Light sheet fluorescence microscope with single light sheet illumination enables rapid 3D imaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along a 45° inclined tube. The element, which is based on a multi-focal diffractive lens and a linear grating, generates five thin light sheets with equal intensities when combined with a refractive cylindrical lens. The generated uniform light sheets can be applied for the scanning of samples in tubes enabling flow-driven 3-dimensional imaging.

In light-sheet fluorescence microscopy, the axial resolution and field of view are mutually constrained. Axially swept light-sheet microscopy (ASLM) can decouple the trade-off, but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest, which sacrifices the photon efficiency. Here, we report a laterally swept light-sheet microscopy (LSLM) scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter. We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved. Moreover, based on the principle of image scanning microscopy, applying the pixel reassignment to the LSLM images, hereby named iLSLM, improves the optical sectioning. Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning. Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.

本文提出了一种无衍射光片荧光显微成像技术，可以方便地对从微米到厘米各种尺寸的多种生物样本进行多尺度三维荧光成像。提出一种双环调控方法以解决传统贝塞尔光片旁瓣过重的问题，该方法能够可调节地产生0.4~5 μm之间不同厚度类型的无衍射光片，同时其旁瓣占比被压低到30%以下。基于这种新的调控手段设计搭建了一套多尺度光片荧光显微成像系统。该系统展示出适用性极强的多尺度成像能力，例如：活细胞双色三维动态成像、膨胀细胞三维超分辨成像以及介观尺度下全器官的高通量三维成像。证明该多尺度成像模式能够显著提升光片荧光显微镜的成像效率，由此可以推进细胞生物学、组织病理学、神经科学等多种生物医学相关研究的发展。

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（~900 μm）比普通艾里光片显微镜的成像视场（~600 μm）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。