Miniature scanning light-sheet illumination implemented in a conventional microscope
Miniature scanning light-sheet illumination implemented in a conventional microscope
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DOI：
10.1364/boe.9.004263
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发表时间：
2018-09-01
影响因子：
3.4
通讯作者：
Rohrbach, Alexander
中科院分区：
文献类型：
--
作者：
Kashekodi, Anjan Bhat;Meinert, Tobias;Rohrbach, Alexander
活细胞是高度动态的系统，在几分钟内对各种生化和机械刺激做出反应，这些刺激由光学镊子等很好地控制。然而，通过荧光显微镜研究活细胞通常不仅受到空间和时间成像分辨率的限制，而且还受到荧光团漂白的限制。因此，我们设计了一种微型光片照明系统，该系统在传统的倒置显微镜上实现，配备光钳和干涉跟踪来捕获减少漂白的活巨噬细胞的3D图像。水平光片由一个0.12 mm的小悬臂镜产生，与检测轴成45度角。物镜发射的照明光束被微镜反射，并垂直于检测轴线照射样品。高斯光束和贝塞尔光束的横向和轴向扫描，加上用于快速对焦的电子可调透镜，可以在不移动样本或物镜的情况下快速捕获3D图像。利用扫描的贝塞尔光束和线共焦探测，获得了平均0.8µm的轴向分辨率，图像对比度提高了10-15倍。(C)OSA开放获取出版协议条款下的2018年美国光学学会。
Living cells are highly dynamic systems responding to a large variety of biochemical and mechanical stimuli over minutes, which are well controlled by e.g. optical tweezers. However, live cell investigation through fluorescence microscopy is usually limited not only by the spatial and temporal imaging resolution but also by fluorophore bleaching. Therefore, we designed a miniature light-sheet illumination system that is implemented in a conventional inverted microscope equipped with optical tweezers and interferometric tracking to capture 3D images of living macrophages at reduced bleaching. The horizontal light-sheet is generated with a 0.12 mm small cantilevered mirror placed at 45 degrees to the detection axis. The objective launched illumination beam is reflected by the micro-mirror and illuminates the sample perpendicular to the detection axis. Lateral and axial scanning of both Gaussian and Bessel beams, together with an electrically tunable lens for fast focusing, enables rapid 3D image capture without moving the sample or the objective lens. Using scanned Bessel beams and line-confocal detection, an average axial resolution of 0.8 mu m together with a 10-15 fold improved image contrast is achieved. (C) 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.