Post-processing strategies in image scanning microscopy.
Post-processing strategies in image scanning microscopy.
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DOI：
10.1016/j.ymeth.2015.05.002
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发表时间：
2015-10
期刊：
影响因子：
4.8
通讯作者：
J. McGregor;C. Mitchell;N. Hartell
中科院分区：
文献类型：
--
作者：
J. McGregor;C. Mitchell;N. Hartell
图像扫描显微镜（ISM）与像素重新分配相结合，提供了一个标准的宽视场成像分辨率提高10.2倍。通过逐点扫描整个样本并捕获在每个扫描位置处生成的荧光信号的图像，记录关于样本结构的附加信息，并且最高可访问空间频率加倍。像素重新分配可以在真实的时间或计算后验中光学地实现，并且经常与物理或数字针孔的使用相结合以拒绝焦点外的光。在这里，我们使用测试图像模拟ISM数据集，并应用标准和非标准处理方法来解决计算像素重新分配和针孔中通常遇到的问题。我们表明，通过将标准像素重新分配到模拟数据集，并探索参考和真实激励位置之间的现实位移的影响，实现了预测的分辨率提高。通过使用定位软件识别检测到的荧光最大值的位置，并在围绕平移的激发位置缩放之前将数字针孔居中在该坐标上，我们可以恢复否则将通过使用与不准确的激发参考对准的针孔而劣化的信号。这一策略证明了使用多光子ISM仪器的实验数据。最后，我们调查的效果，通过组织成像的激发焦点的位置上的深度和观察相对于应用的参考网格的全球缩放。使用模拟和实验数据，我们探讨了全球范围内缩放的参考对ISM图像的影响，并通过在检测到的最大值周围打孔，在整个视场中恢复信号。
Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationallya posterioriand is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view.