基于双螺旋点扩散函数工程的多焦点图像扫描显微

Mutifocal image scanning microscopy based on double-helix point spread function engineering

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,本文提出了一种基于双螺旋点扩散函数工程的多焦点图像扫描显微成像方法和系统.在照明光路中,利用高速数字微镜器件产生周期分布的聚焦点阵对样品进行并行激发和快速二维扫描;在探测光路中,利用双螺旋相位片将激发点荧光信号的强度分布转换为双螺旋的形式;最终,利用后期数字重聚焦处理,从单次样品扫描数据中重构出多个样品层的超分辨宽场图像.在此基础上,利用搭建的系统分别对纤维状肌动蛋白和海拉细胞线粒体进行成像实验,证明了该方法的超分辨能力和快速三维成像能力.

Confocal laser scanning microscopy(CLSM) is a powerful imaging tool providing high resolution and optical sectioning.In its standard optical configuration,a pair of confocal pinholes is used to reject out-of-focus light.The diffraction limited resolution can be broken by reducing the confocal pinhole size.But this comes at the cost of extremely low signal-to-noise ratio(SNR).The limited SNR problem can be solved by image scanning microscopy(ISM),in which the single-point detector of a regular point-scanning confocal microscopy is substituted with an array detector such as CCD or CMOS,thus the two-fold super-resolution imaging can be achieved by pixel reassignment and deconvolution.However,the practical application of ISM is challenging due to its limited image acquisition speed.Here,we present a hybrid microscopy technique,named multifocal refocusing after scanning using helical phase engineering microscopy(MRESCH),which combines the doublehelix point spread function(DH-PSF) engineering with multifocal structured illumination to dramatically improve the image acquisition speed.In the illumination path,sparse multifocal illumination patterns are generated by a digital micromirror device for parallel imaging information acquisition.In the detection path,a phase mask is introduced to modulate the conventional PSF to the DH-PSF,which provides volumetric information,and meanwhile,we also present a digital refocusing strategy for processing the collected raw data to recover the wild-filed image from different sample layers.To demonstrate imaging capabilities of MRESCH,we acquire the images of mitochondria in live HeLa cells and make a detailed comparison with those from the wide-field microscopy.In contrast to the conventional wide-field approach,the MRESCH can expand the imaging depth in a range from -1μm to 1μm.Next,we sample the F-actin of bovine pulmonary artery endothelial cells to characterize the lateral resolution of the MRESCH.The results show that the MRESCH has a better resolution capability than the conventional wide-field illumination microscopy.Finally,the proposed image scanning microscopy can record three-dimensional specimen information from a single multi-spot twodimensional scan,which ensures faster data acquisition and larger field of view than ISM.