在美国能源部基础能源科学办公室的纳米相材料科学用户设备部中心制造和表征微孔阵列。通过橡树岭国家实验室主任的研发基金提供了对这项工作的财政支持。作者还要感谢J.Mougous实验室（华盛顿大学西雅图华盛顿州）提供这些研究中使用的铜绿假单胞菌菌株。

硅微孔阵列制造<ol><li> 聚对二甲苯涂层 <ol><li>根据制造商的规格和说明书（设置:蒸发器设定点= 160℃;炉设定点= 650℃），使用市售的聚对二甲苯涂层系统在硅晶片上沉积1-1.5μm的聚对二甲苯N. 注意:装入室内的聚对二甲苯约6克产生1-1.5微米厚的涂层。 </li></ol></li><li> 光刻 <ol><li>以3,000rpm转速涂覆具有粘合促进剂，20％六甲基二硅氮烷（HMDS）和80％丙二醇单甲醚乙酸酯（PGMEA）（ 参见表材料 ）的聚对二甲苯N-包覆的晶片45秒。用粘合促进剂填充2 mL转移移液管，并将其洒在整个晶片上。让晶片静置约10秒，然后将其干燥。 </li><li>填充2毫升转移移液器与正（参见"材料表" ），并将光致抗蚀剂分配在晶片的中心。以3,000rpm旋转45秒以产生约1.5μm厚的抗蚀剂涂层。 </li><li>将样品在115℃的电热板上软烘烤1分钟。 </li><li>使用具有所需孔图案的接触对准器和光掩模将样品暴露于紫外线。通过图案化的光掩模将旋涂的晶片暴露6秒，给出在365nm处测量的约60-80mJ / cm 2的近似剂量。 </li><li>通过将样品浸在显影剂（&lt;3％四甲基氢氧化铵在水中;参见材料表 ）2分钟来显影图案 。用去离子水冲洗并用干净的氮气干燥。 注意:在开发过程中应清除暴露于紫外线的光致抗蚀剂区域。 </li></ol></li><li> 反应离子蚀刻 <ol><li>使用氧等离子体蚀刻去除暴露的聚对二甲苯一路到硅衬底。 注意:可以调节配方以改变聚对二甲苯的蚀刻速率。对于1和5μm之间的聚对二甲苯厚度，在反应离子蚀刻（RIE）工具上使用60 mTorr，20°C，100 sccm O 2，10 W RF和2,000 W ICP的配方。在蚀刻和去除暴露的聚对二甲苯层之后，图案化区域（ 即暴露的硅）应该看起来有光泽和银色。 </li><li>使用深度RIE（DRIE; 例如 Bosch DRIE）蚀刻工艺来蚀刻到硅中。 注意:蚀刻速率和持续时间将决定井深。 Bosch工艺的一个完整周期（3s沉积步骤:20mTorr，15℃，140sccm C 4 F 8,10W RF和1,750W ICP，随后10秒蚀刻工艺:20mTorr，15℃ ，120sccm SF 6,8W RF和1,750W ICP）对应于约1μm的蚀刻深度。该演示中使用的井深为3 - 3.5μm。 </li><li> V使用物理轮廓测量法蚀刻蚀刻深度。 <ol><li>将样品加载到物理轮廓仪中（参见材料表 ）。 </li><li>打开样品真空并按手动装载按钮。 </li><li>通过按"对焦"按钮将系统对准样品。在视图屏幕上放置适当的测量功能。 </li><li>扫描样品。调整轮廓并测量特征深度。 </li><li>记录蚀刻速率并调制后续蚀刻时间以达到所需的深度。 注意:测量将包括硅阱的深度，沉积的聚对二甲苯的厚度和光致抗蚀剂的厚度。在整个过程中验证每层的厚度是实现精确井深所必需的。 </li></ol></li></ol></li></ol> 2.细菌培养和播种（ 图1a ） <ol><li>在Luria Broth（LB）来自甘油储备的琼脂平板，并在两周内使用。从LB琼脂平板上挑选所需菌株的菌落，并开始铜绿假单胞菌的过夜培养。在37℃下孵育过夜培养约18小时，同时在R2A培养基中以220rpm摇动。 注意:应在接种后两周内挑选菌落，以确保突变和荧光报告基因被保留。所有铜绿假单胞菌工作应在BSL-2条件下进行。 </li><li>使用金刚石刻字将硅晶片分成含有不同尺寸和间距阵列的组合的单个芯片。确保每个芯片包含完整的孔尺寸和间距用于研究。 </li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/55701/55701fig1.jpg" /> 图1:制作和细胞播种程序。 （ a ）微孔阵列a重新蚀刻成涂有薄层聚对二甲苯（i）的硅晶片。为了润湿孔和/或使表面官能化，将蛋白质溶液加入到阵列顶部的液滴中（ii）。去除蛋白质溶液，将晶片干燥，并加入含有所需细菌的新溶液（iii）。在培养期后除去细菌溶液，使晶片干燥，留下孔中和表面上的细菌（iv）。用聚对二甲苯剥离除去表面相关细菌，留下细菌在微孔中干净地播种，由于2％甘油培养基仍然存活，这有助于保持孔水合（v）。然后将硅芯片阵列侧放置在琼脂糖凝胶涂覆的玻璃盖玻片上，其在微孔（vi）中进行细菌生长。 （ b ）在单个硅器件上布置子阵列。每个子阵列包含一组相同的孔。所有sub-a上的微孔的直径阵列的直径范围为5-100μm，组织为井口直径的2x，3x或4x，这在底板示意图上由白色至深灰色表示。当深度浅（&lt;10μm）时，5和10μm的孔直径很少有用，通常是因为缺乏细胞定植这些非常小的孔。在这项工作中，仅分析了直径为15-100μm的井的数据。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55701/55701fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 注意: 如图1b所示 ，一个完整的芯片包含直径范围从5到100μm的孔的子阵列，具有三个不同的间距（ 即 2x，3x和4x直径），重复4次。 <ol start="3"><li> 将500μL/ mL牛血清白蛋白（BSA）的150μL液滴放入PBS溶液中在阵列顶部润湿微孔。在室温下将BSA溶液在芯片上孵育1小时。 <ol><li>通过用磷酸盐缓冲盐水（PBS）填充空的移液器吸头盒的底部来创建腔室。 注意:其他物质，如特异性凝集素，可用于代替BSA功能化微孔表面。 </li></ol></li><li> 在将硅芯片与BSA溶液一起孵育的同时，以2500rpm（相当于950×g的平均值）离心培养物5分钟，然后将其重新悬浮于500μL具有2％甘油的新鲜R2A培养基中。 <ol><li>使用紫外可见分光光度计在600 nm处测定培养物的OD。使用2％甘油R2A培养基将其调节至0.02。 注意:甘油有助于防止在聚对二甲苯剥离过程中油井变干。 </li></ol></li><li>孵育后，取出BSA溶液，用PBS冲洗3次，取出并更换液体喷雾管覆盖硅微孔阵列。在氮气下干燥。 </li><li>向放置在潮湿室中的每个干阵列中加入150μL0.02 OD培养物。在4℃下孵育1小时以使细菌粘附到井壁上。 注意:孵化不需要制冷。 4℃的孵育时间可用于在成像开始之前防止细菌的生长，以便人们可以在生长前观察群落的空间组织。也可以使用室温孵育。两种方案都产生类似的生长曲线。 </li></ol> 3.显微镜设置<ol><li>在硅片开始细菌培养之前，打开舞台顶部的环境控制室（参见表格）并调整控制箱上的设置，使湿度（〜100％）和温度（30-32 °C，参见步骤3.2）可以在添加样品之前平衡。 </li><li>对样品架进行调平并将其插入使用PBS浸泡的实验室擦拭物（参见"材料表" ），在样品周围，将室内的湿度增加到露点。将腔室的温度设置为30°C，将腔室温度设置为32°C，以减少成像平面上的冷凝。 注意:滑块固定器安装在带有约1厘米厚的垫圈的活细胞室中。借助放置在样品架顶部的气泡水平，样品架平整。样品架可以稍微倾斜，并保持密封在垫圈中。 </li><li>当文化孵化在硅芯片上时，在成像前至少30分钟手动打开汞灯的电源开关。手动打开相机和自动显微镜舞台。打开用于控制显微镜和外围设备的软件，并确保软件识别设备。 注意:放大倍数为10倍，NA = 0.3。 </li></ol><p类= "jove_title"&gt; 4。制备琼脂糖玻璃盖玻片<ol><li>微波预先制备的琼脂糖溶液（ 即 R2A培养基中的2％琼脂糖）直到达到液体状态，大约60s。 </li><li>用75毫米x 22毫米的＃1.5玻璃盖玻片用乙醇将背面浸湿，将其纵向放置在2×3"（50 x 75 mm）的玻璃载玻片上，放置两个PDMS垫片（厚度〜1 mm）沿着盖玻片的长边缘移动玻璃盖玻片，使得大约1mm的盖玻片突出于滑块的边缘。 </li><li>将5 mL液体琼脂糖溶液倒入玻璃盖玻片顶部，刚好足以完全覆盖，并将第二个2 x 3"玻璃载玻片放置在组件的顶部，将盖玻片和滑块之间的液体琼脂夹在中间。 注意:这可以控制琼脂糖的深度，使盖玻片和硬化琼脂糖的总厚度与PDMS间隔物的厚度相等。 </li><li>允许玻片载玻片 - 盖玻片 - 琼脂糖凝胶 - 玻璃夹层板，直到琼脂糖溶液开始固化;然后将其转移到冰箱。 15分钟后，取出多余的固体琼脂糖并切割玻璃盖玻片。将其移动到干净的盘子上并放置在冰箱中直到使用。 </li></ol> 5.用琼脂糖涂层盖片和成像法密封孔<ol><li> 细菌孵育完成后，从冰箱中取出琼脂糖包覆的盖玻片，制备硅片，如下。 <ol><li>将硅片浸入超纯水中，每次一片，每次10s。将它们放在实验室擦拭物或组织的边缘上，直到大部分多余的液体从芯片的边缘排出。 </li><li>切割一块胶带以匹配每个硅芯片的边缘长度。将胶带放在覆盖硅的聚对二甲苯上，并用它快速剥离聚对二甲苯涂层。 </li><li> IMMED将每个剥离的芯片反转并放置每个芯片，使得微孔阵列侧面（并与之接触）琼脂糖涂覆的盖玻片的琼脂糖层。在接触琼脂糖后，小心不要移动或移动芯片，以防止孔外的细菌生长。 </li></ol></li><li> 将组装好的微孔阵列/琼脂糖盖玻片放置在扫描仪上的台顶环境控制室的滑块架上。 <ol><li>使用环境光或定向光（ 例如手电筒）来定位感兴趣的阵列。使用控制自动化阶段的商业软件来保存这些职位（见材料表）。储存位置后，请关闭环境或指示灯。 </li><li>在商业软件中，打开"ND采集"面板。 注意:该面板包括一个用于自动保存到特定目录的菜单，以及可编程图像采集。对于这些实验使用"时间"，"XY"和"λ"菜单。 </li><li>要在软件中保存位置，请单击"XY菜单"，然后在需要保存的每个位置上检查左侧的空白框。另外，点击"Include Z"按钮。 </li></ol></li><li> 使用适当的荧光过滤器立方体，在所需波长和10倍放大倍数下随时间获取图像（参见材料表）。 <ol><li>使用控制软件和保存的阵列位置移动到每个保存的位置并专注于井。点击保存的列表中的每个XY位置，并使用绿色荧光蛋白（GFP）过滤器调整焦点。通过单击指向z位置的箭头来保存新的z位置。 注意:此过程可能很耗时。考虑采取增加增益的预防措施，并使用中性密度滤光片降低光线强度，以防止光漂白。 </li><li>通过在聚焦在阵列表面上注意z轴位置的差异来确定每个波长的焦平面之间的z轴距离。使用红色荧光蛋白（RFP）过滤器，从混合的红色/绿色细菌群体中选择2-3个位置，并使用焦点。 <ol><li>使用GFP和RFP荧光滤光器减去焦平面之间的距离，并在"λ"菜单下添加焦平面调整。 注意:例如，如果阵列在50nm的z位置聚焦在GFP通道中，并且相同的阵列在RFP通道中以55μm出现聚焦，则在"λ"中的RFP光学配置旁边加上+5 "菜单。 </li></ol></li><li>开始延时图像采集。 注意:对于本文所示的实验，使用多维图像采集通过控制的商业软件，以30分钟的间隔为每个阵列位置获取RFP和GFP图像相机，快门，滤光轮和电动舞台。 <ol><li>将"间隔"设置为30分钟，将"实验持续时间"设置为"时间"菜单下的24小时。点击"立即运行"。 注意:在"时间"，"XY"和"λ"框被选中的情况下，运行该程序会将舞台移动到每个位置（ 即保存的XYZ位置）上，拍摄一个波长的图像，移动z-位置考虑焦平面差异（ 即 λ或波长控制），取第二个图像，移动到下一个阵列位置（多点），并以30分钟间隔（延时）循环。 </li></ol></li></ol></li><li> 获取照明控制图像。 注意:使用"ND采集"，"时间"和"XY"菜单拍摄4个位置的图像，每个位置为25x。 <ol><li>通过关闭所有光源和摄取，拍摄一系列100"暗场"图像标准幻灯片的"图像"。这些图像将捕获相机噪音。使用时间段内使用的最长曝光时间（步骤5.3.3）。 </li><li>通过在几个不同位置成像标准幻灯片（ 即均匀RFP或GFP强度）来拍摄一系列100"照明场"图像，以捕获在给定实验条件下的不均匀照明。选择最大化信号而不达到饱和的曝光时间。 </li></ol></li></ol>分析<ol><li> 使用图像分析软件（ 例如， ImageJ）处理图像堆栈。 <ol><li>使用商业软件将获取的图像转换为tiff文件格式。通过点击"文件"&gt;"导入"&gt;"图像序列"将图像上传到图像分析软件中。 </li><li>通过对所有"暗场"和"照明场"图像进行平均，创建"校正图像"。 Subtrac通过选择"处理"&gt;"图像计算器"，平均"照明场"图像的平均"暗场"图像。在"操作"字段中选择"Image1"和"Image2"两个图像，然后选择"Subtract"。点击"确定"。 <ol><li>对于平均，加载校正（或暗场）图像，单击"图像"&gt;"堆叠"&gt;"Z项目"&gt;"平均投影"。 </li></ol></li><li>必要时进行图像注册。然后，通过点击"处理"&gt;"减去背景"来执行背景减法。在"半径"字段中输入半径（ 例如 125），然后选择"滑动抛物面"。 </li><li>使用"Process"&gt;"Calculator Plus"进行照明校正。选择以下参数:操作，划分; i1，形象好i2，校正图像; k1，校正图像平均值;和k2，0.单击"Cre"吃新窗口"。 注意:该数据集不需要注册，但在其他工作中，ImageJ插件StackReg与"翻译"转换一起使用。对于背景减法，对每个图像集使用相同的滑动抛物面半径。例如，如果成像的最大的孔的像素半径为100，则对每个图像组使用大于100的半径（ 例如， 125）。 </li></ol></li><li> 确定微孔中每个菌株的生长。 <ol><li>使用ImageJ"MicroArray"插件在所需阵列中的每个微孔周围选择感兴趣区域（ROI）。 <ol><li>在"MAP"菜单中，单击"重置网格"。指定行，列和直径（根据阵列上的孔尺寸和数量），请参见图1b 。从"ROI形状"菜单中选择"圆"。 </li><li>按住"Alt"键，同时用鼠标移动t选择左上角的ROI他的ROI数组。按住"shift"键，同时选择左下角的ROI可以更改阵列的大小。按住"shift"键，同时从阵列右侧选择一个ROI，而不是在角落，以改变ROI的间距。 </li><li>使用上述命令将ROI阵列适合图像中的孔。点击"测量RT"。 注意:插件将从每个ROI导出所需的测量值。使用三个ROI大小，在井周围产生同心环，以收集局部背景信号（ 即，从外环减去的中间环的信号）和荧光测量（ 即来自内环的信号）。 </li></ol></li><li>在电子表格软件中收集数据并计算背景信号。将其导入自定义脚本软件进行进一步分析。 </li></ol></li><li> 数据组织与分析 <ol><li>导入数据并组织数据在ImageJ中以下列顺序收集到ImageJ中的矩阵:列1，子数组号;第2列，井排;第3栏，井柱;第4栏，平均强度;第5栏，背景强度;和第6列，平均强度 - 背景强度。 <ol><li>将mCherry和GFP获取的结果分成不同的矩阵。将每个子阵列和每个颜色的结果存储在单元格阵列中的不同单元格中。 注意:该组织使得在图像数据和测量结果之间来回移动更简单，清理数据并确保测量准确地表示数据。 </li></ol></li><li>调整铜绿假单胞菌的自发荧光 。 注意:在涉及GFP和mCherry菌株的共培养的实验中，应该分析一种仅限于氯化胆碱的芯片，以确定mCherry和绿色自发荧光之间的关系。 <ol><li>绘制所有mCherryΔretS的mCherry-GFP信号，＃916; tse / i1-6所有时间点的孔，以确定mCherry信号和GFP通道中的自发荧光之间的关系。从共培养物中减去自发荧光信号。 </li></ol></li><li>绘制轨迹并将修改后的逻辑方程拟合到每个轨迹，以便使用电子表格软件或自定义脚本软件中的最小二乘法提取参数。 </li><li>寻找GFP和mCherry轨迹参数之间的相关性。 </li></ol></li></ol>

<ol>
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作者没有什么可以披露的。

本文介绍了微孔阵列设备和实验方案，旨在实现基于细胞群发展的高通量和高含量的基于活细胞成像的分析。虽然这里的示范重点是研究接触介导的VI型分泌对社区发育的影响，但阵列被设计为灵活的，适应于广泛的微生物群落和微生物 - 微生物相互作用的研究。这里的工作仅在于使用组成性表达荧光标记物的细菌，以便容易地跟踪成员丰度和位置。然而，通过改变化学环境，通过生长或扰动后来自各个井的生物材料的机会，可以用于确定社区组成和基因表达。 每个硅器件由数十个包含具有各种孔的微孔的阵列组成直径，以2x，3x或4x直径间距组织，并蚀刻到3和3.5μm之间的深度。由于营养物质和次生代谢产物可以自由扩散通过琼脂糖盖，包括不同的沥青子阵列，以便检查微孔或局部营养物质消耗之间的信号传导如何影响社区发展。在这个示范中，增长和社区发展似乎并没有受到阵列内微孔间距或位置的影响。选择浅层深度以简化图像分析，将所有微生物生长和发育限制在单个图像平面上。然而，以前已经使用更深的阱（20μm深），并且可以通过改变硅蚀刻工艺的持续时间来容易地深度地调制。井的长宽比的增加基本上改变了孔内部细胞经历的限制程度，有效地改变了井的总体积到营养物质扩散到井的顶部的区域。这种阵列配置可用于研究井对井或井内信号的影响。 使用硅制造微孔的一个限制是它对可见光是不透明的。因此，依赖于光的传播（ 例如，明场，相位或微分干涉对比成像）的常规分析不能用于监测增长。我们集团正在进行的研究和开发工作着重于适应这种硅微孔配置和实验协议，使用透明阵列来实现更高分辨率的荧光和明场成像，用于监测和定量。通过使用玻璃盖玻片上的诸如SU-8环氧树脂的透明材料制造，可以获得在较短工作距离下工作的目标的亮场和荧光图像。这样就可以通过水浸法或具有≥40倍放大倍率的油物镜获得更好的分辨率，而不像硅阵列那样难以通过琼脂层成像。 如我们以前的工作所述，种子条件和井几何影响井阵列内的细胞接种。在低种子密度或小井中，加载到各个井中的细胞数量和类型的高水平变化允许对实验参数空间进行广泛和快速的探索。更高的播种密度和/或成像较大的孔产生更一致的细胞类型和总接种量的比例，允许分析大量的实验重复。在生长的最初阶段快速查看图像表明细胞主要沿着孔的边缘附着和生长（ 图5a ）。不清楚这是否是与s有关的工件在播种过程中蒸发干燥或暗示细胞附着于多个边缘的偏好。以确定性方式有意地改变孔的形貌或表面化学，可以揭示哪些因素对细胞附着和生物膜形成具有最大的影响。 通过使用注入R2A培养基的琼脂糖凝胶涂覆的玻璃盖玻片来支持这些孔中细菌的生长。用于制造这种琼脂糖涂覆的盖玻片的方法确保了均匀的厚度，通过该厚度进行成像和相当恒定的z位置，从而允许同时成像多个硅芯片。使用超纯琼脂糖作为胶凝剂和基本培养基而不是完整的培养基导致更清洁，更少的自发荧光背景，增加了整体的信噪比。当依靠荧光定量跟踪微生物群落时，必须保持图像条件一致，并且敏锐地意识到自发荧光，光漂白和不均匀照明的贡献。此外，对于需要长时间推移图像采集的实验，需要诸如这里使用的厚层琼脂糖。可以制成较薄的层（约100μm厚）以实现更高倍率的成像。然而，难以将组件保持足够湿度以防止该薄层变干。 虽然对生长参数和初始接种水平的初步分析尚未揭示这里描述的简单系统的任何显着相关性，但是所示的协议和数据强调了随着时间的推移，可以从社区发展图像中提取的信息的深度。随着单一种群殖民地从初始的"成核"地点扩大，井内的空间组织演化。因此，与初始附着密度相关的因素（ 即茎干perhaps从一个物种对井表面的更大偏好相比，另一个）或个体补丁的增长速度可以显着影响社区发展。琐碎的相互作用，例如在生长过程中建立的物种（斑块）之间的接触所介导的相互作用可能需要对图像数据进行更深入的分析。 这里提出的微孔阵列平台和方案有助于微生物群落发育和微生物 - 微生物相互作用的快速组装和高含量分析。通过改变琼脂盖内的培养基组成或通过集成的微流体直接调节和取样来实现对当地化学环境的控制的未来工作应该允许超出模拟自然界限和利基尺寸的实验方案，并开始探索动态变化环境的影响，类似于自然界中发现的环境。未来的工作将会增加具有集成微流体的阵列设计，可用于动态调节局部化学环境并从各个井中采样流体。关于开发可用于更高分辨率和明亮跟踪社区生长的光学透明微孔阵列的其他工作已在其他地方进行了描述。

微生物群落由细胞死亡，分裂，蛋白质浓度，细胞器数目和突变1相关的环境结构和随机过程两个决定因素构成。在自然环境中，几乎不可能分析这些影响对社区组成和活动的个人影响。被自然结构遮蔽，埋在化学和生物环境中，识别社区成员并进一步解决其在自然环境中的时空分布是极具挑战性的。尽管如此，最近的努力已经强调了空间组织对社区功能的重要性，并指出在正在进行的研究2,3,4中需要考虑成员丰富和组织。 它很清楚，当地的化学环境（ 即营养物质和次生代谢物的可获得性），物理结构（ 如土壤结构，植物根系，海洋颗粒或肠道微绒毛），氧气的存在或不存在以及引入致病物种都影响微生物群落5,6,7,8,9,10,11的组成，结构和功能。然而，忽视捕捉这些因素的传统文化技术依然存在。社区组成（ 例如，共同依赖物种的存在），物理附着，信号分子浓度和直接的细胞 - 细胞接触都是形成微生物群落的重要因素，并且可能在c传统文化条件。这些性质难以在大量液体培养物或琼脂平板上复制。然而，允许复制自然环境的关键物理和化学特征的微流体，微图形化和纳米制造技术的可用性使得许多研究人员能够建立细菌群落来研究它们的相互作用12,13,14 ，并开发合成环境模拟自然条件4，15，16，17，18，19，20。 该协议描述了一种制造微孔阵列器件的方法，并提供了可用于功能化th的详细实验程序e井在阵列中生长细菌，作为单一物种殖民地和多成员社区。这项工作还演示了如何使用细菌修饰以产生荧光报告蛋白可以随时间监测孔内的细菌生长。以前提出了类似的阵列，并且显示可以在微孔中跟踪绿脓假单胞菌（ 铜绿假单胞菌）单种群的生长。通过调节孔径和接种密度，数千个生长实验的起始条件可以并行变化，以确定初始接种条件如何影响细菌生长的能力21 。目前的工作使用微型阵列的稍微修改版本，其基于以前的工作，通过实现多个阵列的同时比较和使用更强大的实验协议。此工作中使用的数组包含多个子阵列或阵列包含不同尺寸的直径为15-100μm的孔，其以三个不同的间距（ 即孔径的2x，3x和4x）排列。将阵列蚀刻到硅中，并且通过用已经用中等浸渍的琼脂糖凝胶涂覆的盖玻片密封阵列来实现在硅阵列中接种的细菌的生长。用于研究VI型分泌系统的铜绿假单胞菌突变体用于该示范。 这里提出的结果构建在分析微孔阵列中的多元群落的最终目标，使研究人员能够在控制和探测化学环境的同时监测原位细菌的丰度和组织。这应该最终提供洞察管理社区发展和继承的"规则"。

<table><tbody><tr><td>Parylene N</td><td>Specialty Coating Systems</td><td>CAS NO.:1633-22-3</td><td></td></tr><tr><td>Parylene coater</td><td>Specialty Coating Systems</td><td>Labcoter 2 Parylene Deposition Unit PDS2010</td><td></td></tr><tr><td>Silicon Wafer</td><td>WRS Materials</td><td>100mm diameter, 500-550&amp;mu;m thickness, Prime, 10-20 resistivity, N/Phos&amp;lt;100&gt;,</td><td></td></tr><tr><td>adhesion promoter</td><td>Shin-Etsu Microsci</td><td>MicroPrime P20 adhesion promoter</td><td></td></tr><tr><td>postive tone photoresist</td><td>Rohm and Haas Electronics Materials LLC (Owned by Dow)</td><td>Microposit S1818 Positive Photoresist (code 10018357)</td><td></td></tr><tr><td>Quintel Contact Aligner</td><td>Neutronix Quintel Corp</td><td>NXQ 7500 Mask Aligner</td><td></td></tr><tr><td>Reactive Ion Etching Tool</td><td>Oxford Instruments</td><td>Plasmalab System 100 Reactive Ion Etcher</td><td></td></tr><tr><td>R2A Broth</td><td>TEKnova</td><td>R0005</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Multimode Plate Reader</td><td>Perkin Elmer</td><td>Enspire, 2300-0000</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Nikon</td><td>Eclipse Ti-U</td><td></td></tr><tr><td>Automated Stage</td><td>Prior</td><td>ProScan III</td><td></td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-QiMc</td><td></td></tr><tr><td>Stage-top environmental control chamber</td><td>In Vivo Scientific</td><td>STEV ECU-HOC</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>UltraPure Agarose</td><td>ThermoFisher Scientific</td><td>16500500</td><td></td></tr><tr><td>25 x 75 mm No. 1.5 coverslip</td><td>Nexterion</td><td>High performance #1.5H coverslips</td><td></td></tr><tr><td>Fluorescence Reference Slides</td><td>Ted Pella</td><td>2273</td><td></td></tr><tr><td>Physical Stylus Profilometer</td><td>KLA Tencor</td><td>P-6</td><td></td></tr><tr><td>lab wipes</td><td>Kimberly Clark</td><td>Kimipe KIMTECH SCIENCE Brand, 34155</td><td></td></tr><tr><td>commercial software</td><td>Nikon</td><td>NIS Elements</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>filter cubes</td><td>Nikon</td><td>Nikon FITC (96311), Nikon Texas Red(96313)</td><td></td></tr></tbody></table>

assembly and tracking of microbial community development within a microwell array platform

微生物群落的发展取决于复杂的确定性和随机因素的组合，可以显着改变社区成员的空间分布和活动。我们开发了一个微孔阵列平台，可用于并行快速组装和跟踪数千个细菌群落。该协议强调了该平台的实用性，并描述了其用于光学监控平台内阵列集合内简单的两人社区的发展。该示范使用铜绿假单胞菌的两个突变体，一系列突变体的一部分用于研究VI型分泌致病性。 mCherry或GFP基因的染色体插入片促进具有不同发射波长的荧光蛋白的组成型表达，可用于监测每个微孔内的群落成员丰度和位置。该协议描述了详细的方法d用于将细菌混合物组装到阵列的孔中，并使用延时荧光成像和定量图像分析来测量每个成员群体随时间的相对增长。微孔平台的种植和组装，阵列内微生物群落定量分析所需的成像过程以及可用于揭示微生物物种区域之间相互作用的方法。

这里提出的实验平台是为细菌群落的高通量和高含量研究而设计的。该设计使得能够同时分析成千上万个种植在各种尺寸的井中的社区。通过这种微孔阵列设计，可以确定最终社区组成对初始播种密度，孔尺寸和化学环境的依赖性。这项工作展示了微型阵列中两个成员社区的发展，并提出了分析社区组成和组织的方法。 这里使用的模型系统是在Mougous实验室设计的，以研究铜绿假单胞菌中的 VI型分泌物 。该系统由几种含有GFP或mCherry荧光报告基因的突变菌株组成。在这项工作中使用2种不同的菌株。第一个是GFP标记的ΔretS突变体y表达与VI型分泌相关的毒性效应蛋白，导致敏感细胞中细胞死亡的水平更高。第二个是RFP标记的ΔretSΔtse/ i1-6菌株，其是缺失所有六种已知效应子蛋白的缺失突变体，并且已经显示更易于VI型发病机制22,23,24 。每个物种的增长轨迹单独跟踪两个成员社区（ 即阵列平台内的共同培育）。 将ΔretS和ΔretSΔtse/ i1-6突变体分别接种到三个阵列上，每个阵列分别以1:2的比例混合在一起，然后每30分钟成像20小时。井后细菌生长在6和12小时后停止。荧光图像数据的例子如图2和图3所示。 图2显示了在45μm直径的孔中的ΔretS和ΔretSΔtse/ i1-6菌株随时间的增长， 图3显示了在各种尺寸的孔中大约6小时后期（hps）的这两个相同的两个成员群体的状态。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55701/55701fig2.jpg" /> 图2:时间过程共培养生长实验的样本图像。将表达mCherry （ ΔretSΔtse/ i1-6）（红色）或GFP（ΔretS）（绿色））的铜绿假单胞菌的两个突变体在微孔阵列中以2:1 mCherry:GFP比率接种在一起。这里是在45μm直径的孔中插入后3个不同时间拍摄的复合图像。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55701/55701fig2large.jpg" target="_blank">请点击这里查看大图</a>的这个数字。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55701/55701fig3.jpg" /> 图3:包含在微孔阵列器件中的阱直径的范围。两个成员社区的代表性图像在8小时的生长中捕获。通常，mCherry（ΔretSΔtse/ i1-6）（红色）或GFP（ΔretS）（绿色）菌落显示最小的共定位，保留在微孔中的不同区域内。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55701/55701fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 在获取测量值之前，执行滑动的parabaloid背景减法。采用光照校正步骤，可以定量比较给定实验中成像的每个微孔的信号。这些方法已经是p很明显地描述了25 。使用ImageJ中的微阵列插件，在校正图像中测量红色和绿色细菌的平均强度以及局部背景信号。从井信号中减去局部背景信号，并调整GFP信号以校正铜绿假单胞菌共培养物的自发荧光（从mCherryΔretSΔtse/ i1-6阵列确定的自发荧光） 。一旦进行了所有这些修正，就可以绘制mCherry-和GFP表达细菌的定量生长轨迹。 图4中示出了直径20和50μm的孔的示例生长轨迹。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55701/55701fig4.jpg" /> 图4: 铜绿假单胞菌 VI型分泌突变体的单和共培养物的生长曲线。（ a和d ）分别在20μm和50μm孔中的GFP-ΔretS单次生长曲线。 （ b和e ）分别在20和50μm孔中的mCherry-ΔretSΔtse/ i1-6单生长生长曲线。 （c和f）GFP（绿色）和mCherry（红色） 铜绿假单胞菌菌株的1:2比率混合物的共培养。生长曲线绘制在图像采集开始时，大约2小时后期（hps）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55701/55701fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 来自铜绿假单胞菌菌株的单次和共培养的生长轨迹表明，从井到井的生长有一些变异性，并且在较小尺寸的井中这种变异性增加。然而，平均强度不受井位影响泽。当存在ΔretS应变时，对易感菌株（ΔretSΔtse/ i1-6）的生长似乎没有显着或明显的负面影响。每个菌株的总体强度降低，但这是由于孔中每个菌株的起始浓度较低，因为总体OD在实验中保持一致，而不是每个菌株的个体OD。然而，通过单独观察生长轨迹，很难确定哪些因素对了解这些井中细菌生长最重要。因此，每个轨迹都与修改后的逻辑函数26 （ 图5a ）拟合，从而可以提取相关参数并用于分析细菌生长数据。通过将信号的自然对数除以初始信号来转换每个轨迹。一个改进的逻辑函数，具有对应于最大信号（A）的三个参数，最大值速率（μ）和滞后时间（τ）用于拟合每个轨迹。 鉴于GFP-ΔretS和mCherry-ΔretSΔtse/ i1-6菌株之间的已知相互作用，可以假设在与GFP-ΔretS菌株22,24的共培养物中，mCherry菌株的生长将被抑制。使用提取的参数，可以寻找从GFP和mCherry轨迹提取的参数之间的正相关或负相关。初始参数分析表明，这些物种的共培养对其总体生长影响可以忽略不计。在生长曲线中看到的变异性可能是由于环境因素，例如初始播种密度，其在较小的井直径处变得更可变（ 图5b ）。由于存在而对mCherry菌株产生显着的负面影响<html>未观察到GFP-ΔretS。例如，当绘制最大mCherry信号与初始GFP-RFP信号的比率时，当孔中GFP-ΔretS的比率较高时，没有发现mCherry表达的降低。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55701/55701fig5.jpg" /> 图5:适应改良Logistic方程的荧光增长轨迹。 （ a ）将示例曲线归一化，然后通过调整三个参数，最大强度（A），最大速率（μ）和滞后时间（τ）来修改逻辑方程。 （ b ）各种直径的各个孔中的初始mCherry信号与初始GFP信号。 （ c ）最大mCherry报告信号相对于表达GFP的细菌的初始强度与表达RFP的细菌的比率相比较。ource.jove.com/files/ftp_upload/55701/55701fig5large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 Mougous实验室以前的工作表明，VI型分泌的作用需要分泌细胞和易感细胞之间的直接细胞间细胞接触，并且更长的接触时间会导致更多的细胞裂解23 。因此，我们认为，在这些阵列中，两个突变体的指数增长简直超出了该模型系统中接触介导的致病作用。然而，在各种尺寸的孔中，表达GFP和mCherry的细菌以不同的斑块生长（ 图2 和 3 ）。因此，使用空间分析可以更好地研究含有接触介导的致病因子的细菌群落，而不是关注生长速度。例如，而不是focusi在集成强度上，其表示孔中的红色或绿色细胞的总数，该微孔格式允许计数红色和/或绿色像素的数量和位置。可以分析这些孔内单个菌落或斑块的生长情况，重点是红色和绿色信号重叠的边界（ 图6a ）。然后可以执行量化协同定位事件和最近邻分析，以更仔细地查看各个井内的贴片尺寸，贴片生长速率和贴片重叠等性能。空间分析将通过更高倍率的成像和共焦显微镜来辅助（ 图6b ）。 <img alt="图6" class="xfigimg" src="/files/ftp_upload/55701/55701fig6.jpg" /> 图6:不同物种的空间组织可以使用荧光和共聚焦显微镜进行分析。 （ <strong&gt; a）使用共聚焦显微镜，20倍放大倍数和NA为0.8的一个100μm厚的获取的一系列图像。白色箭头指向黄色区域，红色和绿色菌落重叠或混合。分析在这些地区殖民地生长的方式对于研究基于接触的发病机制将是有趣的。 （ b和c ）15μm直径，〜7.5μm深孔的共焦图像（20倍放大，NA = 0.8）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55701/55701fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

微孔阵列平台内微生物群落发育的组装和跟踪|生物工程|视频

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微生物群落的发展取决于环境结构，成员丰度，特征和相互作用等因素的结合。该协议描述了一种合成的微制造环境，用于同时跟踪包含在femtoliter井中的数千个社区，其中可以近似关键因素，如利基尺寸和约束。

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

assembly-tracking-microbial-community-development-within-microwell

Universidad San Sebastian

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SCIENTISTS IN ACTION

8.9K 次观看.  Oak Ridge National Laboratory. 微生物群落的发展取决于环境结构，成员丰度，特征和相互作用等因素的结合。该协议描述了一种合成的微制造环境，用于同时跟踪包含在femtoliter井中的数千个社区，其中可以近似关键因素，如利基尺寸和约束。 

视频: Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

工程学

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

生物学

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...