Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology.
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-microm-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.
Oberlaender
Marcel
M
Max Planck Institute for Medical Research, Department of Cell Physiology, Jahnstrasse 29, Heidelberg, 69120 Germany.
Bruno
Randy M
RM
Sakmann
Bert
B
Broser
Philip J
PJ
D000818:Animals; D001369:Axons / Q000648:ultrastructure; D048430:Cell Shape; D003712:Dendrites / Q000648:ultrastructure; D017709:Fractals; D021621:Imaging, Three-Dimensional / Q000379:methods* / Q000706:statistics & numerical data; D008239:Lysine / Q000031:analogs & derivatives; D008853:Microscopy / Q000379:methods* / Q000706:statistics & numerical data; D008959:Models, Neurological; D009474:Neurons / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar; D012984:Software
C013411:biocytin; D008239:Lysine