基于双螺旋点扩展函数工程的三维多焦点双光子激光扫描显微技术

Three-Dimensional Multifocal Two-Photon Laser Scanning Microscopy Based on Double-Helix Point Spread Function Engineering

为了进一步提高成像速度和分辨率,提出了基于双螺旋点扩展函数(DH-PSF)工程的多焦点双光子激光扫描显微成像方法和系统(DH-MTPLSM)。在激发光路中,通过高速相位型空间光调制器(SLM)同时实现了三维多焦点阵列的产生和在样品面上的高精度并行数字寻址扫描;在探测光路中,通过双螺旋相位片将系统探测PSF调制为DH-PSF,从而提供样品的轴向信息,减少轴向扫描层数,进而提高三维成像速度;结合基于DH-PSF的数字重聚焦算法,恢复出不同深度样本的宽场图像,通过单次二维扫描获得样品的三维光切片信息。在此基础上,利用搭建的DH-MTPLSM系统开展了小鼠肾组织切片的双光子成像实验,验证了该方法的快速三维高分辨成像能力,这对于MTPLSM的发展具有重要的意义。

We present a method and a system of multifocal two-photon laser scanning microscopy(MTPLSM) based on double-helix point spread function(DH-PSF) engineering(DH-MTPLSM) to improve the imaging speed and resolution.In the excitation light path, a high-speed phase-only spatial light modulator(SLM) is employed to generate threedimensional(3D) multifocal arrays and implement high-precision parallel digital addressing scanning of the sample surfaces. In the detection light path, a double-helix phase plate is inserted to modulate the system detection PSF to DHPSF, which provides axial information of the sample. The aim is to reduce the number of layers scanned axially and ultimately enhance the speed of 3D imaging. Moreover, with a DH-PSF-based digital refocusing algorithm, we reconstruct the wide-field images of the sample at different depths and obtain the 3D optical sections of the sample by single2D scanning. On this basis, we build a DH-MTPLSM system, carry out two-photon imaging experiments on mouse kidney sections with this system, and verify the ability of the proposed method in fast 3D high-resolution imaging, which is of great significance for the development of MTPLSM.