视场增强的平板艾里光片显微镜研究(特邀)

Research on field-enhanced planar-Airy light-sheet microscopy(invited)

光片显微镜是近些年来研究较多的生物成像技术,相较于传统的激光共聚焦扫描显微镜而言,光片显微镜能够实现快速、低光毒性的体积成像。光片显微镜的照明光束可以选择高斯光束或其他无衍射光束(如贝塞尔光束、艾里光束等)。艾里光片显微镜是目前研究较多的技术,但是普通的艾里光片显微镜存在一个较大的问题,艾里光束具有自弯曲的特性,导致艾里光片在视场的两端超出探测物镜的景深范围,无法发挥出最优的成像效果。将艾里光束旋转45°形成平板艾里光片,使艾里光片不超出探测物镜的景深,以增大光片显微镜的成像视场。并利用双光子荧光激发技术,免除图像的后处理过程,大大提高了成像的效率。利用Matlab进行光学仿真,得到平板艾里光片显微镜的成像视场(~900μm)比普通艾里光片显微镜的成像视场(~600μm)增加了50%。搭建的平板艾里光片显微镜利用荧光微球进行校正实验,得到成像系统的横向分辨率为(1.93±0.17)μm,轴向分辨率为(3.19±0.41)μm。对斑马鱼脑出血模型的实时观测中,可以得到时间分辨率为x×y×z=0.60 mm×0.60 mm×0.40 mm/60 s的成像结果,并可以对局部血管的生长和发育进行实时监测,有利于脑出血疾病的机制探究。

Light-sheet microscopy is a biological imaging technology that has been studied a lot in recent years.Compared with traditional confocal laser scanning microscopy,light-sheet microscopy can achieve rapid volume imaging with low phototoxicity.The illumination beam of light-sheet microscope can choose Gaussian beam or other non-diffracting beams(such as Bessel beam,Airy beam,etc.).Airy light-sheet microscopy is the most researched technology at present,but there is a big problem with ordinary Airy light-sheet microscopy.Airy beam has the characteristic of self-bending,which causes Airy beam to exceed detection at both ends of the field of view.The depth of field of the objective lens cannot produce the best imaging effect.The Airy beam was rotated by 45°to form a planar-Airy light-sheet,so that the Airy light-sheet did not exceed the depth of field of the detection objective,so as to increase the imaging field of view of the light-sheet microscope.And using twophoton fluorescence excitation technology,the post-processing process of the image was eliminated,greatly improving the efficiency of imaging.In this study,Matlab was used for optical simulation,and the imaging field of view(~900μm)of the planar-Airy light-sheet microscope was increased by 50%compared with the imaging field of view(~600μm)of the ordinary Airy light-sheet microscope.The constructed planar-Airy light-sheet microscope was calibrated with fluorescent microspheres,and the lateral resolution of the imaging system was(1.93±0.17)μm and the axial resolution was(3.19±0.41)μm.In the real-time observation of the zebrafish intracerebral hemorrhage model,imaging results with a temporal resolution of x y z=0.60 mm 0.60 mm 0.40 mm/60 s can be obtained,and the growth and development of local blood vessels can be monitored in realtime to explore the mechanism of cerebral hemorrhage disease.