Neuronal morphometry directly from bitmap images

TA Ferreira, AV Blackman, J Oyrer, S Jayabal… - Nature …, 2014 - nature.com
Nature methods, 2014nature.com
Neuroscientists measure the tree-like structures of neurons in order to better understand
how neural circuits are constructed and how neural information is processed. In 1953,
Donald Sholl published his well-known technique for quantitative analysis of the complex
arbors of dendrites and axons 1, but conventional methods still require reconstruction of
arbors via time-consuming manual or semi-automated tracing from microscopy images. To
bypass this reconstruction step and perform the Sholl technique directly on images instead …
Neuroscientists measure the tree-like structures of neurons in order to better understand how neural circuits are constructed and how neural information is processed. In 1953, Donald Sholl published his well-known technique for quantitative analysis of the complex arbors of dendrites and axons 1, but conventional methods still require reconstruction of arbors via time-consuming manual or semi-automated tracing from microscopy images. To bypass this reconstruction step and perform the Sholl technique directly on images instead, we developed Sholl Analysis (http://fiji. sc/Sholl), an open-source program for ImageJ/Fiji 2 (Supplementary Fig. 1). The plug-in employs an improved algorithm to retrieve data from two-or three-dimensional (2D or 3D) bitmap images in any format supported by the Bio-Formats library (Supplementary Methods). It pairs this data retrieval with curve-fitting, regression analysis and statistical inference so that users can automatically extract a collection of Sholl-based metrics of arborization 1, 3 (Supplementary Note).
Using individual cortical pyramidal neurons in 3D images, we found Sholl Analysis to be accurate when benchmarked against corresponding manual reconstructions (Supplementary Fig. 2). The method was also resilient to image degradation by simulated shot noise (Supplementary Fig. 3 and Supplementary Software). To further assess accuracy, and to explore the utility of Sholl Analysis in tackling neurons that are particularly slow to reconstruct manually, we studied cerebellar Purkinje cells in mice, which have large and intricate dendritic arbors. From tiled 3D image stacks of cerebellum (Fig. 1a), we selected seven Brainbow2. 1-expressing Purkinje neurons and isolated their morphologies (Fig. 1b and Supplementary Note). We then used the Sholl Analysis software to retrieve ten metrics and found they were indistinguishable from those retrieved from manual reconstructions of the same 7 cells (Fig. 1c, d and Supplementary Methods).
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