结合虚拟单像素成像解卷积的双边照明光片荧光显微技术

Dual-sided illumination light-sheet fluorescence microscopy with virtual single-pixel imaging deconvolution

光片荧光显微术(light-sheet fluorescence microscopy,LSFM)采用薄片光束从侧面激发样品,在垂直于光片方向上进行成像,具有成像速度快、光学层析能力强以及光漂白和光毒性低等优点,适用于对较大活体生物样品进行高质量、长时间三维动态观测.然而,传统高斯光束LSFM存在分辨率低和成像视场小的问题.本文在双边照明LSFM的基础上,结合虚拟单像素成像解卷积技术,提出了一种大视场高分辨双边照明LSFM,实现了视场和分辨率的同时提升.设计和搭建了双边照明LSFM,开展了荧光珠和转基因斑马鱼样品的三维光切片显微成像实验,实验结果证明了系统的三维高分辨成像能力,对于大视场、高分辨LSFM的发展和应用具有重要意义.

In light-sheet fluorescence microscopy(LSFM)a thin light sheet is used to excite the specimen from the side and imaging is performed in the direction perpendicular to the light-sheet.It has the advantages of fast imaging speed,high optical sectioning capability and low photobleaching and phototoxicity to samples.Therefore,it is suitable for high-quality,long-term three-dimensional dynamic observation of large living biological samples.However,the traditional Gaussian light sheet illumination microscopy technology has the problems of small imaging field of view and low spatial resolution.Based on the existing dual-sided illumination LSFM,a large field of view and high resolution LSFM combined with virtual single-pixel imaging deconvolution is presented in this paper,which improves the field of view and resolution of LSFM simultaneously.The relevant microscope is designed and built,and three-dimensional optical sectioning imaging experiments on fluorescent beads and transgenic zebrafish standard samples are carried out.The experimental results prove the three-dimensional high resolution imaging capability of the microscope,which is of great significance in developing the large field of view and high resolution LSFM.