Automated correction of fast motion artifacts for two-photon imaging of awake animals
Automated correction of fast motion artifacts for two-photon imaging of awake animals
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DOI：
10.1016/j.jneumeth.2008.08.020
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发表时间：
2009-01-15
影响因子：
3
通讯作者：
Kerr, Jason N. D.
中科院分区：
文献类型：
--
作者：
Greenberg, David S.;Kerr, Jason N. D.
体载钙染料的双光子成像可以同时记录几十个空间分辨神经元的动作电位。然而，将这项技术扩展到清醒的动物身上，由于大脑运动引起的人工制品，在技术上仍然具有挑战性。由于在双光子激发显微镜中，图像像素是通过扫描聚焦脉冲激光在大脑内感兴趣的小区域中顺序捕获的，因此成像区域的快速位移可能会不均匀地扭曲图像。如果不加以纠正，清醒动物的大脑运动将引起人工荧光变化，掩盖与AP放电相关的小的功能性荧光增加。因此，我们提出了一种在清醒动物的双光子成像中检测和校正快速和缓慢位移的程序。我们的算法基于Lucas-Kanade框架，直接对运动失真的成像数据进行操作，既不需要心跳等外部信号，也不需要无失真的模板图像。运动校正精度测试在硅在广泛的简化和现实的位移轨迹和多个水平的荧光噪声。通过比较同时成像的红色和绿色荧光团获得的溶液，在体内证实了准确性。最后，我们评估了运动位移体载钙成像在运动校正和不运动校正的情况下检测AP的准确性，我们得出结论，通过该程序实现的精确运动校正对于清醒动物的单次AP检测是必要和充分的。(C) 2008 Elsevier BY。版权所有。
Two-photon imaging of bulk-loaded calcium dyes can record action potentials (APs) simultaneously from dozens of spatially resolved neurons in vivo. Extending this technique to awake animals, however, has remained technically challenging due to artifacts caused by brain motion. Since in two-photon excitation microscopes image pixels are captured sequentially by scanning a focused pulsed laser across small areas of interest within the brain, fast displacements of the imaged area can distort the image nonuniformly. If left uncorrected, brain motion in awake animals will cause artifactual fluorescence changes, masking the small functional fluorescence increases associated with AP discharge. We therefore present a procedure for detection and correction of both fast and slow displacements in two-photon, imaging of awake animals. Our algorithm, based on the Lucas-Kanade framework, operates directly cn the motion-distorted imaging data, requiring neither external signals such as heartbeat nor a distortion-free template image. Motion correction accuracy was tested in silica over a wide range of simplified and realistic displacement trajectories and for multiple levels of fluorescence noise. Accuracy was confirmed in vivo by comparing solutions obtained from red and green fluorophores imaged simultaneously. Finally, the accuracy of AP detection from motion-displaced bulk-loaded calcium imaging is evaluated with and without motion correction, and we conclude that accurate motion correction as achieved by this procedure is both necessary and sufficient for single AP detection in awake animals. (C) 2008 Elsevier BY. All rights reserved.