超短脉冲激光多光子电离显微术研究

Unstained high resolution or ultra-high resolution optical microscopic imagining technologies are of essential importance to the development of cell biology and biophotonics, and are also critical for new breakthroughs in life science and modern medicine researches. Based on the ionization characteristics of different substances and material structures, a new type of nonlinear microscopic imagining method can be formed, which is the so-called ultrashort pulse laser induced ionization microscopy. Such a new nonlinear imagining method is fluorophore-free and ubiquitous for a broad variety of applications. This current currently proposed project aims at further study of the fundamental scientific principles and applicability of the femtosecond laser multiphoton ionization microscopy so that a solid foundation for the instrumentation of this novel technique can be established for microscopic imagining of biological cells and analysis of micro- and nano- materials. In the proposed project, both experimental and theoretical investigations of laser ionization microscopy applied to different types of biological cells and micro- nano-materials will be performed. It is expected that a complete evaluation of the key technical advantages and application potentials in terms of molecule specialty, detection sensitivity, and spatial resolution of ultrashort pulse laser multiphoton ionization microscopy will be obtained.

无标记、高分辨或超高分辨光学显微成像技术对细胞生物学和生物光子学的发展、实现生命科学和现代医学研究的新突破具有重大意义。基于物质不同结构或不同组份所表现出的不同电离特性来实现显微成像的超短脉冲激光电离显微术是一种新型的、无需荧光标记物从而能够具有材料普适性的非线性光学显微成像方法。本项研究旨在对由本课题组提出的飞秒激光多光子电离显微术的科学原理和适用范围开展深入的研究和探索，为下一步构建可直接用于生物细胞成像和微纳米尺度材料分析的超短脉冲激光电离显微镜实用化仪器打下一个牢固的前期基础。项目将通过开展不同类型的生物细胞和微纳结构材料的激光电离成像实验以及相关理论分析实现对超短脉冲激光多光子电离显微术的特异性、灵敏度和空间分辨率等核心技术指标的一个全面的评估和界定。

利用超短脉冲激光高峰值功率的特点，在待测样品表面或大带隙的透明材料内部通过多光子电离过程产生空气或样品电离，记录不同位置处的电离信号，即可获得与待测样品表面形貌、化学组分相关的显微图像。本项目在对超短脉冲激光多光子电离显微系统优化设计的基础上，成功搭建了一台由计算机程序控制的电离显微成像样机。利用该成像系统样机，通过实验研究发现，多光子电离显微成像的横向分辨率可达100 nm量级（金属样品），并且具有远大于聚焦脉冲瑞利长度的景深，可在保持高的横向分辨率的同时对透明样品不同深度处的细节实现同时成像。实验还发现，超短脉冲激光的色散，即脉冲时域强度分布对电离显微成像质量具有重要影响，具有残余二阶和正三阶色散的激光脉冲具有更好的电离成像结果。利用搭建的电离显微成像系统样机，对包括电介质材料、金属材料以及生物细胞进行了优于光学显微的清晰成像，还可对透明样品进行不同深度处的层析成像。本项目研究的超短激光脉冲多光子电离显微技术因其明显优于传统光学显微技术的分辨率、大景深、优秀的化学组分分辨能力以及可在液体/气体环境下工作的特点，必将在显微成像和微纳尺度材料特性的研究方面发挥重要作用。