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中生长和基因表达的平台。对生长中的细菌进行延时显微镜观察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，细菌的指数生长迅速破坏固体支持物，耗尽局部营养环境，拥挤细胞，并限制测量持续时间。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规避这种测量的低通量，以创建一个大规模并行，高通量，单细胞“芯片”-DNA微阵列的活体模拟物。 在我们的第一个目标中，我们将继续构建单细胞恒化器-一种微型图案垫，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化的水凝胶垫，将细菌限制在高密度的线性轨道上。缓冲液流过凝胶中的微流控管线，输送新鲜的营养缓冲液并冲走多余的细胞。柔软多孔的水凝胶以非扰动压力将细胞固定在适当位置，并允许不受限制的扩散，以保持均匀的营养环境。我们将专注于创造一种设备，允许实时控制营养环境和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大大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化水凝胶上的方法。我们将采用两种方法将细胞打印到图案化的焊盘上，这两种方法都已通过原理验证实验进行了验证。这一目标的进展将允许在单个图案化水凝胶上并行运行多个实验，从而显著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，以在纳米图案化垫上进行高速，高通量，延时显微镜生长，并且我们将开发自动图像分析软件，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夜之间监测数百万个细胞的整个生命周期。 这些进展将使我们实验室的高通量单细胞研究成为可能。我们将能够表征调控复杂的多蛋白机器（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。 公共卫生相关性：我们将开发一种单细胞恒化器，它可以高通量测量在恒定营养条件下生长的单个活细胞的生长和基因表达。我们将开发将数千种不同菌株打印到单个设备上的方法，以及收集和分析在该设备中生长的细菌的大量延时数据集的方法。这个实验平台将使我们能够在单细胞水平上研究全基因组转录对广泛的环境压力的反应，包括多种抗生素暴露和营养饥饿。

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. PUBLIC HEALTH RELEVANCE: We will develop a single-cell chemostat that allows the high-throughput measurement of growth and gene expression of single living cells growing under constant nutrient conditions. We will develop methods to print thousands of distinct strains to a single device and methods to collect and analyze massive time-lapse data sets of bacteria growing in this device. This experimental platform will allow us to study genome-wide transcriptional response to a wide range of environmental stresses, including multiple antibiotic exposure and nutrient starvation, at the single-cell level.