Broser Philip J, Schulte R, Lang S, Roth A, Helmchen Fritjof, Waters J, Sakmann Bert, Wittum G
Max-Planck-Institut für Medizinische Forschung, Abteilung Zellphysiologie, Jahnstr. 29, D-69120, Heidelberg, Germany.
J Biomed Opt. 2004 Nov-Dec;9(6):1253-64. doi: 10.1117/1.1806832.
Two-photon microscopy in combination with novel fluorescent labeling techniques enables imaging of three-dimensional neuronal morphologies in intact brain tissue. In principle it is now possible to automatically reconstruct the dendritic branching patterns of neurons from 3-D fluorescence image stacks. In practice however, the signal-to-noise ratio can be low, in particular in the case of thin dendrites or axons imaged relatively deep in the tissue. Here we present a nonlinear anisotropic diffusion filter that enhances the signal-to-noise ratio while preserving the original dimensions of the structural elements. The key idea is to use structural information in the raw data-the local moments of inertia-to locally control the strength and direction of diffusion filtering. A cylindrical dendrite, for example, is effectively smoothed only parallel to its longitudinal axis, not perpendicular to it. This is demonstrated for artificial data as well as for in vivo two-photon microscopic data from pyramidal neurons of rat neocortex. In both cases noise is averaged out along the dendrites, leading to bridging of apparent gaps, while dendritic diameters are not affected. The filter is a valuable general tool for smoothing cellular processes and is well suited for preparing data for subsequent image segmentation and neuron reconstruction.
双光子显微镜与新型荧光标记技术相结合,能够对完整脑组织中的三维神经元形态进行成像。原则上,现在可以从三维荧光图像堆栈中自动重建神经元的树突分支模式。然而在实际操作中,信噪比可能较低,尤其是在对组织中相对较深部位的细树突或轴突进行成像时。在此,我们提出一种非线性各向异性扩散滤波器,它在保持结构元素原始尺寸的同时提高了信噪比。关键思路是利用原始数据中的结构信息——局部惯性矩——来局部控制扩散滤波的强度和方向。例如,圆柱形树突仅在与其纵轴平行的方向上得到有效平滑,而不是垂直方向。这在人工数据以及来自大鼠新皮质锥体细胞的体内双光子显微镜数据中都得到了验证。在这两种情况下,噪声都沿着树突被平均掉,导致明显间隙的弥合,而树突直径不受影响。该滤波器是用于平滑细胞突起的一种有价值的通用工具,非常适合为后续的图像分割和神经元重建准备数据。