title: Semi-automated counting method of axons in transmission electron microscopic images.
abstract: OBJECTIVE
  To evaluate the accuracy of a new semi-automated method for counting axons in transmission electron microscopic (TEM) images.
  
  
  METHODS
  Optic nerve cross sections were obtained from both eyes of Sprague Dawley rats after unilateral induction of chronic ocular hypertension. TEM images (3000× magnification) of cross sections were evaluated by both semi-automated and manual counting methods. The semi-automated counting method was performed using ImageJ software after simple image optimization, and the resulting estimate of axon damage was compared with semiquantitative damage grading scale from light microscopic (LM) images.
  
  
  RESULTS
  Axon counts obtained from the semi-automated method were strongly correlated with those obtained from the manual counting method (Pearson's correlation coefficient r = 0.996, P < 0.001) and from the full manual count from LM images (Spearman's ρ = 0.973, P < 0.001). The semi-automated method measured axonal damage with an error of 0.94 ± 3.16% (mean ± standard deviation), with worse axonal damage associated with greater error. Interobserver and intra-observer variability in axons counts were low (Spearman's ρ = 0.999, P < 0.005). The results of the semi-automated counting method were highly correlated with semiquantitative damage grading scale (Spearman's ρ = 0.965, P < 0.001).
  
  
  CONCLUSIONS
  Results of our semi-automated method for counting axons in TEM images were strongly correlated with those of conventional counting methods and showed excellent reproducibility.
authors: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors: Chan Yun
authors: CY
authors: Kim
authors: Department of Ophthalmology, CHA Bundang Medical Center, CHA University, 59 Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-712, Korea.
authors: Seungsoo
authors: http://orcid.org/0000-0001-9520-4089
authors: S
authors: Rho
authors: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors: Naeun
authors: N
authors: Lee
authors: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors: Chang-Kyu
authors: CK
authors: Lee
authors: Department of Ophthalmology, CHA Bundang Medical Center, CHA University, 59 Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-712, Korea.
authors: Youngje
authors: Y
authors: Sung
mesh_terms: D000818:Animals; D001331:Automation; D007091:Image Processing, Computer-Assisted; D007429:Intraocular Pressure; D046529:Microscopy, Electron, Transmission; D008863:Microspheres; D015588:Observer Variation; D009900:Optic Nerve / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
keywords: axon; experimental model; glaucoma; ocular hypertension; semi-automated counting; transmission electron microscopy