3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.
3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.
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DOI：
10.1371/journal.pone.0030826
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lichtman J
中科院分区：
文献类型：
--
作者：
Lakadamyali M;Babcock H;Bates M;Zhuang X;Lichtman J
神经元之间的连接是理解大脑功能的关键。在脑回路中映射神经连接需要具有高空间分辨率的成像技术以促进神经元追踪和高分子特异性以标记不同的细胞和分子群体。在这里，我们测试了一个三维（3D），超分辨率成像方法，随机光学重建显微镜（STORM），用于跟踪神经连接使用培养的海马神经元从野生型新生大鼠胚胎作为模型系统。使用膜特异性标记的方法，提高标记密度相比，细胞质标记，我们成像神经过程在44 nm的二维和116 nm的三维分辨率，通过考虑两个定位精度的荧光探针和奈奎斯特标准的基础上标记密度。与共聚焦图像的比较表明，与目前达到的分辨率，我们可以区分和跟踪更多的神经元过程中的超分辨率图像。利用超分辨成像技术进一步提高了追踪精度。这里获得的分辨率在很大程度上受到标记密度的限制，而不是荧光探针的定位精度。因此，通过进一步提高标记浓度，原则上可以实现更高的图像分辨率，从而实现更高的跟踪精度。
The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.
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