干细胞向心肌细胞的分化是心肌再生医学领域中的重大科学问题。研究干细胞到心肌细胞的功能性分化，尤其是分化心肌细胞的收缩功能目前面临两大困境：1）没有合适的光学成像方法；2）没有合适的细胞培养模型。我们首次利用非线性光学显微成像技术揭示了鼠原生心肌细胞的肌原纤维形成机制。本项目提出并发展一种具有多目标寻址和细胞区域匹配面激发功能的三维时空同步聚焦视频SHG/TPEF复合成像技术和系统；利用多光束激光诱导细胞微排列技术在弹性基底上规则排列细胞，构建类在体的心肌细胞-干细胞共培养模型；通过对共培养的干细胞的分化和收缩过程的高速三维立体光学成像记录，研究在机械拉伸和电刺激条件下干细胞分化的心肌细胞和原生心肌细胞的肌原纤维的形成过程以及收缩功能相关参数，揭示干细胞功能性分化为心肌细胞的机理。本项目研究将为细胞相互作用过程的研究提供重要光学手段，为发展新一代的心肌再生医学治疗方案提供重要的科学依据。

The proposed research addresses a significant problem in cardiac regenerative medicine, the contractile properties of stem cell-derived cardiomyocytes. The proposed technique has unique capabilities for establishing an in vivo-like cardiac-cell construct in an in vitro cocultural environment and capturing green-fluorescence protein and second harmonic generation images in real time to visualize the formation of myofibrils. The technique to be developed in the proposed research provides a platform technology for any biological problem where real-time visualization of structure and marker expression in multicellular tissue constructs is essential. The objective of the proposed research is to explore whether stem cell-derived cardiomyocytes will have the same pattern of contractile-mechanism formation as a native cardiomyocyte and/or the ability to generate a contraction force that is similar to that generated by a native cardiomyocyte. The specific aims are: 1) To develop a spatial light modulator-based rapid scanning, three-dimensionally addressable, two photon and second harmonic generation confocal microscope for dynamic myofibril imaging; 2) To utilize a novel laser cell patterning and a surface patterning techniques to build tissue constructs in vitro on a distensible substrate; and 3) To combine the cell patterning and rapid scanning techniques to investigate myofibrillogenesis in both stem cell-derived and native cardiomyocytes under mechanical stretching and electrical triggering. The success of the proposed research will provide data for the solution of the stem cell-based cardiac regenerative medicine.