便的方式来消除发射光谱检测窗口，通过调整交叉。然而，它可以在宽视场显微镜进行类似的实验，通过使用自定义过滤器或消除交叉计算。 ntent"&gt;在这种类型的成像实验的最常见的困难是不足的信号。成像硬件（尤其是物镜和检测器），收集足够的信号，以对数据的关键。 阈值的步骤的结果有很大的影响，列入背景像素可以打击任何趋势得到的比率。自动的方法是优选的，但是由于有限的信号，手动阈值通常是最为有效的方法。如果手动阈值被使用时，所使用的标准应该尽可能好地阐述，并分析可能需要执行失明或由不同的实验者展示再现性。可用于抗霉素A（为mitGO Ateam2）和H 2 O 2或DTT（线粒体roGFP）的对照实验以确认以荧光比预测的变化。 美图roGFP和mitGO ATeam2非侵入性，比例探头监控三刀在活酵母细胞的线粒体健身。这里使用的有针对性的比例传感器线粒体线粒体特定信号序列融合。其他细胞区室，可以研究与另外的合适的靶序列的原始传感器。此外，它是可能的结合，这些传感器中的任何一个与互补的荧光标记物（ 例如，细胞器或信号因子）或传感器（ 如钙），在活细胞中，同时测量多个过程。

线粒体ATP的产生，氨基酸，脂肪酸，血红素，铁硫簇和嘧啶的生物合成的细胞器是必不可少的。线粒体发挥钙稳态的关键角色，并在细胞凋亡的调控。1增加的证据链接线粒体老化和与年龄有关的疾病，包括帕金森氏症，阿尔茨海默氏病，肌萎缩性侧索硬化症，亨廷顿氏病。2虽然个人住他们的整个生活与神经退行性疾病都与线粒体蛋白质的突变，这种疾病的症状只出现在以后的生活。这表明发生变化，随着年龄的增长，使疾病的病理出现线粒体。事实上，与整体细胞的健康和寿命在酵母和哺乳动物细胞线粒体健身3,4在这里，我们介绍了如何使用基因编码的荧光生物传感器，比例来评估两个关键功能mitoc活酵母细胞hondria:氧化还原状态和ATP水平。 线粒体功能的有氧能量动员是公认的。线粒体的氧化还原状态的物种减少和氧化的细胞器，包括NAD + / NADH，FAD / FADH 2，NADP + / NADPH，谷胱甘肽/氧化型谷胱甘肽（GSH / GSSG）和活性氧（ROS）的产物。线粒体解偶联或缺氧影响线粒体呼吸活动，改变比例的NAD + NADH在细胞器。活性氧，所生产，从低效内线粒体膜的电子传递链的复合物，以及胺的脱氨作用，通过线粒体外膜的单胺氧化酶的5星 ，损害脂质，蛋白质和核酸之间的电子转移，并有被认为与老化和年龄相关的神经退行性疾病6,7,8。 ROS信号转导中也发挥了作用，在mitochondria，通过氧化作用的谷胱甘肽。例如，NADH脱氢酶不仅有利于活性氧的产生，但也调节通过与谷胱甘肽池9,10相互作用。 α-酮戊二酸脱氢酶，乌头酸，TCA循环的组成部分，展览活动减少，在氧化环境11,12。 事实上，乌头酸活性的氧化还原依赖调节是保守的，从细菌到哺乳动物13,14。因此，监测的氧化还原状态和线粒体ATP水平是至关重要的，以了解它们的功能和作用，在疾病的病理。 生化的方法已被用来评估整个细胞的氧化还原状态或ATP水平或分离的线粒体。广泛使用的方法，以评估氧 ​​化还原状态的全细胞或分离线粒体基于测量的氧化还原对的GSH / GSSG 15的水平。荧光素 - 荧光素酶系统通常用于测量线粒体在任一透整个细胞或分离线粒体的ATP水平。16,17,18,19,20在该试验中，荧光素酶的ATP结合和催化从荧光素的氧化和化学发光。21所发出的光的强度的量成比例在反应混合物中的ATP 22 这些方法已揭示的基本信息，对线粒体的功能，包括发现，神经变性疾病，如阿尔茨海默氏病，患者具有异常低的ATP水平。[23]然而，它们不能被用于图像的生活，完整的细胞。此外，全细胞分析方法的基础上提供了在所有车厢的细胞的氧化还原状态或ATP水平平均。在孤立的细胞器是测量潜在的问题，因为在亚细胞分离线粒体氧化还原状态或ATP水平可能会改变。最后，从我们的实验室和其他最近的研究表明，异构单个细胞内线粒体的功能，这反过来又影响母亲和女儿细胞的寿命。 因此 ，有必要在活细胞与亚细胞分辨率来衡量线粒体ATP水平和氧化还原状态。 这里描述的线粒体功能的生物传感器都是基于GFP。氧化还原敏感的GFP（roGFP）24,25的表面暴露的半胱氨酸被添加到该分子的GFP变异体。 roGFP，像野生型GFP，有两个激发峰（〜400纳米〜480纳米）和1〜510 nm的发射峰。在roGFP〜400 nm的激发波长的增加导致氧化的半胱氨酸残基。这些半胱氨酸的减少有利于激发〜480纳米。因此，比例为510 nm发射峰在480nm和400 nm的激发roGFP时揭示的相对量减少，氧化roGFP的，这反映了荧光基团的氧化还原状态的环境。 两个自愿减排量离子roGFP广泛应用于:roGFP1 roGFP2。两者都包含相同的半胱氨酸插入。是根据roGFP1野生型GFP roGFP2的是根据S65T的GFP，它具有更有效的激励wtGFP 24相比，在480nm和在400nm处的激发效率较低。 roGFP1 pH敏感比roGFP2，其动态范围进一步扩展到范围减小。因此，roGFP1可能是更有益的监测更减少车厢如线粒体或胞液中，并具有可变的pH值，如内涵体的隔间。 roGFP2提供明亮的信号，并在一些研究中，动态范围更大比roGFP1 24,26。在拟南芥（Arabidopsis thaliana）的研究表明，类似的两个传感器（吨半氧化，65和95秒和吨半减少，272和206秒，roGFP1和roGFP2，分别）的氧化还原状态的变化所需的时间。26 是一种微创，可靠MitGO ATeam2传感器，用来测量在萌芽酿酒酵母线粒体ATP。 GO-ATEAM的一个福斯特共振能量转移（FRET）探针，由ε亚基的F o的F 1-ATP合酶之间夹着27 FRET的供体和受体荧光蛋白（GFP和橙色荧光蛋白（OFP）的，分别）。的ATP结合ε亚基蛋白质的构象变化，带来的结果FRET的供体受体靠近，并允许从供体到受体的能量转移。有两个变种GO-ATEAM的，GO-ATeam1和GO-ATeam2的。 GO-ATeam2具有较高的亲和力MgATP GO-ATeam1相比的，使其更适合用于测量通常较低[27]在线粒体中ATP的细胞质。 为了探讨线粒体氧化还原状态，我们构建了融合蛋白（美图roGFP1）由roGFP1，融合到线粒体前导序列一ND表示从着丝粒为主（低拷贝数）控制下的强烈甘油醛-3 - 磷酸脱氢酶（GPD）的子（p416GPD Addgene）酵母表达质粒。我们使用roGFP1老化模型真菌酿酒酵母（Saccharomyces cerevisiae）的上下文中，以探测线粒体氧化还原状态。我们发现，roGFP1可以检测线粒体氧化还原状态的变化过程中发生的老化和营养可用的，但没有明显的不利的影响酵母细胞。我们也看到个别活酵母细胞内线粒体的氧化还原状态的变化，这一发现强调了重要性的生物传感器与亚细胞空间分辨率。 MitGO ATeam2的一个变种GO ATeam2，其中有在氨基端的GO-ATeam2的线粒体信号序列插入细胞色素c氧化酶亚基VIIIA 27我们修改了mitGO ATeam2的探头（请提供实验室H.野地研究所Øf科学和工业研究，大阪大学，日本）使用亚克隆酵母，通过Xba1和HindⅢ网站，进入酵母表达载体pAG415GPD的CCDB（Addgene，剑桥，MA，USA），这是一个低拷贝质粒含有强组成GPD启动子。我们在芽殖酵母表达mitGO ATeam2，并发现，通过复染的DNA结合染料DAPI，它完全定位到线粒体，它作为一个有效的探针测量生理变化线粒体ATP水平。 roGFP和GO-ATEAM的基因编码。其结果是，它们可以被引入，并稳定地保持在完整的细胞中，并提供个人，活细胞的氧化还原状态或ATP水平的信息。此外，这两种生物传感器监测在生理条件下发生的氧化还原状态或ATP水平的变化，也是比例28两种探头。其结果是，与这些探针的测量不会受到茶生物传感器浓度或样品照明或厚度的民营企业。最后，无论是生物传感器提供的亚细胞空间分辨率。事实上，已针对roGFP线粒体，ER，内涵体和过氧化物酶体24，而且可以检测到这些细胞器的氧化还原状态的变化，基本上与pH值无关。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich (St. Louis, MO)</td>
			<td>1397-94-0</td>
			<td>Dissolved in ethanol to a 2 mg/ml stock solution.</td>
		</tr>
		<tr>
			<td>SGlyc (synthetic glycerol-based) yeast growth medium *omit for SGlyc-Ura 
			**omit for SGlyc-Leu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Dissolve in H2O. Adjust pH to 5.5 with NaHCO3. Autoclave. 
			 
			Ingredients: 
			0.67% Yeast nitrogen base without amino acids 
			3% Glycerol 
			0.05% Glucose 
			2 mg/ml adenine 
			2 mg/ml uracil* 
			1 mg/ml L-arginine 
			1 mg/ml L-histidine 
			1 mg/ml L-leucine** 
			3 mg/ml L-lysine 
			2 mg/ml L-methionine 
			4 mg/ml L-phenylalanine 
			2 mg/ml L-tryptophan 
			3 mg/ml L-tyrosine</td>
		</tr>
		<tr>
			<td>SC (synthetic complete, glucose-based) yeast growth medium *omit for SGlyc-Ura 
			**omit for SGlyc-Leu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Dissolve in H2O. Adjust pH to 5.5 with NaHCO3. Autoclave. Ingredients: 
			0.67% Yeast nitrogen base without amino acids 
			3% Glucose 
			2 mg/ml adenine 
			2 mg/ml uracil* 
			1 mg/ml L-arginine 
			1 mg/ml L-histidine 
			1 mg/ml L-leucine** 
			3 mg/ml L-lysine 
			2 mg/ml L-methionine 
			4 mg/ml L-phenylalanine 
			2 mg/ml L-tryptophan 
			3 mg/ml L-tyrosine</td>
		</tr>
		<tr>
			<td>Valap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Combine ingredients in a 1:1:1 (w:w:w) ratio. Melt by submerging in a 70 &deg;C H2O bath. Aliquot into glass petri dishes. Store at room temperature. Ingredients: 
			Vaseline petroleum jelly, hard paraffin, lanolin</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment and Software</td>
		</tr>
		<tr>
			<td>Precleaned Gold Seal Rite-on Micro Slides</td>
			<td>Thomas Scientific (Swedesboro, NJ)</td>
			<td>3050</td>
			<td>Size: 25 x 75 mm; Thickness: 0.93 to 1.05 mm</td>
		</tr>
		<tr>
			<td>High-performance coverslips, No. 1.5, 18x18 mm</td>
			<td>Zeiss (Thornwood, NY)</td>
			<td>474030-9000-000</td>
			<td>These are less variable in thickness (170&plusmn;5 &mu;m) than standard coverslips, reducing spherical aberration and improving 3D imaging performance</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Cover Glass, No. 1.5</td>
			<td>Fisher Scientific (Pittsburgh, PA)</td>
			<td>12-545E</td>
			<td>Size: 22 x 22 mm, No. 1.5 thickness (170 &mu;m)</td>
		</tr>
		<tr>
			<td>A1 laser scanning confocal microscope with spectral detector and 100x/1.49 NA Apo-TIRF objective</td>
			<td>Nikon (Melville, NY)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AxioObserver.Z1 microscope equipped with a 100x/1.3NA EC Plan-Neofluar objective (Zeiss) and Orca ER cooled CCD camera (Hamamatsu) and controlled by Axiovision software</td>
			<td>Zeiss (Thornwood, NY); Hamamatsu (Hamamatsu City, Japan)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis software</td>
			<td>Perkin Elmer (Waltham, MA)</td>
			<td>&nbsp;</td>
			<td>Restoration module for deconvolution; Quantitation module for ratio calculation and measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health (Bethesda, MD)</td>
			<td>&nbsp;</td>
			<td><a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a></td>
		</tr>
	</tbody>
</table>

ratiometric biosensors that measure mitochondrial redox state and atp in living yeast cells

线粒体有许多细胞过程中的角色，从能量代谢和钙稳态控制细胞的寿命和程序性细胞死亡。这些过程的影响和受氧化还原状态和线粒体ATP生产。在这里，我们描述了使用两个比例，基因编码生物传感器的活酵母细胞可以检测线粒体氧化还原状态和ATP的水平，在亚细胞分辨率。线粒体氧化还原状态是​​用氧化还原敏感绿色荧光蛋白（roGFP）的线粒体基质中，有针对性地计量。水户roGFP包含半胱氨酸残基的位置147和204的GFP，经历可逆的，依赖于环境的氧化和还原反应，这反过来又改变的蛋白质的激发光谱。 MitGO ATEAM是一个福斯特共振能量转移（FRET）探针，其中F o的ε亚基的F 1-ATP合酶被夹持在FRET的供体和受体荧光蛋白rescent。 ATPε亚基中的蛋白质构象变化带来的结果结合FRET供体和受体在接近并允许从捐赠者到受体荧光共振能量转移。

测量线粒体氧化还原状态与线粒体roGFP 在这里，我们表明，线粒体roGFP1的动态范围来检测线粒体氧化还原状态的变化，从完全氧化在活酵母细胞减少，而不会影响酵母细胞的生长或线粒体的形态。首先，我们发现，细胞线粒体有针对性的GFP和roGFP1的增长，正常率（ 图1A）。最大生长速率，最大坡度为日志相生长期间生长曲线和时间达到最大生长速率，是类似表达线粒体GFP和线粒体roGFP1的的细胞中。此外，线粒体在酵母中表达线粒体roGFP1的展览野生型形态（ 图1B）。具体来说，他们是管状的，沿母芽轴对齐，并积聚在母亲和女儿细胞的提示。由于线粒体功能障碍往往导致碎裂，这是正常的形态支持的想法线粒体roGFP1不扰乱线粒体功能。 为了评估线粒体roGFP1的动态范围，我们对待中旬日志相野生型酵母细胞的过氧化氢 ​​（H 2 O 2）和二硫苏糖醇（DTT），分别测得的平均线粒体R / O比评估线粒体氧化还原状态（ 图2）。用H 2 O 2的滴定或DTT从0到10毫米的查询结果的测量范围从0.6（在氧化条件下）为1.23（在还原条件下）以剂量依赖的细胞平均R / O比的变动。因此，线粒体​​roGFP1的是一种有效的生物传感器在活细胞中的线粒体氧化还原状态的分析。 美图roGFP1还提供亚细胞线粒体的氧化还原状态的决议。亚的决议表明，单个酵母细胞内线粒体相对氧化还原状态不同。如果在一个单一的酵母细胞的线粒体功能上是不同的，这是有可能的y是被动地或主动隔离（ 图3）。这种隔离可以母女年龄不对称和子细胞的复兴做出贡献。该发现强调了需要的生物传感器与线粒体的氧化还原状态的亚细胞分辨率。 长期以来，人们一直称为31的光能够诱发GFP生色团的变化。当暴露于高强度的400纳米的光，光转化roGFP1经历一个物种具有不同的发射光谱26。光电转换发生时，有绿光发光氧化线粒体roGFP1的减少和绿色发光激发减少线粒体roGFP1，改变的R / O线粒体roGFP1的（ 图4B）的比例增加。使用低强度的激励（ 例如，在40％的照明科利柏光源使用400nm的LED），没有显着的光转化，在分析期间（ 图4C）。在p<html>称为方法，以减少光转化是在365nm处激发的氧化形式的roGFP（见讨论）。 测量线粒体ATP与mitGO ATeam2 MitGO ATeam2定位在哺乳动物细胞中表达时，27位到线粒体我们发现，mitGO ATeam2共定位在酵母线粒体DNA用DAPI染色，并且被发现在野生型酵母线粒体（ 图5A）的典型的管状结构。因此，我们证实线粒体定位序列从哺乳动物细胞色素c氧化酶亚基VIIIA而不破坏线粒体的形态在酵母线粒体定位探头。此外，我们发现，细胞的生长速度表达mitGO ATeam2类似细胞表达的线粒体定位的绿色荧光蛋白在葡萄糖和甘油媒体的（ 图5B）。因此，表达的mitGO ATeam2并不会出现有害细胞或线粒体。 的_content"&gt;接下来，我们测试是否的mitGO ATeam2 FRET响应变化在线粒体ATP水平。要做到这一点，我们处理的细胞与抗霉素A，细胞色素C还原酶剂结合，抑制电子传递链的氧化辅酶Q破坏质子梯度，抑制线粒体ATP生产的20位数FRET抗霉素治疗的细胞中的比例下降（ 图6C）。 给出的证据表明，mitGO-ATeam2是一种有效的生物传感器的线粒体ATP，我们使用这种探头监控在酵母线粒体ATP水平，使用发酵的或非发酵的碳源（ 图6A和6B）传播。在葡萄糖生长细胞的总荧光面积的减少证实，减少线粒体丰富的葡萄糖阻遏结果。我们注意到在水平的mitGO ATeam2细胞与细胞之间的变化。有趣的是，我们的初步统计数据表明，有马<html> y是同一细胞内ATP水平的差异，在不同的线粒体（ 图6D）。 <img alt="图1" src="/files/ftp_upload/50633/50633fig1hires.jpg" fo:content-width="6in"/> 图1。美图roGFP1检测线粒体氧化还原状态，而不影响细胞的生长率或线粒体形态（A）生长的酵母表达线粒体有针对性的GFP或RO-GFP1的测定在600 nm处光密度的变化作为时间的函数的增长SC-URA液体培养基中，在30°C（B）上板:原始图像的正常线粒体形态和本地化渠道。较低的面板比率图像显示的减少通道除以氧化通道（右）的颜色参考。图像显示效果的阈值选择的结果。最优阈值包括线粒体的结构和排除背景。jove.com/files/ftp_upload/50633/50633fig1hires.jpg"目标="_blank"&gt;点击这里查看大图。 <img alt="图2" src="/files/ftp_upload/50633/50633fig2hires.jpg" fo:content-width="4in" fo:src="/files/ftp_upload/50633/50633fig2.jpg"/> 图2。美图roGFP1检测线粒体氧化还原状态的变化在治疗与过氧化氢 ​​或二硫苏糖醇（AB）中秋节日志相酵母细胞表达线粒体roGFP1的孵育在SC-市建局媒体与不治疗（0）或指定的浓度H 2 O 2或DTT 20分钟，在30°C（A）R / O线粒体roGFP1的比例图像叠加在透射光图像，显示细胞轮廓的最大强度的预测。右上角显示线粒体roGFP1的的颜色参考。酒吧:5微米（B） </R / O线粒体比例roGFP1的每一个实验条件下的强&gt;定量。错误酒吧平均标准误差。结果获得至少20个细胞，每个条件下， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50633/50633fig2hires.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="3.5in" src="/files/ftp_upload/50633/50633fig3.jpg" /> 图3。美图roGFP1提供的亚细胞线粒体的氧化还原状态的决议。最大强度投影R / O的线粒体roGFP1比通道的透射光图像上叠加。右上角显示线粒体roGFP1的的颜色参考。酒吧:5微米。这种特殊的细胞的平均R / O的线粒体roGFP1比为0.88。然而，个别细胞内线粒体的氧化还原状态显示不同。所显示的数字计算出的R / O线粒体比例roGFP1的不同的线粒体重新的gions <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50633/50633fig3.jpg" target="_blank">点击这里查看大图。</a> <img alt="图4" src="/files/ftp_upload/50633/50633fig4.jpg" fo:src="/files/ftp_upload/50633/50633fig4hires.jpg" /> 图4。高强度的激励导致光电转换和改变的R /的线粒体roGFP1比中秋节日志相酵母表达线粒体roGFP1的40％（A）或100％（B）LED光源的功率从400纳米的光照射在从减少和氧化线粒体roGFP1的，荧光被抓获每4秒。图片显示的是最大的预测，其颜色代表的R / O比线粒体roGFP1的（见色彩右下规模）。酒吧:5微米（C）R / O线粒体roGFP1的比例超过40％的LED功率照明时成像期间保持不变。然而，在LED照明100％力量，我们观察时间依赖性变化的R / O比线粒体roGFP1，它反映的荧光光控。黑色的正方形，40％的LED电源;灰色钻石，100％LED功率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50633/50633fig4hires.jpg" target="_blank">点击此处查看大图。</a> <img alt="图5" fo:content-width="4.5in" src="/files/ftp_upload/50633/50633fig5.jpg" /> 图5。定位于MitGO ATeam2在酵母线粒体，不会影响酵母细胞的生长率（A）酵母细胞表达mitGO ATeam2的生长数中期，固定使用多聚甲醛和与DNA结合的染料DAPI染色，如前所述。 19所示的图像的最大强度的预测反褶积的Z系列。为简单起见，的mitGO的ATeam2图像获得使用常规GFP过滤器，所以它们仅表示对ATP的人口有未结合的。细胞出线示于白色。 N:细胞核DNA。线粒体DNA:线粒体DNA。酒吧:为1μm（B）野生型酵母细胞中的生长曲线表达线粒体定位的GFP或中mitGO ATeam2葡萄糖基（SC）和基于甘油（SGlyc）的液体培养基中在30℃下这一数据是平均每一株在每个时间点的一式四份，代表两个独立的实验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50633/50633fig5.jpg" target="_blank">点击这里查看大图。</a> <img alt="图6" fo:content-width="5.5in" src="/files/ftp_upload/50633/50633fig6.jpg" /> 图6。 - ATeam2 MitGO措施的变化在酵母线粒体ATP水平亚细胞和suborganellar分辨率（AB）排放mitGO ATeam2 GFP（510纳米）和OFP（560纳米），和定量的酵母的560/510 nm处的发射比细胞培养过夜葡基于SE-（SC）（A）或基于甘油（SGlyc）的（B）媒体。 560/510的比例通道显示在右下方的颜色参考。酒吧:1微米（C）560/510的比例为细胞定量在SGlyc媒体传播带或不带抗霉素A（2微克/毫升）1小时30°C。在ATP水平下降后，抗霉素A治疗统计学显着性（秩瓦利斯意义测试）（D）560 nm/510纳米比的mitGO ATeam2中旬日志相野生型细胞SC媒体上传播。 560/510的比例通道显示在右下方的颜色参考。以白色示出的细胞轮廓。在整个小区560/510 nm处的平均比例为0.43。显示的数字是560/510 nm处比线粒体内的特定区域。酒吧:1微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50633/50633fig6.jpg" target="_blank">点击这里查看大图。</a>

Watch this Scientific Journal Video about Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells at JoVE.com

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

我们描述了使用两个比例，基因编码生物传感器的，这是基于GFP，监测线粒体的氧化还原状态和细胞内ATP水平分辨率在活酵母细胞。

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

ratiometric-biosensors-that-measure-mitochondrial-redox-state-atp

Nanyang Technological University

Research

JoVE Journal

Bioengineering

21.0K 次观看.  Columbia University. 我们描述了使用两个比例，基因编码生物传感器的，这是基于GFP，监测线粒体的氧化还原状态和细胞内ATP水平分辨率在活酵母细胞。

视频: Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

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.

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

由共轭聚合物活细胞电活动的光控制

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

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...