An automated platform for kinase assays on patient cells

描述(由申请人提供)： 为研究激光诱导的组织损伤，提出了一种具有时间分辨成像能力的集成激光微束显微镜系统。激光微束为细胞微操作提供了一种快速、自动化和非接触的手段，因为它具有高空间特异性的能量沉积能力和有限的附带损害。人们对它们在组织微处理和单细胞分析中的应用越来越感兴趣。然而，由于激光-细胞相互作用发生的空间尺度小、时间尺度快，对其基本机制的研究受到限制。我们提出的实验平台将在模拟体内系统的组织工程样本中以高空间和时间分辨率成像激光微束诱导的损伤的动力学。聚焦的纳秒激光脉冲将被传送到样本，在特定位置造成伤害，这个过程将在纳秒到微秒的时间尺度上使用时间分辨成像进行成像。从这些图像可以确定高温等离子体、冲击波传播和空化气泡膨胀和坍塌对细胞损伤的贡献。时间分辨图像还将用于定量估计空化气泡参数，如气泡大小、坍塌时间和气泡能量。气泡动力学将被用来估计组织部位的流体速度和剪应力。激光脉冲的生物效应将使用荧光分析进行监测。一个主要目标将是将时间分辨成像的物理效应与观察到的生物反应联系起来。流体动力学模拟将在快速时间尺度上提供细胞对高剪切场的响应，以提供损伤过程的生物物理特征。该平台将被设计成可扩展用于激光微束和生物学中的空化现象的一般研究。这项研究将主要在印度班加罗尔的国家生物科学中心与Kaustubh Rau博士合作进行，作为NIH赠款R01-EB004436的延伸。

DESCRIPTION (provided by applicant): An integrated laser microbeam-microscope system with time resolved imaging capabilities is proposed for studying laser induced tissue injury. Laser microbeams offer a fast, automated and non-contact means for cellular micromanipulation due to their ability to deposit energy with high spatial specificity and limited collateral damage. There is growing interest in their use for applications in tissue microprocessing and single cell analysis. However, research on basic mechanisms of laser-cell interactions is limited due to the small spatial scales and fast time scales over which they occur. The experimental platform we propose will image the dynamics of laser microbeam-induced injury with high spatial and temporal resolution in tissue engineered samples that mimic in-vivo systems. Focused nanosecond laser pulses will be delivered to the sample to cause injury at specific sites and the process will be imaged using time-resolved imaging on nanosecond to microsecond timescales. The contributions of the high-temperature plasma, Shockwave propagation and cavitation bubble expansion and collapse to cell injury will be determined from these images. Time-resolved images will also be used for quantitative estimation of cavitation bubble parameters such as bubble size, collapse time and bubble energy. The bubble dynamics will be used to estimate fluid velocity and shear stress at the tissue site. The biological effects of laser pulses will be monitored using fluorescence assays. A major goal would be to relate the physical effects from time-resolved imaging to the observed biological response. The hydrodynamic modeling will provide insights on cellular response to high shear fields on fast time scales to provide a biophysical characterization of the damage process. The platform will be designed to be scalable for general studies of laser microbeams and cavitation phenomena in biology. This research will be done primarily in Bangalore, India at the National Centre for Biological Sciences in collaboration with Dr. Kaustubh Rau as an extension of NIH grant R01-EB004436.