An automated platform for kinase assays on patient cells

描述（由申请人提供）： 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，用于研究激光诱导的组织损伤。激光微束提供了一种快速，自动化和非接触的手段，细胞显微操作，由于他们的能力，存款能量与高空间特异性和有限的附带损害。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，激光与细胞相互作用的基本机制的研究是有限的，由于小的空间尺度和快速的时间尺度上，他们发生。我们提出的实验平台将在模拟体内系统的组织工程样品中以高空间和时间分辨率成像激光微束诱导损伤的动态。聚焦的纳秒激光脉冲将被传递到样品以在特定部位造成损伤，并且该过程将使用纳秒至微秒时间尺度上的时间分辨成像来成像。高温等离子体、冲击波传播和空化泡膨胀和破裂对细胞损伤的贡献将从这些图像中确定。时间分辨图像也将用于定量估计空化气泡参数，如气泡大小，崩溃时间和气泡能量。气泡动力学将用于估计组织部位的流体速度和剪应力。激光脉冲的生物学效应将使用荧光分析进行监测。一个主要目标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。流体动力学建模将提供关于细胞对快速时间尺度上的高剪切场的响应的见解，以提供损伤过程的生物物理表征。该平台将被设计为可扩展的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Kaujih Rau博士合作进行，作为NIH资助R 01-EB 004436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.