A Cellular Chip for High-Throughput Measurements of Single, Growing Cells

描述（由申请人提供）：在本提案中，我们将开发一个用于高通量测量单个细菌细胞生长和基因表达的平台。细菌生长的延时显微镜是一项非常成功的技术，它揭示了单细胞生长和基因表达的自然异质性。然而，细菌的指数增长迅速压倒了固体支持，耗尽了当地的营养环境，拥挤细胞，并限制了测量时间。在这里，我们将通过结合微流控技术、微阵列技术和自动图像分析来创建大规模并行、高通量、单细胞“芯片”——DNA微阵列的活体模拟物，从而规避此类测量的低通量。在我们的第一个目标中，我们将继续构建单细胞化学抑制剂——一种微图案垫，可用于长时间培养高密度细胞，同时允许单个细胞成像。我们使用软光刻技术来制造纳米图案的水凝胶垫，将细菌限制在高密度的线性轨道上。缓冲液流经凝胶中的微流体线，提供新鲜的营养缓冲液，并冲洗掉多余的细胞。柔软和多孔的水凝胶以非扰动压力将细胞固定在适当的位置，并允许无限制的扩散以保持均匀的营养环境。我们将专注于创造一种设备，可以实时控制营养环境，并精确控制容纳细胞的水凝胶的机械性能。这种单细胞恒化器将极大地增加延时测量的持续时间和可以在视场中成像的细胞数量。在我们的第二个目标中，我们将开发利用现有微阵列技术将数千种不同的细菌菌株打印到单个图案水凝胶上的方法。我们将采用两种方法将细胞打印到有图案的衬垫上，这两种方法都已通过原理验证实验进行了验证。这方面的进展将允许在单一图案的水凝胶上并行进行多个实验，从而大大增加可以同时表征的不同细菌菌株的数量。在我们的最终目标中，我们将开发必要的基于图像的技术，以在纳米图案垫片上执行高速，高通量，延时显微镜的生长，我们将开发自动化图像分析软件，以从这些大型数据集中提取细胞谱系内生长，分裂和荧光基因表达的完整历史。这些技术将使我们能够在一次夜间测量中监测数百万个细胞的整个生命。这些进步将使我们实验室的高通量单细胞研究成为可能。我们将能够描述调节复杂的多蛋白机器（如鞭毛）组装的转录网络；并研究对多种抗生素压力的转录反应——这是一个与人类健康极其相关的问题。此外，通过提供高通量单细胞测量平台，我们的细胞芯片将被证明是回答微生物学中许多紧迫问题的有用工具。

DESCRIPTION (provided by applicant): In this proposal, we will develop a platform for the high-throughput measurement of growth and gene expression in single bacterial cells. Time-lapse microscopy of growing bacteria has been an extremely successful technique, revealing the natural heterogeneity that underlies growth and gene expression in single cells. However, the exponential growth of bacteria quickly overwhelms the solid support, depleting the local nutrient environment, crowding cells, and limiting measurement duration. Here, we will circumvent the low throughput of such measurements by combining microfluidic techniques, microarray technology, and automated image analysis to create a massively parallel, high-throughput, single-cell 'chip'-a living analog of the DNA microarray. In our first aim, we will continue our construction of a single-cell chemostat-a micro-patterned pad that can be used to cultivate a high density of cells for long durations while allowing individual cells to be imaged. We use soft-lithography to create nano-patterned hydrogel pads that constrain bacteria to high-density linear tracks. Buffer flow through microfluidic lines in the gel delivers fresh nutrient bufer and washes away excess cells. Soft and porous hydrogels hold cells in place with a non-perturbative pressure and allow unrestricted diffusion to maintain a uniform nutrient environment. We will focus on creating a device that allows real-time control over the nutrient environment and precise control over the mechanical properties of the hydrogel that holds the cells. This single-cell chemostat will dramatically increase both the duration of time-lapse measurements and the number of cells that can be imaged in a field-of-view. In our second aim, we will develop methods to leverage existing microarray technology to print thousands of distinct bacterial strains onto a single patterned hydrogel. We will pursue two approaches for printing cells to patterned pads, both of which have been validated by proof-of-principle experiments. Advances in this aim will allow multiple experiments to be run in parallel on a single patterned hydrogel, providing a dramatic increase in the number of distinct bacterial strains that can be characterized simultaneously. In our final aim, we will develop the necessary image-based techniques to perform high-speed, high- throughput, time-lapse microscopy of growth on a nano-patterned pad, and we will develop the automated image analysis software needed to extract the full history of growth, division, and fluorescent gene expression within cellular lineages from these large data sets. These techniques will allow us to monitor the entire life of millions of cells in a single overnight measurement. These advances will make high-throughput, single-cell studies possible in our laboratory. We will be able to characterize the transcriptional network that regulates the assembly of complex, multi-protein machines such as the flagella; and investigate the transcriptional response to multiple antibiotic stresses-a proble that is extremely relevant to human health. Moreover, by providing a platform for high-throughput single-cell measurements, our cellular chip will prove a useful tool for answering many pressing questions in microbiology.