基于时空聚焦光场的三维多光子显微原理与技术研究

Multiphoton microscopy employing ultrafast laser sources can provide better penetration depths for imaging biotissues. Therefore, this technique is of vital importance for life science. However, in the conventional multiphoton bioimaging, it is inevitable for the excitation beam to generate out-of-focus fluorescence signal, which reduces the signal-to-noise ratio and sets a limit for imaging deeply inside the biotissues. Here we propose to resolve this issue of noise background using a spatiotemporal focusing technology originally developed by our group. In addition, the spatiotemporal focusing technique has been further studied by some research groups in the US and Europe. By looking at the characteristic behaviors of the spatiotemporally focused beams propagating in biotissues and by developing methodologies for compensating aberration and dispersion, we will verify the feasibility and performance of this approach, and experimentally demonstrate a scanning multiphoton bioimaging system with improved penetration depths in various biotissues. Besides, we will explore the principle and mechanism for substantially improving the imaging resolution by utilizing spatiotemporally focused structured light fields. Our group has been focusing on ultrafast laser technology and ultrafast light field manipulation, femtosecond laser 3D micro- and nanophotonics, and multiphoton imaging. The expertise provides solid foundation for the research proposed herein.

基于超快激光的多光子荧光显微技术能够提供更好的生物组织穿透深度，对生命科学研究具有重要意义。然而，传统多光子显微成像过程中产生的显著的焦点外荧光激发形成了较强的背景噪声，从而降低了成像信噪比并限制成像深度。本课题采用课题组自行研发，具有自主知识产权并现已被欧美同行研究组借鉴采用的飞秒激光时空聚焦新技术，为解决上述的背景荧光噪声问题提供一条新的途径。通过对时空聚焦光束在生物体中的传输行为研究，以及发展时空聚焦光场相差与色散补偿方法，对该技术途径进行系统验证，并最终通过实验来演示具有更好穿透深度的多光子扫描生物成像。此外，本课题还将探索通过结构光场与时空聚焦光场的融合，来实质性地提升成像分辨率的原理与机制。本课题研究组在超快激光技术与超快光场调控、超快激光三维微纳光学、多光子成像等领域持续积累，为本项目的顺利执行奠定了技术与理论基础。

由于超快激光能够在透明材料（如生物组织、玻璃等）中形成高度局域化的三维相互作用，从而开辟了三维显微成像与三维微加工特色领域。特别是对超快激光进行时空聚焦与光束整形还可以进一步增强在传输方向的局域能力，从而提升成像与加工的三维分辨率。本项目研究了时空聚焦光束的理论传输行为，得到时空聚焦光场对脉冲啁啾的依赖关系，体现出与标准高斯分布超快光场显著不同的非线性传输行为特征；通过优化脉冲时空整形，实现了大尺度透明材料的高精度三维制备并进行成像分析；获得了具有单模光场的低损耗三维流体光波导，以及从单模光波导逐渐转化为模场尺寸更大的多模光波导的模斑转换器件，同时对上述器件形貌进行三维成像观测并对性能进行系统检测。项目成果表明，采用时空聚焦及时空整形光场可突破三维非线性相互作用中的衍射极限瓶颈，有望突破厚样品材料的三维高分辨成像难题。以上成果在项目执行期间发表5篇SCI论文。