【作者】 李宏伟；

【导师】 柳鹏飞；

【作者基本信息】 天津大学 ， 生物医学工程， 2023， 硕士

【摘要】 激光光片荧光显微成像技术作为一种新型的荧光显微成像方法,因其正交的照明及探测光路,具有成像速度快、三维分辨率高、光漂白及光毒性弱等优势,可广泛应用于脑科学、类器官等多个生命科学领域,助力基础研究和临床应用。一般,光片显微镜以高斯光束作为照明光束,其成像视场和轴向分辨率相互制约。以艾里光束为代表的无衍射光束可一定程度上平衡这种制约关系,保持高轴向分辨率的同时,可获得较大成像视场。但是,艾里光束除具有无衍射特性外,还具有自弯曲性,而探测物镜的景深有限,视场两侧光片会超出探测物镜的景深范围,激发的荧光信号难以被收集,这限制了艾里光片的视场最大化。基于此,本文提出光片显微成像的视场增强研究,主要包括以下两个方面:第一,在照明光路中,提出“类艾里”光片成像方法,降低照明光束的自弯曲度,令其更大比例处于景深限制以内,从而实现视场的增大;第二,在探测光路中,提出相位调制元件辅助的景深扩展方法,用以收集视场两侧更多的荧光信号,最终增大光片显微成像系统的视场。为开发视场增强的光片显微成像系统,本文主要工作如下:1.激发光片的理论分析与仿真及“类艾里”光片研究。本文基于Richards-Wolf矢量衍射积分理论,建立激发光片的矢量衍射积分数学模型,并应用啁啾Z变换进行积分数值求解,以立方相位的光瞳函数为基础,建立了艾里光片电场分布仿真模型。利用“幂次叠加”的相位调制方式生成“类艾里”光片。以上述电场仿真为依据,从理论分析的角度对比了不同参数条件下的光片类型,相位公式为exp[2παi[(u~2+v~2)+(u~3+v~3)+(u~4+v~4)+(u~5+v~5)+(u~6+v~6)+(u~4+v~4)]]的类艾里光束相较于同等轴向分辨率的NA=0.175和α=25的艾里光束能够提升7%和5%的成像视场。且类艾里光片的强度聚焦在视场两侧,能够有效减弱生物组织的散射吸收作用,更适宜大体积生物样本成像。2.基于景深扩展的艾里光片显微成像方法研究。探讨了锥透镜和三次相位掩模板两种光学器件对荧光信号的调制作用。本文基于菲涅尔衍射积分构建探测光路的电场分布数学模型,并通过快速傅里叶变换(FFT)方法对电场分布进行了数值求解,实现了锥透镜和三次相位掩模板扩展探测光路景深的方法进行理论仿真。从仿真分析出发,探究了锥透镜的位置、底角、形状等因素对探测光路点扩散函数的影响;研究了三次相位掩模板的位置、比例因子对探测光路点扩散函数的影响,最终利用USFA分辨率测试图进行仿真成像,验证两种方法扩展景深的可行性。并对扩展景深后的照明光路点扩散函数进行理论计算,验证扩展景深对艾里光片显微成像系统的视场扩展作用。3.视场增强的艾里光片显微成像系统的搭建与校正。该系统利用类艾里光片较缓的自弯曲特性实现了视场增强作用,并利用锥透镜扩展景深实现了成像视场的增大。使用荧光微球对上述理论方法进行了标定实验。4.视场增强的艾里光片显微成像系统的生物学应用。基于此系统,对创伤性脑损伤小鼠的大脑、肝脏、肾脏器官的组织病变进行三维显微观测,比较正常小鼠与病变小鼠组织的病理区别,为创伤性脑损伤的治疗提供一定的理论支撑。基于以上研究,视场增强的艾里光片显微成像技术具有大视场的成像优势。根据荧光微球标定实验,高斯光片能够获得70μm的成像视场,艾里光片能够获得360μm的成像视场,视场提升约414%。类艾里光片在保证高轴向分辨率(4.35μm)的同时,可获得890μm的单次扫描视场。相较于同轴向分辨能力的艾里光片,能够获得7%和5%的视场增强。景深扩展的艾里光片能够获得490μm的成像视场,相较于艾里光片视场增强约36%。景深扩展的类艾里光片能够获得1300μm的成像视场,相较于类艾里光片视场增强约46%。利用类艾里光片显微成像系统可以对创伤性脑损伤小鼠的大脑及全身器官进行三维显微成像,观察脑出血位置的血管破裂及血液淤积现象,及脑损伤对身体器官的影响,有望为创伤性脑损伤的预后治疗提供新的方法。更多还原

【Abstract】 Light-Sheet Fluorescence Microscopy(LSFM)imaging technology is a new type of fluorescence microscopy imaging method that has the advantages of fast imaging speed,high three-dimensional resolution,and weak photobleaching and phototoxicity due to its orthogonal illumination and detection optical paths.It can be widely applied across the disciplines of organoid,brain,and other life sciences,facilitating both fundamental research and practical applications.Typically,the LSFM uses a Gaussian beam as its illumination beam,which results in mutual restrictions on its imaging field of view(FOV)and axial resolution.Airy beam,which represents a Non-diffracted beam,can partially alleviate this constraint relationship and enable larger imaging FOV while maintaining high axial resolution.However,Airy beam not only has Non-diffraction characteristic,but also has self-bending property,and the depth of field(DOF)of the detection objective is limited.As a result,the FOV of the Airy beam is also limited.The light-sheet on both sides of the FOV will exceed the DOF range of the detection objective,and the excited fluorescence signal is difficult to be collected,which limits the maximum imaging FOV of the Airy beam.Based on this,this paper presents a study on FOV enhancement for Airy light-sheet fluorescence microscopy,which primarily focuses on the following two aspects:Firstly,to expand the FOV,we suggest using an"Airy-like"imaging method in the illumination path,which reduces the self-bending of the illumination beam and ensures that a larger portion of it falls within the DOF limit.This leads to improved image quality and FOV.Secondly,in the detection path,a method of enhancing DOF assisted by phase modulation element is proposed to collect more fluorescence signals on both sides of the FOV,and finally increase the FOV of the light-sheet fluorescence microscopy.In order to develop the light-sheet fluorescence microscopy with field of view enhancement,the main work of this paper is as follows:1.Theoretical analysis and simulation of illumination light-sheet,and investigation of"Airy-like"light-sheet.This paper establishes a mathematical model using Richards-Wolf vectorial integral theory to analyze the properties of illumination light-sheets.The proposed method utilizes Chirped Z-transform for integration and numerical solutions.A simulation model for the electric field distribution of an Airy light-sheet is established based on the cubic phase pupil function.The phase modulation method of"power superposition"is used to generate"Airy-like"light-sheet.Based on the electric field simulation,we compared different types of light-sheets from a theoretical perspective,while varying the parameters under different conditions.The phase formula of Airy-like beam is exp[2παi[(u~2+v~2)+(u~3+v~3)+(u~4+v~4)+(u~5+v~5)+(u~6+v~6)+(u~4+v~4)]]In comparison to an Airy light-sheet with the same axial resolution parameters(NA=0.175 andα=25),the application of an Airy-like light-sheet can result in a 7%and5%improvement in FOV.Additionally,the Airy-like light-sheet exhibits a more highly focused intensity on both sides of the FOV,effectively reducing the scattering and absorption effects of biological tissue.This makes it particularly well-suited for imaging larger biological samples.2.Study on Airy light-sheet microscopic imaging using DOF extension.Investigation of the modulation effects of axicon lenses and cubic phase masks on fluorescence signals.This paper presents a mathematical model of the electric field distribution,which is based on the Fresnel diffraction integral.The electric field distribution is then numerically solved using the fast Fourier transform(FFT)method.The proposed model enables the theoretical simulation of axicon lenses and cubic phase masks,leading to the extension of detection path DOF.Through simulation analysis,this paper explores how the position,angle,and shape of the axicon lens impact the point spread function(PSF)of the detection path.The effect of the position and scaling factor(α)of the cubic phase mask on the PSF of the detection path was analyzed.Finally,the USFA resolution test image was utilized to simulate imaging,and the feasibility of the two methods for extending the DOF was confirmed.Additionally,the illumination path PSF with extended DOF was theoretically calculated to confirm the effect of the FOV expansion of the Airy light-sheet imaging system.3.A FOV-enhanced Airy light-sheet imaging system was constructed and calibrated for optimal performance.The system utilizes the slow self-bending characteristics of the Airy light-sheet to enhance the FOV,and the DOF is increased by the addition of a axicon lens.Calibration was conducted using fluorescent microsphere.4.Biological application of FOV-enhanced Airy light-sheet imaging system.Based on this system,three-dimensional microscopic observation was conducted on the tissue lesions of the brain,liver,and kidney organs of mice with traumatic brain injuries.The pathological differences between the normal mice and the diseased mice were compared,providing certain theoretical support for the treatment of traumatic brain injury(TBI).Based on the aforementioned research,the FOV-enhanced Airy light-sheet microscopic imaging technology provides the advantage of a larger FOV.The fluorescence microsphere calibration experiment revealed that the imaging FOV obtained by Gaussian light-sheet was 70μm,while that of Airy light-sheet was 360μm,improving the FOV by approximately 414%.The Airy-like light-sheet can also maintain high axial resolution(4.35μm)while obtaining a single scanning FOV of 890μm.When compared with Airy light-sheet with the same axial resolution,the FOV can be enhanced by 7%and 5%.Additionally,the image FOV obtained by the extended DOF with Airy light-sheet is 490μm,representing a 36%enhancement compared to the Airy light-sheet.With the extension of DOF,the Airy-like light-sheet’s imaging FOV can be 1300μm,resulting in a 46%increase compared to the Airy-like light-sheet.To observe the phenomenon of blood vessel rupture and blood stasis in the location of cerebral hemorrhage,as well as the influence of brain injury on body organs,three-dimensional microscopic imaging was performed on the brain and whole-body organs of mice with traumatic brain injury using the Airy-like microscopic imaging system.This provides a new method for the prognosis and treatment of traumatic brain injury.更多还原