Automated three-dimensional detection and counting of neuron somata

J Neurosci Methods. 2009 May 30;180(1):147-60. doi: 10.1016/j.jneumeth.2009.03.008. Epub 2009 Mar 21.

Abstract

We present a novel approach for automated detection of neuron somata. A three-step processing pipeline is described on the example of confocal image stacks of NeuN-stained neurons from rat somato-sensory cortex. It results in a set of position landmarks, representing the midpoints of all neuron somata. In the first step, foreground and background pixels are identified, resulting in a binary image. It is based on local thresholding and compensates for imaging and staining artifacts. Once this pre-processing guarantees a standard image quality, clusters of touching neurons are separated in the second step, using a marker-based watershed approach. A model-based algorithm completes the pipeline. It assumes a dominant neuron population with Gaussian distributed volumes within one microscopic field of view. Remaining larger objects are hence split or treated as a second neuron type. A variation of the processing pipeline is presented, showing that our method can also be used for co-localization of neurons in multi-channel images. As an example, we process 2-channel stacks of NeuN-stained somata, labeling all neurons, counterstained with GAD67, labeling GABAergic interneurons, using an adapted pre-processing step for the second channel. The automatically generated landmark sets are compared to manually placed counterparts. A comparison yields that the deviation in landmark position is negligible and that the difference between the numbers of manually and automatically counted neurons is less than 4%. In consequence, this novel approach for neuron counting is a reliable and objective alternative to manual detection.

MeSH terms

  • Animals
  • Cell Count / instrumentation
  • Cell Count / methods*
  • Glutamate Decarboxylase / metabolism
  • Image Cytometry / methods
  • Image Processing, Computer-Assisted / methods
  • Imaging, Three-Dimensional / methods*
  • Immunohistochemistry / methods
  • Male
  • Microscopy, Confocal / methods*
  • Nervous System / cytology*
  • Neurons / cytology*
  • Neurons / physiology
  • Pattern Recognition, Automated / methods*
  • Rats
  • Rats, Wistar
  • Software
  • Staining and Labeling / methods
  • gamma-Aminobutyric Acid / metabolism

Substances

  • gamma-Aminobutyric Acid
  • Glutamate Decarboxylase
  • glutamate decarboxylase 1