- 1. 上海光学精密机械研究所强场激光激光物理国家重点实验室
- 2. 上海光机所
摘要: 光片荧光显微镜(Light-sheet fluorescence microscopy)以高分辨率、低漂白性和低光毒性成为近年国内外研究的热点，但对于观察一些生物体内较快的生命过程如神经元的活动、血液的流动等，光片荧光显微成像装置尚存在着成像面固定、成像速度慢、成像深度小或残留伪像等问题。 本文研究搭建了一种基于电动可调焦透镜(electrically tunable lens)的大范围快速光片荧光显微成像系统. 通过引入电动可调焦透镜与一维振镜以实现成像物平面和光片位置的快速移动，再结合高速sCMOS完成快速光片荧光显微成像。另外实验中通过改善光路与提升动态成像质量，实现了大范围扫描并减少了伪像。最终对成像性能进行测试，本系统的纵向分辨率和横向分辨率分别达到 ~5.5μm和 ~0.7μm，单幅图像稳定成像的速度为 ~275 frames/s，成像深度可超过138μm，能满足对具有一定尺寸的生物样本进行实时清晰成像的需求。
High-speed and large-scale light-sheet microscopy with electrically tunable lens
- Received Date:
17 December 2019
Abstract: Fluorescence microscopic imaging technology realizes specific imaging by labeling biological tissue with fluorescence molecules. Which has a high signal-to-noise ratio and has been widely used in the field of medical biology research. Some typical fluorescence microscopy techniques, such as confocal microscopy or two-photon microscopy, have high fluorescence intensity, long exposures can cause phototoxicity and photobleaching of biological tissues, which are difficult to meet the demands of long-time observation or noninvasive imaging. Then, light sheet fluorescence microscopy (LSFM) has become hot topics in fluorescence micro-imaging in recent years due to its fast speed, high resolution, low photobleaching and low phototoxicity. The imaging speed of a typical light sheet microscopy is not fast enough to observe fast biological activities such as transmission of neural signals, blood flow, heart beats. At present, many reported light-sheet fluorescence microscopies still have problems such as fixed imaging surface, slow imaging speed, small imaging depth or residual artifacts. Therefore, in this paper, a rapid light-sheet fluorescence microscopy based on electrically tunable lens is built. To achieve rapid movement of the focal plane of the detection objective lens, the electrically tunable lens is introduced to meet the fast changing of the diopter. Similarly, rapid movement of light sheet is achieved by introducing one-dimensional galvanometer to change the rotation angle. Fast imaging requires keeping the light sheet and focal plane overlapping in real time. Which is then combined with a high-speed sCMOS receiving fluorescence to complete the whole imaging. In the experiment, the vertical depth is significantly increased by modifying the optical path, and the LABVIEW programming is used to coordinate and improve the dynamic imaging quality, which effectively reduced the artifacts generated in rapid imaging. Finally, an imaging speed of 275 frames/s with a lateral resolution of ~0.73 μm, vertical resolution of ~5.5 μm, and an imaging depth of ~138 μm was achieved. This is of significance for the development of real-time and non-invasive imaging of living biological tissues.