Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet
Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet
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DOI：
10.1364/oe.23.006102
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发表时间：
2015-03-09
期刊：
影响因子：
3.8
通讯作者：
Gao, Liang
中科院分区：
文献类型：
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作者：
Gao, Liang
选择性平面照明显微术（SPIM）以其能够获得具有高三维空间分辨率、良好的光学切片能力和高成像速度的三维图像的能力而吸引人。然而，当需要大视场（FOV）时，必须做出权衡，导致较低的轴向分辨率或较差的光学切片能力。在这里，我们提出了一种新的方法，通过SPIM的三维成像，能够保持其高的三维空间分辨率和良好的光学切片能力在一个大的FOV。代替试图生成大且均匀厚度的激发光片，该方法通过使用于创建光片的激发光束散焦来将相对小的光片快速平铺到图像平面内的多个位置，并且在每个位置处拍摄一个附加图像，使得可以通过重复该过程并将所有图像拼接在一起来对大FOV进行成像。通过实施该方法，具有薄厚度和良好的激发光限制的光片可以用于SPIM成像，具有略微妥协的成像速度。通过数值模拟和实验研究了该方法的成像性能，并通过对琼脂糖凝胶中的荧光颗粒和活的C.线虫胚胎(C)2015美国光学学会
Selective Plane Illumination Microscopy (SPIM) is attractive for its ability to acquire 3D images with high 3D spatial resolution, good optical sectioning capability and high imaging speed. However, tradeoffs have to be made when a large field of view (FOV) is required, results in lower axial resolution or worse optical sectioning capability. Here, we present a novel method for 3D imaging by SPIM that is capable to maintain its high 3D spatial resolution and good optical sectioning capability within a large FOV. Instead of trying to generate a large and uniformly thick excitation light sheet, the method tiles a relative small light sheet quickly to multiple positions within the image plane by defocusing the excitation beam used to create the light sheet, and takes one additional image at each position, so that a large FOV can be imaged by repeating this process and stitching all images together. By implementing this method, light sheets with thin thickness and good excitation light confinement can be used for SPIM imaging with slightly compromised imaging speed. The method was investigated through both numerical simulation and experiments, and the imaging performance was demonstrated by imaging fluorescent particles embedded in agarose gel and live C. elegans embryos. (C) 2015 Optical Society of America