title: A Novel ImageJ Macro for Automated Cell Death Quantitation in the Retina.
abstract: OBJECTIVE
  TUNEL assay is widely used to evaluate cell death. Quantification of TUNEL-positive (TUNEL+) cells in tissue sections is usually performed manually, ideally by two masked observers. This process is time consuming, prone to measurement errors, and not entirely reproducible. In this paper, we describe an automated quantification approach to address these difficulties.
  
  
  METHODS
  We developed an ImageJ macro to quantitate cell death by TUNEL assay in retinal cross-section images. The script was coded using IJ1 programming language. To validate this tool, we selected a dataset of TUNEL assay digital images, calculated layer area and cell count manually (done by two observers), and compared measurements between observers and macro results.
  
  
  RESULTS
  The automated macro segmented outer nuclear layer (ONL) and inner nuclear layer (INL) successfully. Automated TUNEL+ cell counts were in-between counts of inexperienced and experienced observers. The intraobserver coefficient of variation (COV) ranged from 13.09% to 25.20%. The COV between both observers was 51.11 ± 25.83% for the ONL and 56.07 ± 24.03% for the INL. Comparing observers' results with macro results, COV was 23.37 ± 15.97% for the ONL and 23.44 ± 18.56% for the INL.
  
  
  CONCLUSIONS
  We developed and validated an ImageJ macro that can be used as an accurate and precise quantitative tool for retina researchers to achieve repeatable, unbiased, fast, and accurate cell death quantitation. We believe that this standardized measurement tool could be advantageous to compare results across different research groups, as it is freely available as open source.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Maidana
authors.forename: Pavlina
authors.initials: P
authors.lastname: Tsoka
authors.forename: Bo
authors.initials: B
authors.lastname: Tian
authors.forename: Bernard
authors.initials: B
authors.lastname: Dib
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Matsumoto
authors.forename: Keiko
authors.initials: K
authors.lastname: Kataoka
authors.forename: Haijiang
authors.initials: H
authors.lastname: Lin
authors.forename: Joan W
authors.initials: JW
authors.lastname: Miller
authors.forename: Demetrios G
authors.initials: DG
authors.lastname: Vavvas
mesh_terms: D000818:Animals; D001322:Autoanalysis / Q000379:methods; D002452:Cell Count / Q000379:methods; D016923:Cell Death; D020287:In Situ Nick-End Labeling; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D012160:Retina / Q000166:cytology / Q000473:pathology*