title: Calibration of histological retina specimens after fixation in Margo's solution and paraffin embedding to in-vivo dimensions, using photography and optical coherence tomography.
abstract: BACKGROUND
  The extent of retinal tissue deformation by histological processing needs to be separately measured for every workup protocol. This work presents a simple approach for its quantitative assessment, and shows lateral and axial scaling factors for a common protocol. We calibrated histological measurements by in-vivo photographic and optical coherence tomographic (OCT) measurements, using retinal photocoagulation lesions as calibration markers.
  
  
  METHODS
  We evaluated four rabbit eyes that were examined histologically after fixation in Margo's solution (1 % paraformaldehyde:1.25 % glutaraldehyde), isopropanol dehydration, paraffin embedding and hematoxylin and eosin staining. Distances between 51 pairs of laser lesions were compared in photographs and on histological slides. Retinal thickness measurements were performed at 15 anatomically defined sites in these eyes, and related to anatomically matched OCT thickness measurements of six different rabbit eyes.
  
  
  RESULTS
  We found that the ratio of histological over photographic lesion distances was 1.17 (95 % CI 1.13-1.22), indicating 17 % lateral retinal stretching or expansion by the processing. Thickness measurements in histology were 65.6 % of the in-vivo thickness as measured in OCT, indicating 1/3 axial tissue compression or shrinkage.
  
  
  CONCLUSIONS
  We provide an analysis of retinal tissue deformation after fixation in Margo's solution and paraffin embedding. In spite of protocol optimization for reduced tissue deformation, the workup caused 1/3 axial compression/shrinkage and 17 % lateral elongation, which was unexpected. We show a simple way how to calibrate retina specimens by fundus photography and OCT, two methods that are readily available to most ophthalmologists. Our findings underline the necessity to calibrate specimens prior to morphometry.
authors.affiliation: Department of Ophthalmology, University Hospital of Schleswig-Holstein, Campus Kiel, House 25, Arnold-Heller-Str. 3, 24105, Kiel, Germany, koinzer@auge.uni-kiel.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Koinzer
authors.forename: Sandra
authors.initials: S
authors.lastname: Bajorat
authors.forename: Carola
authors.initials: C
authors.lastname: Hesse
authors.forename: Amke
authors.initials: A
authors.lastname: Caliebe
authors.forename: Marco
authors.initials: M
authors.lastname: Bever
authors.forename: Ralf
authors.initials: R
authors.lastname: Brinkmann
authors.forename: Christoph
authors.initials: C
authors.lastname: Roecken
authors.forename: Johann
authors.initials: J
authors.lastname: Roider
mesh_terms: D000818:Animals; D002138:Calibration; D006652:Histological Techniques; D017075:Laser Coagulation; D016612:Paraffin Embedding; D010781:Photography / Q000379:methods*; D011817:Rabbits; D012160:Retina; D016707:Tissue Fixation; D041623:Tomography, Optical Coherence / Q000379:methods*
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