基于受激发射损耗原理的超分辨成像与纳米加工关键技术的理论模拟与实验研究
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摘要
本论文“基于受激发射损耗原理的超分辨成像与纳米加工关键技术的理论模拟与实验研究”,将矢量衍射积分理论运用于连续光受激发射损耗(CW STED)显微系统的研制中。并在此基础上,探讨了CW STED显微系统在远场光学超分辨成像技术、宏观纳米结构制备技术、流体动力学等研究领域中的基础性科学性与技术问题。该研究工作,为进一步探索具有更高空间分辨能力的超分辨光学技术,并拓展该项技术的在相关学科领域的应用奠定了理论与实验基础
STED显微技术的核心思想是利用具备一定空间结构的消激发光束选择消除荧光激发,从而压缩了荧光点扩散函数达到突破衍射极限的目的。因此,在STED技术中,消激发光的空间结构、光强分布等因素极大的影响了STED显微技术的分辨率。论文首先阐述了利用矢量衍射积分理论描述高数值孔径汇聚光系统光线的方法,并在此基础上建立了计算连续激光CW STED显微系统焦点光场分布的理论模型。通过使用多种光束偏振态配合多种光学相位调制方案,计算获得了焦平面处消激发光的仿真图案,并且对能够有效压缩激点扩散函数的消激发光空间结构的特点,以及它们在STED系统的分辨率改进方而做了详细分析与讨论。此外,由于光学系统的初级像差的存在会对消激发光的分布造成影响,造成STED系统分辨率的下降。本文利用焦点光场的计算模型,模拟了初级像差,特别是多重初级像差同时存在时激发光、消激发光在焦点的光强分布的空间特点,并进行了具体系统的分析。这些工作对于更深入的认识STED显微成像技术的关键技术具有理论指导意义。
STED显微技术为远场纳米加工提供了新的理念,在本论文中,基于自己搭建的CW STED系统,我们探索了使用具有环形结构的可见光加工纳米柱/纳米孔技术。在我们搭建的可见光激光直写系统中,采用了中心波长为532nm的CW绿光,通过相位为0-2Pi旋转变化的涡旋相位板对激光波前进行调制来获得环形结构的曝光光束。当曝光光束照射到正光刻胶表面时,由于这种中空的环形光束结构使得光照区域的中央不能被曝光从而形成柱状的纳米结构；当使用负性光刻胶时,经曝光后产生的是纳米孔结构。实际制备实验中,我们在一类正光刻胶薄膜表面成功的制造出了纳米柱阵列,AFM显微镜测量结果显示所制造的纳米柱直径大小可以达到1/11入射波长—远小于衍射极限尺寸。实验制造中存在的图案的变形,通过比较多像差衍射积分理论进行模拟计算,能够得到合理可信的解释。
本论文的第三部分工作,基于我们搭建的CW STED系统,开展了激光与多种荧光试剂的相互作用的研究。实验测试了多种荧光试剂的激发与消激发效果,并对某些试剂在强的激发光与消激发光共同作用下所引发的独特的非线性效应做了分析与探讨。研究结果为进一步提高CW STED技术分辨能力、研究荧光分子的非线性效应提供了重要的参考。我们搭建的这套CW STED显微系统,对涂覆了荧光颗粒的样品成像,具有70nm的分辨力。基于这套CW STED显微系统结合激光诱导荧光漂白测速技术可以实现对纳米通道内流体速度剖面的测量,这将为芯片实验室、纳米传感器等的研究做出重要贡献。
本博士论文中,首先通过数值模拟的方法,研究了STED显微技术中影响分辨率的关键技术,为进一步改进STED显微技术奠定了理论模拟基础。其次,基于CWSTED系统,提出了一种新型、简单、经济的利用CW可见光制造纳米尺度以下纳米柱与纳米孔的方法。此外,利用自建的CW STED系统,研究了多种荧光试剂的受激发射损耗的效果。本论文的工作为完善远场光学超分辨成像的理论与技术、进一步提高其分辨能力、扩展其在相关研究领域的应用提供了一些新的思路与方法。
A number of techniques of nano imaging, fabricating, fluid velocity measuring based on the continuous wave (CW) stimulated emission depletion (STED) system have been studied by using vectorial diffraction integral theory and a self-made CW STED system in this Ph D project. It lays theoretical and experimental foundation for resolution improving and new application developing of the far-field super resolving technology.
The core ideology of STED technology is to confine the point spread function (PSF) of the fluorescence to a sub-diffraction size with a certain structural depletion beam. Therefore,the spatial structure and the intensity distribution properties are a kind of dominating factors of the resolution.In this project, the theoretical model for focal field calculation of CW STED system has been built up based on vectorial diffraction integral theory which is applied for focusing light investigating in high numerical aperture system.The depletion focal patterns engineered by applying the variety of phase plates with different polarized laser are obtained in the calculation; the characters of the efficient PSF compressing STED structures, as well as the improvements on resolution, are analyzed and discussed in detail.. In addition, the primary aberrations cause the decline of the resolution. Therefore, the primary aberration effects, especially the combined influence of the primary aberrations on depletion beam have been studied by utilizing the theoretical model. The study is instructive for the insightful understanding of the key element of the STED technology.
The STED technology has inspired new approaches for the far-field nano fabrication. In this project, a nanopillar/pore fabrication method based on a CW visible laser direct writing system derived from a CW STED system is proposed. In the experiment, a visible laser with center wavelength at532nm is modulated by a0-2Pi spiral phase plate to generate a donut-shaped exposure beam. The photoresist at the center of the focal spot will not be exposed due to the zero intensity dark core of the donut-shaped pattern. A kind of nanopillar structure will be obtained when a positive-tone photoresist is adopted, while nanopore structure will be fabricated when using a negative-tone photoresist in lithography. In the experimental fabrication, a kind of nanopillar array has been obtained successfully on a positive film with the pillar's diameter can achieve1/11of the incident wavelength, far smaller than the diffraction limitation. The transformation of the experiment results are explained reasonably with the multiple aberrations theory. This study has proposed and realized a novel, simple, efficient and cost-saving nanopillar/pore fabricating method with CW visible light.
In the third part of the thesis, a STED system using CW lasers is built up and applied for the fluorescence depletion efficiency study. Moreover, the nonlinearly phenomenon that might be induced by the strong excitation and depletion field in some reagents are analyzed and discussed; it provides valuable references for resolution improving and the nonlinearly fluorescence effect studying in STED technology. In addition, the imaging of fluorescent particles has been obtained based on the CW STED system with the resolution achieving70nm. The velocity profile in nano channels also been measured by combining the system with laser induced photobleaching method, which will make a great contribution for the key technologies of lab-on-chip and nano sensing.
In this project, the theoretical model for CW STED system has been built up for the study of the key techniques of the STED technology firstly. Then, based on CW STED system, a novel, simple and cost-saving nanopillar/pore fabrication method has been proposed. In addition, the depletion efficiency of variety dyes has been studied with a self-made CW STED system. The super resolution imaging and micro fluid measuring has been presented. In sum, this doctoral project provides some new thoughts and approaches for further improving the resolution and exploring the new applications of the far-field super-resolution technology.
STED显微技术的核心思想是利用具备一定空间结构的消激发光束选择消除荧光激发,从而压缩了荧光点扩散函数达到突破衍射极限的目的。因此,在STED技术中,消激发光的空间结构、光强分布等因素极大的影响了STED显微技术的分辨率。论文首先阐述了利用矢量衍射积分理论描述高数值孔径汇聚光系统光线的方法,并在此基础上建立了计算连续激光CW STED显微系统焦点光场分布的理论模型。通过使用多种光束偏振态配合多种光学相位调制方案,计算获得了焦平面处消激发光的仿真图案,并且对能够有效压缩激点扩散函数的消激发光空间结构的特点,以及它们在STED系统的分辨率改进方而做了详细分析与讨论。此外,由于光学系统的初级像差的存在会对消激发光的分布造成影响,造成STED系统分辨率的下降。本文利用焦点光场的计算模型,模拟了初级像差,特别是多重初级像差同时存在时激发光、消激发光在焦点的光强分布的空间特点,并进行了具体系统的分析。这些工作对于更深入的认识STED显微成像技术的关键技术具有理论指导意义。
STED显微技术为远场纳米加工提供了新的理念,在本论文中,基于自己搭建的CW STED系统,我们探索了使用具有环形结构的可见光加工纳米柱/纳米孔技术。在我们搭建的可见光激光直写系统中,采用了中心波长为532nm的CW绿光,通过相位为0-2Pi旋转变化的涡旋相位板对激光波前进行调制来获得环形结构的曝光光束。当曝光光束照射到正光刻胶表面时,由于这种中空的环形光束结构使得光照区域的中央不能被曝光从而形成柱状的纳米结构；当使用负性光刻胶时,经曝光后产生的是纳米孔结构。实际制备实验中,我们在一类正光刻胶薄膜表面成功的制造出了纳米柱阵列,AFM显微镜测量结果显示所制造的纳米柱直径大小可以达到1/11入射波长—远小于衍射极限尺寸。实验制造中存在的图案的变形,通过比较多像差衍射积分理论进行模拟计算,能够得到合理可信的解释。
本论文的第三部分工作,基于我们搭建的CW STED系统,开展了激光与多种荧光试剂的相互作用的研究。实验测试了多种荧光试剂的激发与消激发效果,并对某些试剂在强的激发光与消激发光共同作用下所引发的独特的非线性效应做了分析与探讨。研究结果为进一步提高CW STED技术分辨能力、研究荧光分子的非线性效应提供了重要的参考。我们搭建的这套CW STED显微系统,对涂覆了荧光颗粒的样品成像,具有70nm的分辨力。基于这套CW STED显微系统结合激光诱导荧光漂白测速技术可以实现对纳米通道内流体速度剖面的测量,这将为芯片实验室、纳米传感器等的研究做出重要贡献。
本博士论文中,首先通过数值模拟的方法,研究了STED显微技术中影响分辨率的关键技术,为进一步改进STED显微技术奠定了理论模拟基础。其次,基于CWSTED系统,提出了一种新型、简单、经济的利用CW可见光制造纳米尺度以下纳米柱与纳米孔的方法。此外,利用自建的CW STED系统,研究了多种荧光试剂的受激发射损耗的效果。本论文的工作为完善远场光学超分辨成像的理论与技术、进一步提高其分辨能力、扩展其在相关研究领域的应用提供了一些新的思路与方法。
A number of techniques of nano imaging, fabricating, fluid velocity measuring based on the continuous wave (CW) stimulated emission depletion (STED) system have been studied by using vectorial diffraction integral theory and a self-made CW STED system in this Ph D project. It lays theoretical and experimental foundation for resolution improving and new application developing of the far-field super resolving technology.
The core ideology of STED technology is to confine the point spread function (PSF) of the fluorescence to a sub-diffraction size with a certain structural depletion beam. Therefore,the spatial structure and the intensity distribution properties are a kind of dominating factors of the resolution.In this project, the theoretical model for focal field calculation of CW STED system has been built up based on vectorial diffraction integral theory which is applied for focusing light investigating in high numerical aperture system.The depletion focal patterns engineered by applying the variety of phase plates with different polarized laser are obtained in the calculation; the characters of the efficient PSF compressing STED structures, as well as the improvements on resolution, are analyzed and discussed in detail.. In addition, the primary aberrations cause the decline of the resolution. Therefore, the primary aberration effects, especially the combined influence of the primary aberrations on depletion beam have been studied by utilizing the theoretical model. The study is instructive for the insightful understanding of the key element of the STED technology.
The STED technology has inspired new approaches for the far-field nano fabrication. In this project, a nanopillar/pore fabrication method based on a CW visible laser direct writing system derived from a CW STED system is proposed. In the experiment, a visible laser with center wavelength at532nm is modulated by a0-2Pi spiral phase plate to generate a donut-shaped exposure beam. The photoresist at the center of the focal spot will not be exposed due to the zero intensity dark core of the donut-shaped pattern. A kind of nanopillar structure will be obtained when a positive-tone photoresist is adopted, while nanopore structure will be fabricated when using a negative-tone photoresist in lithography. In the experimental fabrication, a kind of nanopillar array has been obtained successfully on a positive film with the pillar's diameter can achieve1/11of the incident wavelength, far smaller than the diffraction limitation. The transformation of the experiment results are explained reasonably with the multiple aberrations theory. This study has proposed and realized a novel, simple, efficient and cost-saving nanopillar/pore fabricating method with CW visible light.
In the third part of the thesis, a STED system using CW lasers is built up and applied for the fluorescence depletion efficiency study. Moreover, the nonlinearly phenomenon that might be induced by the strong excitation and depletion field in some reagents are analyzed and discussed; it provides valuable references for resolution improving and the nonlinearly fluorescence effect studying in STED technology. In addition, the imaging of fluorescent particles has been obtained based on the CW STED system with the resolution achieving70nm. The velocity profile in nano channels also been measured by combining the system with laser induced photobleaching method, which will make a great contribution for the key technologies of lab-on-chip and nano sensing.
In this project, the theoretical model for CW STED system has been built up for the study of the key techniques of the STED technology firstly. Then, based on CW STED system, a novel, simple and cost-saving nanopillar/pore fabrication method has been proposed. In addition, the depletion efficiency of variety dyes has been studied with a self-made CW STED system. The super resolution imaging and micro fluid measuring has been presented. In sum, this doctoral project provides some new thoughts and approaches for further improving the resolution and exploring the new applications of the far-field super-resolution technology.
引文
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