In Situ Single Cell Laser Lysis and Downstream qRT-PCR Profiling

描述（由申请人提供）：目前的单细胞RT-PCR分析严格限于分离细胞的分析，使其无法与原位细胞状态分析兼容，并且丢失了原始组织中细胞初始位置和形态的信息。我们建议开发一种创新的基于微流体的工具，用于临床和科学用户分析基因表达异质性，原位使用单细胞mRNA表达分析。该设备使用双光子激光在三维组织内已知坐标连续分解单个细胞。与传统的单光子激光裂解不同，双光子激光裂解依靠超快脉冲光源与生物材料之间的非线性相互作用，在纳米尺度的焦体积内实现能量向细胞的精确传递。裂解液立即运输到基于乳化的（油滴）qRT-PCR模块来分析mRNA的表达。通过优化激光功率和流体动力学聚焦以及精确的流量控制，可以最大限度地减少顺序裂解细胞之间的残留污染。由于微流控通道的规模小，样品处理的总体积通量减少到微升，细胞裂解和裂解物包封之间的时间间隔约为秒，qRT-PCR完成时间约为一小时。该技术非常适合基础生物医学研究和临床应用，如评估单细胞基因表达中的肿瘤细胞群异质性。此外，该技术也适用于未来的发展，以增加可以量化的基因数量。最终的实现将是一个高度复用的平台，能够检测从样品中洗脱的每个初始液滴的数十个mRNA序列。

DESCRIPTION (provided by applicant): Current RT-PCR analysis of single-cells has been strictly limited to analysis of disassociated cells, preventing its compatibility with in-situ analsis of cell states and loses information of initial cell location and morphology within the native tissue. We propose to develop an innovative microfluidic based tool for clinical and scientific users to analyze gene expression heterogeneity, in situ, using single-cell mRNA expression analysis. The device uses a two-photon laser to serially lyse individual cells at known coordinates within a 3D tissue. Differing from conventional single-photon laser lysis, two-photon laser lysis relies on the nonlinear interaction between an ultrafast pulsed light source and the biological material to achieve an energy transfer to the cell precisely within the nanometer-scale focal volume. The lysate is immediately transported to an emulsion-based (oil-droplet) qRT-PCR module to profile mRNA expression. Carryover contamination between sequentially lysed cells is minimized by optimizing laser power and by using hydrodynamic flow focusing with precise flow rate control. Because of the small scale of the microfluidic channels, the total volume flux for sample processing is reduced to microliters, the elapsed time interval between cell lysing and lysate encapsulation is on the order of seconds, and completion of qRT-PCR is on the order of one hour. This technology is well suited to basic biomedical research and clinical applications such as assessing tumor cell population heterogeneity in single-cell gene expression. Additionally, the technology is also amenable to future developments to increase the number of genes that can be quantified. The ultimate implementation would be a highly multiplexed platform capable of detecting dozens of mRNA sequences for each initial droplet eluted from the sample.