High-speed and large-scaled light-sheet microscopy with electrically tunable lens
High-speed and large-scaled light-sheet microscopy with electrically tunable lens
复制标题
具有电动可调镜头的高速、大规模光片显微镜
DOI：
10.7498/aps.69.20191908
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发表时间：
2020-04
影响因子：
1
通讯作者：
Jun Liu
中科院分区：
文献类型：
--
作者：
Hu Yu-Yao;Dong Liang;Jing Wang;Jun Liu
荧光显微成像技术通过荧光分子标记生物组织实现特异性成像，具有较高的信噪比，在医学生物学研究领域得到了广泛的应用。一些典型的荧光显微技术，如共聚焦显微镜、双光子显微镜等，荧光强度高，但长时间曝光会引起生物组织的光毒性和光漂白，难以满足长时间观察或无创成像的需求。近年来，薄片荧光显微术（LSFM）以其快速、高分辨率、低光漂白、低光毒性等优点成为荧光显微成像领域的研究热点。 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约5.5 μm和约0.7 μm, 单幅图像稳定成像的速度约为275 frames/s, 成像深度可超过138 μm, 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.目前，许多已报道的光幕荧光显微镜仍存在成像面固定、成像速度慢、成像深度小或残留伪影等问题。因此，本文建立了一种基于电调透镜的快速光幕荧光显微镜。为了实现探测物镜焦平面的快速移动，引入了电调透镜，以满足快速改变屈光度的要求。同样地，通过引入一维振镜来改变旋转角度，实现了光幕的快速移动。快速成像要求光幕与焦平面真实的重叠，然后与高速sCMOS接收荧光相结合，完成整个成像。实验中，通过修改光路使垂直深度显著增加，并利用LABVIEW编程进行协调，提高了动态成像质量，有效减少了快速成像中产生的伪影。最终获得了275帧/s的成像速度、0.73 μm的横向分辨率、5.5 μm的纵向分辨率和138 μm的成像深度。这对于发展活体生物组织的实时、无创成像具有重要意义。
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 and two-photon microscopy, have high fluorescence intensity, but the long exposure can cause phototoxicity and photobleaching of biological tissue, which is difficult to meet the demand for long-time observation or noninvasive imaging. Then, light sheet fluorescence microscopy (LSFM) has become a hot research topic 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, and heart beats. At present, many reported light-sheet fluorescence microscopies still have some 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 the rapid movement of the focal plane of the detection objective lens, the electrically tunable lens is introduced to meet the reqirement for fast changing of the diopter. Similarly, the rapid movement of light sheet is achieved by introducing one-dimensional galvanometer to change the rotation angle. Fast imaging requires the light sheet and focal plane to overlap 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 significantly increases by modifying the optical path, and the LABVIEW programming is used to coordinate and improve the dynamic imaging quality, which effectively reduces 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 is achieved. This is of significance for developing the real-time and non-invasive imaging of living biological tissues.