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

描述（由申请人提供）：目前单细胞的RT-PCR分析已严格限于分离细胞的分析，阻止了其与细胞状态原位分析的兼容性，并丢失了天然组织内初始细胞位置和形态的信息。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使用单细胞mRNA表达分析来原位分析基因表达异质性。该设备使用双光子激光连续裂解三维组织内已知坐标处的单个细胞。与传统的单光子激光裂解不同，双光子激光裂解依赖于超快脉冲光源与生物材料之间的非线性相互作用，以实现能量精确地在纳米尺度的聚焦体积内传递到细胞。将裂解物立即转移至基于乳液的（油滴）qRT-PCR模块以分析mRNA表达。通过优化激光功率和使用具有精确流速控制的流体动力学流动聚焦，最大限度地减少连续裂解细胞之间的残留污染。由于微流体通道的小规模，用于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经过的时间间隔大约为秒，并且qRT-PCR的完成大约为一小时。该技术非常适合基础生物医学研究和临床应用，例如评估单细胞基因表达中的肿瘤细胞群体异质性。此外，该技术也适合未来的发展，以增加可以量化的基因数量。最终的实现将是一个高度多路复用的平台，能够检测从样品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.