该项目由国家卫生研究院资助, GM018568。该协议的一部分是在纳米系统中心 (CNS), 这是国家纳米技术协调基础设施网络 (NNCI) 的成员, 得到国家科学基金会的支持下, NSF 奖1541959。CNS 是哈佛大学的一部分。许多感谢是由于托马斯诺曼和内森勋爵的工作, 在构思和制作的主模板使用的设备, 在这里显示和建设的原始版本的设备。我们还感谢约翰 Paulsson 的宝贵合作意见, 感谢他的实验室成员的意见和不断改进的细菌微流控装置。

1. 硅橡胶器件铸件
<ol><li>在无尘环境中, 将聚氧烷聚合物和固化剂以10:1 的比例混合, 在真空下至少10分钟。</li>
	<li>在聚苯乙烯培养板上的一块完整的铝箔上放置一个硅母 (或其环氧或聚氨酯复制品)。倒入脱的硅橡胶混合物在大师到深度大约5毫米. 加气培养皿至少10分钟. 使用柔和的气流来打破表面气泡。 注意: 所使用的双层设备的尺寸在随附的 CAD 文件5中提供 (请参阅补充文件 1)。在脱气过程中, 塑料复制主可以部分浮动;如果发生这种情况, 用一个干净的塑料涂抹器向下推复制副本。</li>
	<li>在60° c 的烤箱中固化至少1小时, 冷却至室温。从培养皿中取出含有该母材的铝箔和硫化的硅橡胶。小心剥去主人背上的铝箔, 然后小心地剥去主人的皮。 注: 另外, 在室温下, 可以在一夜之间治愈。硅晶片的相对脆弱性可以克服使用环氧树脂胶粘剂永久债券的硅片, 以1/16 英寸厚的铝盘大小相同的硅片。</li>
	<li>由于晶圆通常包括几种微流体设备, 尺寸略有不同 (请参阅补充文件 1), 使用干净、锋利的手术刀将单个设备切割成尺寸。 注意: 对于例程B. 枯草, 请使用具有1.0 µm 宽的单元格沟槽的设备, 在随附的 CAD 文件中用 C 或 F 标记。</li>
</ol>2. 设备冲压和粘合
<ol><li>在设备的图案一侧放置一条半透明的办公室磁带 (请参见材料表), 以增强图案化功能的可见性。入口和出口端口被确定为可见的射流通道两端的圆形区域 (图 1)。为了提高入口和出口引脚在设备上冲孔时的能见度, 请使用细尖标记标记入口和网点。 注意: 为了确保入口和出口引脚不拥挤, 每个其他通道都被打上, 从该设备的字母指示符下面的通道开始。</li>
	<li>翻转的硅橡胶设备, 使图案的一面是下来, 并通过该设备打孔通过0.75 毫米活检冲床的标记点;丢弃 punchings。 注: 由于活检穿孔所产生的孔通常在穿孔进入聚合物的地方略有爆发, 所以设备被打成图案的一侧。以倒置的方向冲孔设备可确保图案一侧的孔具有锋利的边框。</li>
	<li>使用显微镜, 确保穿孔孔与设备的入口和出口区域重叠。使用细尖的镊子, 以消除任何外来的碎片, 从穿孔孔。</li>
	<li>使用胶带去除设备两侧的灰尘, 放入干净的聚苯乙烯培养板。用100% 异丙醇润湿22毫米 x 40 mm 盖玻璃, 并用无尘擦拭擦;用无尘的空气或氮气流干。</li>
	<li>将穿孔的硅橡胶设备功能端与清洗过的盖玻璃一起放到氧气等离子清洗器中。<ol>
<li>等离子处理设备的设置如下: 真空, 70 mTorr;O2压力, 200 mTorr;期间, 十五年代;电源, 30 w 在等离子处理期结束后立即取出设备并将玻璃从等离子清洗器上盖上。</li>
		<li>倒置的硅橡胶, 并把它放在封面玻璃的中心, 使图案的一面接触玻璃;确保进料通道 (在入口和出口区域之间运行) 与盖玻璃的长轴对齐。按下非常轻轻, 以确保对玻璃的硅橡胶密封。</li>
	</ol>
</li>
	<li>在烤箱中烘烤至少1小时60° c 的组装装置。烘烤后, 通过在设备的一个角落轻轻推送, 以手动验证该产品是否与玻璃结合。 注: 一旦设备粘合, 应在24小时内使用, 因为聚合物将变得越来越疏水性随着时间的推移, 等离子体处理。</li>
</ol>3. 微流体管道的制备
<ol><li>聚合物管材的切割长度 (内径 (ID) 0.02 英寸, 外径 (外径) 0.06 英寸) 到适当的长度, 以达到从注射器泵到开关设备 (如果使用) 和设备时, 安装在显微镜上。切割尽可能多的长度, 因为有微流体通道。</li>
	<li>通过切割和附加2厘米长的挠性硅胶油管 (0.03 英寸 ID, 0.065 英寸外径) 到 "y" 和1厘米长的硅胶油管到底部的 "y" 的倒钩上, 为夹紧阀装配 y 接头 (如果使用)。</li>
	<li>切割10厘米 (或适当) 长度的聚合物管材;通过将它们插入到 "Y" 的底部倒钩上的1厘米硅管段, 将它们一端连接到开关接合处。连接他们在另一端的21G 针已经从他们的塑料注射器适配器和弯曲大约90°大约9毫米从针末端被去除。</li>
	<li>连接21G 钝针到油管段的一端, 从注射器到开关装置, 将针头插入油管。将各油管长度的另一端插入硅胶导管段, 在 Y 形路口的入口倒钩上。 注: 使用某种仪器来控制夹紧阀和接合处;这可以很容易地从一个微提示框 (图 2D)。</li>
	<li>切断聚合物导管的长度, 将废料从装置的出口处运到废物烧杯。连接他们在一端与弯曲的21G 针如上所述准备。把管子的另一端带到一个废弃的烧杯里。</li>
	<li>准备适当数量的培养基, 含0.1 毫克/毫升牛血清白蛋白 (BSA), 并填充所需的注射器大小和数量。将 BSA 加载的注射器连接到所准备的21G 针上, 并将注射器装入注射器泵中。 注: 对于典型的实验, 使用了5通道, 每台都有20毫升的注射器。一个实验, 其中的媒介是交换将需要2银行5注射器每。在这里, 包含第一个银行的泵被称为 phase-1 泵, 包含第二个银行的泵, 与后交换介质, 被称为 phase-2 泵。</li>
	<li>通过在高流速下运行注射器泵 (> 500 µL/分钟), 从入口管道中清除空气, 首先将注射器垂直定向, 然后敲击注射器, 使气泡顶部。一旦空气已从注射器中清除, 放置注射器泵水平, 并逐步轻拍或轻弹的聚合物线从注射器通过开关装置, 以消除和清除气泡。对第二个注射器 (如果切换介质) 重复此过程。</li>
	<li>清除气泡后, 设置 phase-2 注射器泵 (即, 用将在开关后使用的介质) 转换为1.5 µL/分钟, 然后暂停流。将小活页夹夹在与第二介质相对应的 Y 接合处的挠性油管段上。继续运行 phase-1 注射器泵在一个适度的流速 (例如, 10 µL/分钟) 至少30分钟, 以清除第二个介质从进口油管下游的 Y 交界处。</li>
</ol>4. 细胞培养制剂
<ol><li>在一夜之间, 在所需的培养基中生长出培养的兴趣, 到固定相。细菌的菌株应包含一个突变, 使细胞不动, 使细胞不游出的渠道的设备。 注: 在本协议中, 细菌菌株为B. 枯草3610, 包含巫婆A233V替换 (此突变导致鞭毛为直而不是螺旋状, 防止细胞运动), PrsbVmNeonGreen记者的细胞压力, 和本组织的 Phyperspank-mNeptune (红色) 记者突出细胞。在示例协议中使用了 LB 蓝诺克斯介质。典型的微实验菌株包含一个或多个荧光转录的记者和一个组成产生的细胞质荧光蛋白, 以促进细胞的自动检测。当使用了富培养基时, 未观察到生长缺陷, 但在微流体条件下, 由于分泌的铁被介质流冲走, 所以在最小介质中的枯草芽孢杆菌的生长可能受到抑制。在这种情况下, 可以通过添加柠檬酸钠和氯化铁来恢复培养基, 如 S750介质11所报告的那样。</li>
	<li>在实验的早晨, 将枯草芽孢杆菌细胞稀释1:50 成一个250毫升的、含有25毫升生长培养基的瓶子。在37° c 的震动培养中, 将细胞生长为 5-6 h, 光学密度大于1。 注: 稠密的细胞培养是可取的, 因为固定阶段的B. 枯草细胞更小, 在细胞链中发现较少, 便于设备加载。不同的细菌种类可能需要其他培养基或生长条件以获得最佳的负荷。</li>
	<li>使用装有5µm 孔径过滤器的注射器, 将大约15毫升的细胞培养物过滤到15毫升的锥形管中, 以移除B. 枯草杆菌细胞链 (对于大肠杆菌是不必要的, 而且可能不需要与其他物种)。 注: 应删除相对较少的细胞;滤液应该混浊。</li>
	<li>离心过滤培养10分钟在室温 4000 x g。倒入上清液, 重残留在管中的剩余上清液体中的细胞颗粒, 如有必要, 增加大约500µL 的新鲜培养基, 以促进移细胞悬浮。</li>
</ol>5. 设备加载
<ol><li>轻轻地将空的薄凝胶加载微提示 (请参见材料表) 放入微流体设备的出口孔中。</li>
	<li>使用 P200 微的薄凝胶加载技巧, 钝化该装置通过注入含有1毫克/毫升 BSA 的介质进入入口孔, 观察出口孔中的提示, 以监测微流控通道的填充 (图 2A)。在室温下孵育设备约5分钟。 注意: BSA 通常用作阻挡或钝化剂, 它将结合到聚脂聚合物的疏水性表面, 增加其亲水性, 减少其他蛋白质或细胞 (通过细胞表面分子) 的结合。</li>
	<li>使用薄的凝胶加载技巧, 加载每个通道与悬浮单元 (从4节), 使用插座通道中的提示, 通过设备监视单元的进度 (图 2B)。 注: 通过将 P200 微设置为其最大 200-µL 体积, 然后在吸进小体积 (大约一半的吸管尖端薄部分的体积) 之前, 将空气中的气垫吸进。悬浮细胞的体积不需要精确, 因为该设备的体积相对于微。我们知道没有上限的密度的悬浮细胞, 只要细胞悬浮可以 pipetted 到设备。细胞团块可以堵塞的设备, 应避免通过彻底移和/或涡混合。</li>
	<li>轻轻地从出水孔中取出凝胶加载的小贴士, 一次工作一次以防止损坏设备。</li>
	<li>离心机在一个适当的转子适配器在一个合适的离心在大约 6000 x g 10 分钟 (图 2C) 的设备。 注: 采用定制加工的铝转子适配器, 设计成适合离心转子 (图 2C);不同的离心机模型可以使用适当的转子适配器。该适配器的重要特点是, 它提供了横向力量的方向, 封闭两端的细胞战壕, 从而迫使细胞进入侧通道。</li>
	<li>验证显微镜下的成功加载 (图 3), 但不将设备粘贴到幻灯片持有者/阶段插入。 注: 在这个协议中, 一个 60X, 1.4 NA Ph3 油浸泡目标是用于在这一阶段的验证和后续成像。</li>
</ol>6. 设备装配、平衡和安装
<ol><li>小心地将已加载的设备安装到舞台插入上, 方法是: 将该系统的两侧的盖玻璃贴在舞台插入的底部 (即, 在附加设备之前反转舞台插入)。反转舞台插入设备组件, 使其在软表面上朝向并放置, 如无尘擦拭 (图 2D)。</li>
	<li>将 phase-1 注射器泵的流量调整到35µL/分钟, 每次使用一条车道, 插入进口针, 然后将出口针 (即, 运行到废物烧杯;图 2E)。</li>
	<li>用干净的无尘擦拭擦去多余的介质, 并目测检查设备是否有泄漏。检查排气管的外观过剩的细胞 (通常出现的混浊条纹) 和中型半月板, 这将慢慢地向废物烧杯。</li>
	<li>允许设备运行在35µL/分钟约 15-30 分钟, 直到所有连接的车道排入废物烧杯。</li>
	<li>设置 phase-1 注射器泵到1.5 µL/分钟, 并暂停流。将整个泵和装置设备带到显微镜 (这一过程是通过把它全部放在一个滚动的推车), 并重新启动 phase-1 介质流量在1.5 µL/分钟. 小心地安装在倒置荧光显微镜上的舞台插入装置上, 使用必要的胶带, 以路线的进口和出口油管。</li>
	<li>在设备上定位所需位置以进行成像, 并根据需要开始成像。请注意, 细胞通常需要几个小时 (大约10代) 达到稳态指数阶段的增长, 并开始的图像采集可能会延迟的期望, 使成像从平衡期之后。 注意: 在随后的图像分析中, 应通过跟踪细胞分裂时间和确保它们达到恒定的最小值来验证在指数阶段保持的细胞的稳态生长。</li>
</ol>7. 中型开关
<ol><li>在连续的一轮成像, 暂停流动的 phase-1 注射器泵。仔细松开的粘合剂剪辑从 phase-2 硅胶油管, 移动每一个硅胶管在 phase-1 分支的 Y 交界处。暂停流动的 phase-2 注射器泵 (这是设置为1.5 µL/分钟在步骤 3.8), 并继续成像。 注: 在1.5 µm/分钟与10厘米长的聚合物油管的 Y 结的下游, 第二阶段的介质到达设备在大约50分钟。该设备的时间可以通过使用含有荧光示踪粒子的介质进行经验测试。</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+microfluidic+tracking+of+coccoid+cyanobacterial+cells+reveals+robust+control+of+division+timing.">Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing.</a> BMC Biol. 15 (1), 11 (2017).</li><li>Campos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+constant+size+extension+drives+bacterial+cell+size+homeostasis.">A constant size extension drives bacterial cell size homeostasis.</a> Cell. 159 (6), 1433-1446 (2014).</li><li>Taheri-Araghi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-size+control+and+homeostasis+in+bacteria.">Cell-size control and homeostasis in bacteria.</a> Curr Biol. 25 (3), 385-391 (2015).</li><li>Siryaporn, A., Kim, M. K., Shen, Y., Stone, H. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization,+competition,+and+dispersal+of+pathogens+in+fluid+flow+networks.">Colonization, competition, and dispersal of pathogens in fluid flow networks.</a> Curr Biol. 25 (9), 1201-1207 (2015).</li><li>Connell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+prokaryotic+social+behaviors+with+bacterial+"lobster+traps".">Probing prokaryotic social behaviors with bacterial "lobster traps".</a> MBio. 1 (4), (2010).</li><li>Long, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bacterial+adaptation+dynamics+at+the+single-cell+level+using+a+microfluidic+chemostat+and+time-lapse+fluorescence+microscopy.">Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy.</a> Analyst. 139 (20), 5254-5262 (2014).</li><li>Okumus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+slowing-down+of+cytoplasmic+diffusion+allows+in+vivo+counting+of+proteins+in+individual+cells.">Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.</a> Nat Commun. 7, 11641 (2016).</li><li>Teng, S. W., Mukherji, S., Moffitt, J. R., de Buyl, S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+circadian+oscillations+in+growing+cyanobacteria+require+transcriptional+feedback.">Robust circadian oscillations in growing cyanobacteria require transcriptional feedback.</a> Science. 340 (6133), 737-740 (2013).</li><li>Baker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable,+Pneumatically+Actuated+Microfluidic+Device+with+an+Integrated+Nanochannel+Array+To+Track+Development+of+Individual+Bacteria.">Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria.</a> Anal Chem. 88 (17), 8476-8483 (2016).</li><li>Russell, J. R., Cabeen, M. T., Wiggins, P. A., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+in+a+phosphorelay+drives+stochastic+entry+into+sporulation+in+Bacillus+subtilis.">Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis.</a> EMBO J. 36 (19), 2856-2869 (2017).</li><li>Cabeen, M. T., Russell, J. R., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+microfluidic+platform+to+uncover+basic+features+of+energy+and+environmental+stress+responses+in+individual+cells+of+Bacillus+subtilis.">Use of a microfluidic platform to uncover basic features of energy and environmental stress responses in individual cells of Bacillus subtilis.</a> PLoS Genet. 13 (7), e1006901 (2017).</li></ol>

作者没有什么可透露的。

这个微协议是灵活的, 因为许多步骤可以修改, 以优化其使用特定的物种或应变或为特定目的。实际上, 在本协议中, 我们对原始的 "母机器" 概念4进行了修改, 以优化其与B. 枯草-枯草杆菌的使用。通常, 细胞被限制的沟槽构成一个单层特征, 而在这个协议中我们使用两层细胞沟, 在细胞周围有一个浅通道。两层设计被引入, 作为一种方法, 最大限度地从B. 枯草细胞的养?...

地球上最引人注目的生命特征之一就是它的韧性和多样性。分子生物学的一个中心目标是理解细胞利用基因和蛋白质在各种各样的环境条件下最大化生长和适应的逻辑。为了实现这一目标, 科学家必须能够自信地观察单个细胞在特定条件下如何生长、分裂和表达基因, 并注意到细胞对其环境的后续变化的反应。然而, 传统的微生物学分析方法有技术上的局限性, 影响到可以解决的问题的类型。例如, 基于批量区域性的分析在过去的几年中非常有用, 但它们只提供了能够掩盖有意义的细胞间变异或细胞在总人口中的较小的子群的行为的人口级数据。基于光显微镜的活细菌单细胞分析揭示单细胞行为, 但在技术上也是有限的。细菌通常固定在含有生长培养基的琼脂糖垫上, 但细胞生长和分裂会使显微镜下的观察和消耗的养分在短短的几个细胞周期后耗尽, 极大地限制观测时间1, 2。此外, 由于细胞生长而导致的营养物质的局部耗竭和代谢副产物的积累, 不断地改变着当地细胞生长的环境, 难以测量或预测。使用琼脂糖垫的这种环境变化对稳态行为的研究或对生长条件下的特定变化的细胞反应都是一个挑战3。
微流体技术, 其中一个液体介质是不断流经 microfabricated 设备, 提供了一个经典的实验限制的解决方案。微流控装置可以保持单个细胞在活细胞显微镜中的位置, 而生长介质的流动不断地为细胞提供新鲜的养分, 洗去代谢副产物和多余的细胞, 从而产生高度均匀的生长环境.在恒定生长条件下, 细胞行为可以从环境因素的影响中分离出来, 从而使细胞内部逻辑得以不受阻碍的观察。当流体流动防止微流控装置变得拥挤与细胞时, 单细胞血统观察为数以万计或数百个世代成为可能4,5。如此长的观察时间允许检测到其他不可察觉的长期或稀有的细胞行为。最后, 通过该装置流动的培养基的组成可以随意改变, 使细胞在对压力的发生或对某一化合物的引入或去除作出反应时能够被观察到。
微已经享受了许多重要的应用。例如, 它已被用于组织, 器官, 或身体上的芯片设备, 其中多个人类细胞类型的共同培养, 以模拟在体内条件6;微环境中线虫运动的研究7;检查细菌生物膜之间的相互作用 (例如, 8);并用于封装和操作微小体积的单元或化学物质 (例如, 9)。微流控器件在微生物学领域也越来越受欢迎 (对于优秀的评论, 请参见10和11), 特别是由于它们的物理和流特性与自然微生物龛位的匹配很好,12。例如, 微最近被微生物学用于精确测量细胞生长和分裂13,14,15, 分析病原体移动16,监视仲裁传感17和生理过渡18, 以及蛋白质计数19, 在许多其他示例中。这里提出的方法是专门为分析单一细菌细胞的血统, 而不是组合的菌株或物种。此处演示的微流控装置利用了 "母机器" 设计的一个变体4, 其中的单元格在一个封闭端和一个开放端的微流控槽内的单文件中生长;细胞生长和分裂将子代细胞向上和流出的开放端推向流体流动。我们的分析通常只着眼于 "母亲" 的细胞, 被限制在沟的封闭端。我们认为这种方法是对以往基于光镜的单细胞分析技术的进步, 例如琼脂糖垫上的细胞固定。在这里, 当枯草芽孢杆菌被用作模型时, 该方法也适用于其他细菌种类 (大肠杆菌是另一种常见的模型; 某些具有不同细胞大小或形态的物种可能需要制造新的设备不同的尺寸)。使用荧光记者来标记细胞和可视化基因表达的变化需要使用基因驯服的物种;然而, 即使没有荧光标记, 对细胞生长和形态学的分析也是可能的。
本议定书不包括使用光刻技术制作硅母版的过程, 它在别处被广泛描述为5;硕士也可以很容易地外包的微细加工设施。它包括从增强硅母料中成型一个多晶硅装置;将装置与盖板玻璃接合;组装微流控入口和出水管道, 包括夹紧阀, 允许中型开关;钝化该装置, 制备细菌细胞, 并将该装置装入细菌细胞;将管道连接到设备上并均衡电池;并将该装置装入荧光显微镜进行成像。由于许多不同的图像采集和处理软件工具可以用来可视化和分析不同的数据的兴趣4,5, 示例图像显示, 但图像捕获方法不包括在此协议中。

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">(240)4019862</td>
			<td style="width:167px;">This is referred to as PDMS in the protocol. There are 2 components that are mixed at a 10:1 ratio</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">0.75-mm biopsy punch</td>
			<td style="width:71px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">22 x 40 mm No. 1.5 cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48393 172</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Plasma Etch PE-50</td>
			<td style="width:71px;">Plasma Etch Inc.</td>
			<td style="width:119px;">PE-50</td>
			<td>Instrument used to bond PDMS to glass using oxygen plasma treatment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Tygon flexible tubing ID 0.02&#34;, OD 0.06&#34;</td>
			<td style="width:71px;">Saint-Gobain PPL Corp.</td>
			<td>AAD04103</td>
			<td>For the main part of the tubing, e.g. attached to the needles</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:223px;">Silicone Tubing, 0.04&#34; ID, 0.085&#34; OD</td>
			<td style="width:71px;">HelixMark Standard Silicone Tubing</td>
			<td style="width:119px;">60-795-05</td>
			<td>For pinch valves and Y-junction connection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Bovine Serum Albumin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906-100G</td>
			<td>Used as passivation agent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">ART Gel Pipet Tips (P200)</td>
			<td style="width:71px;">Molecular BioProducts</td>
			<td align="right" style="width:119px;">2155</td>
			<td>For loading the device with medium and cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Acrodisc 32-mm syringe filter with 5-&#956;m Supor membrane</td>
			<td style="width:71px;">Pall Life Sciences</td>
			<td align="right" style="width:119px;">4560</td>
			<td>For filtering cultures before device loading</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">21-ga blunt needles, 1&#34;</td>
			<td style="width:71px;">McMaster-Carr</td>
			<td style="width:119px;">75165A681</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Y connector with 200-series barbs, 1/16&#34; ID tubing, polypropylene</td>
			<td style="width:71px;">Nordson Medical</td>
			<td style="width:119px;">Y210-6005</td>
			<td>The company is AKA Value Plastics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eclipse Ti-E inverted microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>This unit has been discontinued by the manufacturer and is replaced by the Ti 2 E.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">LB Lennox</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Microfuge 18</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td align="right">367160</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Bacillus subtilis strain NCIB3610</td>
			<td style="width:71px;">Bacillus Genetic Stock Center</td>
			<td style="width:119px;">3A1</td>
			<td>wild-type parental strain modified for use in the experiments shown in this protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gravity convection oven</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">414005-106</td>
			<td>for curing PDMS and baking assembled devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch-Weld Epoxy Kit</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">2216 B/A</td>
			<td>may be used to bond a silicon wafer to an aluminum backing plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch Magic tape, 3/4&#34; width</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;"></td>
			<td>to clean dust from PDMS devices and to place over features to increase visibility (denoted &#34;office tape&#34; or &#34;adhesive tape&#34; in protocol)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Stereomicroscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
			<td>listed here as an example; nearly any stereomicroscope will do</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isopropyl alcohol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">W292907</td>
			<td>To clean cover glass</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Syring pump, 6 channel</td>
			<td style="width:71px;">New Era Pump Systems Inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Kimwipes</td>
			<td style="width:71px;">Kimberly-Clark</td>
			<td align="right" style="width:119px;">34120</td>
			<td>a brand of dust-free wipes</td>
		</tr>
	</tbody>
</table>

single-cell microfluidic analysis of bacillus subtilis

微流控技术克服了传统的微生物学分析方法的许多局限性。与大块培养方法不同, 它提供单细胞分辨率和长达数百代的长观测时间;不同于琼脂糖垫基显微镜, 它有统一的生长条件, 可以严格控制。由于生长培养基的连续流动使微流控装置中的细胞与细胞生长和新陈代谢引起的局部化学环境的不可预知的变化隔绝, 基因表达和细胞生长的真实变化反应具体的刺激可以更自信地观察。枯草芽孢杆菌在这里用作细菌模型, 以演示 "母机器" 型细胞分析方法。我们展示了如何构造和铅化一个微流体设备, 加载它与细胞, 启动显微成像, 并暴露细胞的刺激, 从一个生长媒介切换到另一个。一个压力响应的记者被用来作为一个例子来揭示这种方法可以获得的数据类型。我们还简要地讨论了这种方法在其他类型的实验, 如细菌产分析的进一步应用。

在将微流体管道连接到设备之前, 通过相衬显微镜评估的成功的初始细胞负荷, 将被认为具有包含一个或多个细菌细胞的所有或几乎所有的微流体侧通道 (图 3A)。最佳加载将显示每个通道中的多个单元格, 但是通道将在平衡期间 (图 3B) 中由于单元格的增长而填充单元格。有较差负荷的车道, 其中相对较少的 (< 50%) 侧通道装有细胞,...

Watch this Scientific Journal Video about Single-cell Microfluidic Analysis of Bacillus subtilis at JoVE.com

Single-cell Microfluidic Analysis of Bacillus subtilis

单细胞微流控芯片分析枯草芽孢杆菌

本文以枯草芽孢杆菌为例, 对单个细菌细胞谱系的微流体分析方法进行了研究。该方法克服了传统分析方法在微生物学中的不足, 允许在严格控制和均匀生长条件下观察几百个细胞世代。

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

single-cell-microfluidic-analysis-of-bacillus-subtilis

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Single-cell Microfluidic Analysis of Bacillus subtilis

11.9K 次观看.  Oklahoma State University. 本文以枯草芽孢杆菌为例, 对单个细菌细胞谱系的微流体分析方法进行了研究。该方法克服了传统分析方法在微生物学中的不足, 允许在严格控制和均匀生长条件下观察几百个细胞世代。

视频: Single-cell Microfluidic Analysis of Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

科研

生物学

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用:根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

单细胞微流控芯片分析枯草芽孢杆菌

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成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌