Research Papers

Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology

[+] Author Affiliations
Marcel Oberlaender, Randy M. Bruno, Bert Sakmann, Philip J. Broser

Max Planck Institute for Medical Research, Department of Cell Physiology, Jahnstraße 29, Heidelberg, 69120 Germany

J. Biomed. Opt. 12(6), 064029 (December 28, 2007). doi:10.1117/1.2815693
History: Received April 02, 2007; Revised June 19, 2007; Accepted July 09, 2007; Published December 28, 2007
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A fundamental challenge in neuroscience is the determination of the three-dimensional (3D) morphology of neurons in the cortex. Here we describe a semiautomated method to trace single biocytin-filled neurons using a transmitted light brightfield microscope. The method includes 3D tracing of dendritic trees and axonal arbors from image stacks of serial 100-μm-thick tangential brain sections. Key functionalities include mosaic scanning and optical sectioning, high-resolution image restoration, and fast, parallel computing for neuron tracing. The mosaic technique compensates for the limited field of view at high magnification, allowing the acquisition of high-resolution image stacks on a scale of millimeters. The image restoration by deconvolution is based on experimentally verified assumptions about the optical system. Restoration yields a significant improvement of signal-to-noise ratio and resolution of neuronal structures in the image stack. Application of local threshold and thinning filters result in a 3D graph representation of dendrites and axons in a section. The reconstructed branches are then manually edited and aligned. Branches from adjacent sections are spliced, resulting in a complete 3D reconstruction of a neuron. A comparison with 3D reconstructions from manually traced neurons shows that the semiautomated system is a fast and reliable alternative to the manual tracing systems currently available.

© 2007 Society of Photo-Optical Instrumentation Engineers

Citation

Marcel Oberlaender ; Randy M. Bruno ; Bert Sakmann and Philip J. Broser
"Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology", J. Biomed. Opt. 12(6), 064029 (December 28, 2007). ; http://dx.doi.org/10.1117/1.2815693


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