Calibration of histological retina specimens after fixation in Margo’s solution and paraffin embedding to in-vivo dimensions, using photography and optical coherence tomography

  • Stefan KoinzerEmail author
  • Sandra Bajorat
  • Carola Hesse
  • Amke Caliebe
  • Marco Bever
  • Ralf Brinkmann
  • Christoph Roecken
  • Johann Roider



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.


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.


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.


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.


Morphometry Histology Deformation Artifact OCT Optical coherence tomography Retina Rabbit 



The authors declare that they have no conflict of interest.

The data were presented at the following conference:

• Annual meeting of the German-speaking Ophthalmopathologists, Erlangen, Germany; October 26/27, 2012.

The authors gratefully acknowledge grant support for this collaborative research project by the German Ministry of Education and Research (BMBF) according to the Innovation Award for Advancing Medical Technology 2006, grant #01EZ0734 (Dept. of Ophthalmology, University hospital of Schleswig-Holstein, Campus Kiel), #01EZ0732 (Medical Laser Center Lübeck), #01EZ0733 (Institute of Biomedical Optics Lübeck) and #01EZ0735 (Carl Zeiss Meditec AG).

Technical assistance by Monika Marquardt, Serap Luick, and Barbara Fluke is also gratefully acknowledged.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Stefan Koinzer
    • 1
    Email author
  • Sandra Bajorat
    • 1
  • Carola Hesse
    • 1
  • Amke Caliebe
    • 2
  • Marco Bever
    • 3
  • Ralf Brinkmann
    • 3
  • Christoph Roecken
    • 4
  • Johann Roider
    • 1
  1. 1.Department of OphthalmologyUniversity Hospital of Schleswig–HolsteinKielGermany
  2. 2.Institute of Medical Informatics and StatisticsUniversity Hospital of Schleswig–HolsteinKielGermany
  3. 3.Medical Laser Center Lübeck GmbHLübeckGermany
  4. 4.Institute of PathologyUniversity Hospital of Schleswig–HolsteinKielGermany

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