双光子亚衍射多焦点结构光照明显微研究  

Two-Photon Sub-Diffraction Multifocal Structured Illumination Microscopy

作　　者：权小娟 张晨爽 林丹樱[1] 于斌[1] 屈军乐[1] Quan Xiaojuan;Zhang Chenshuang;Lin Danying;Yu Bin;Qu Junle(Shenzhen Key Laboratory of Photonics and Biophotonics,Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province,College of Physics and Optoelectronic Engineering,Shenzhen University,Shenzhen 518060,Guangdong,China)

机构地区：[1]深圳大学物理与光电工程学院,深圳市光子学与生物光子学重点实验室,光电子器件与系统教育部/广东省重点实验室,广东深圳518060

出　　处：《中国激光》2023年第15期22-30,共9页Chinese Journal of Lasers

基　　金：国家重点研发计划(2022YFF0712500,2022YFF0706001);国家自然科学基金(61975131,62175166,62235007,62275165);深圳市科技计划(JCYJ20200109105411133,ZDSYS20210623092006020)。

摘　　要：为进一步提高双光子多焦点结构光照明显微技术(2P-MSIM)的空间分辨率,笔者提出并发展了一种双光子亚衍射多焦点结构光照明显微成像方法(2P-sMSIM).首先,通过改进的Gerchberg-Saxton(GS)相位恢复算法设计亚衍射聚焦点阵,生成相位图,利用高速相位型空间光调制器产生亚衍射聚焦点阵.通过计算机模拟的仿真实验,探究算法的可行性,并通过对荧光染料溶液的激发成像,证明了每个亚衍射聚焦点阵的平均尺寸为正常衍射受限点阵聚焦点尺寸的80%.其次,将该点阵引入2P-MSIM系统,对固定在BS-C-1细胞内的微管和商用线粒体切片分别进行了超分辨成像实验,证明了在亚衍射聚焦点阵激发下,2P-MSIM的分辨率和成像质量得到了进一步提高,这对于2P-MSIM的发展具有重要意义.Objective The multifocal structure light microscope(MSIM)can achieve an imaging depth of 50μm and a diffraction limited resolution that is two times that of the traditional wide-field structure light technique.However,factors such as fluorescence noise,scattering,and aberration limit its chromatographic capability and imaging depth when conducting super-resolution imaging of thick samples.To overcome these limitations,the MSIM technique combined with two-photon excitation,is implemented to obtain a twophoton multifocus structured light illumination microscope(2P-MSIM),and the imaging depth and resolution of MSIM are thus improved.The MSIM has great application potential in the field of fast super-resolution microscopic imaging of deep tissues in vivo.However,owing to the diffraction limit,the enhancement of the existing 2P-MSIM spatial resolution is limited.To further improve the spatial resolution of 2P-MSIM,a two-photon sub-diffraction multifocal structure illumination microscopy(2P-sMSIM)is proposed in this paper.Methods Using the improved Gerchberg-Saxton(GS)phase recovery algorithm,the conditions restricting the amplitude and the phase were enhanced in specific locations on the spectrum plane(the input plane reached the output plane after Fourier transform);subsequently,the amplitude and phase were constrained.After several iterations,the phase diagram of the sub-diffraction spot arrays(SSAs)was calculated,and the initial SSAs were obtained.The sidelobe intensity was controlled at approximately one-tenth of the center intensity(the sidelobe effect can be eliminated by the subsequent image-processing algorithm),and the algorithm parameter optimization was completed to obtain an SSA suitable for system imaging.Raw data for SSA and normal diffraction limited spot arrays were obtained.Next,Gaussian pinhole filtering,pixel relocation,and deconvolution algorithms were used to obtain the SSA and normal diffraction limited spot array images.The resolution of 2P-MSIM imaging was improved under the excitation of the

关 键 词：生物光学 多焦点结构光照明显微 亚衍射聚焦点阵 空间光调制器 相位恢复 

分 类 号：TH742[机械工程—光学工程]