Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology. The setup can also be integrated with complex growth chambers, and can be used to enrich or sort the imaged cells. Furthermore, MACS facilitates the visualization of individual cytoplasmic fluorescent proteins (FPs) in E. coli, allowing low-abundance proteins to be counted using standard total internal reflection fluorescence (TIRF) microscopy. Finally, MACS can be used to impart mechanical pressure for assessing the structural integrity of individual cells and their response to mechanical perturbations, or to make cells take up chemicals that otherwise would not pass through the membrane. This protocol describes the assembly of electronic control circuitry, the construction of liquid-handling components and the creation of the MACS microfluidics chip. The operation of MACS is described, and automation software is provided to integrate MACS control with image acquisition. Finally, we provide instructions for extending MACS using an external growth chamber (1 d) and for how to sort rare cells of interest.

中文翻译：

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使用微流控辅助细胞筛选平台的悬浮培养物的单细胞显微镜检查。

依赖于悬浮培养的非贴壁细胞（如大肠杆菌）的荧光成像的研究通常受到数据吞吐量和成像分辨率之间必须权衡的阻碍。我们开发了一种用于微流控辅助细胞筛选（MACS）的平台，该平台通过将悬浮细胞临时固定在微流控芯片中而克服了这一折衷。这使高通量和自动化的单细胞显微镜检查适用于各种细胞类型和大小。由于可以直接从悬浮培养物中快速采样细胞，因此MACS无需进行样品制备，因此可以进行测量而不会干扰天然细胞的生理状态。该装置还可以与复杂的生长室集成在一起，并且可以用于富集或分选成像的细胞。此外，MACS促进了大肠杆菌中单个细胞质荧光蛋白（FPs）的可视化，从而允许使用标准的全内反射荧光（TIRF）显微镜对低丰度蛋白进行计数。最后，MACS可用于施加机械压力，以评估单个细胞的结构完整性及其对机械扰动的响应，或使细胞吸收原本不会通过膜的化学物质。该协议描述了电子控制电路的组装，液体处理组件的构造以及MACS微流体芯片的创建。描述了MACS的操作，并提供了自动化软件以将MACS控制与图像采集集成在一起。最后，