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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
Pathology

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.

Keywords

Morphometry Histology Deformation Artifact OCT Optical coherence tomography Retina Rabbit 

Notes

Acknowledgements

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.

References

  1. 1.
    Oberholzer M (1983) Morphometrie in der klinischen Pathologie. Allgemeine Grundlagen. Springer, Berlin Heidelberg New YorkCrossRefGoogle Scholar
  2. 2.
    Quintyn JC, Brasseur G (2004) Subretinal fluid in primary rhegmatogenous retinal detachment: physiopathology and composition. Surv Ophthalmol 49:96–108PubMedCrossRefGoogle Scholar
  3. 3.
    Margo CE, Lee A (1995) Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact. Graefes Arch Clin Exp Ophthalmol 233:366–370PubMedCrossRefGoogle Scholar
  4. 4.
    Koinzer S, Schlott K, Ptaszynski L, Bever M, Kleemann S, Saeger M, Baade A, Caliebe A, Miura Y, Birngruber R, Brinkmann R, Roider J (2012) Temperature-controlled retinal photocoagulation — a step toward automated laser treatment. Invest Ophthalmol Vis Sci 53:3605–3614PubMedCrossRefGoogle Scholar
  5. 5.
    Schlott K, Koinzer S, Ptaszynski L, Bever M, Baade A, Roider J, Birngruber R, Brinkmann R (2012) Automatic temperature-controlled retinal photocoagulation. J Biomed Opt 17:061223PubMedCrossRefGoogle Scholar
  6. 6.
    Prince JH (1964) The rabbit in eye research. Charles Thomas, SpringfieldGoogle Scholar
  7. 7.
    Stefánsson E, Wilson CA, Lightman SL, Kuwabara T, Palestine AG, Wagner HG (1987) Quantitative measurements of retinal edema by specific gravity determinations. Invest Ophthalmol Vis Sci 28:1281–1289PubMedGoogle Scholar
  8. 8.
    Watson PG, Young RD (2004) Scleral structure, organisation and disease. A review. Exp Eye Res 78:609–623PubMedCrossRefGoogle Scholar
  9. 9.
    Yu AK, Merrill KD, Truong SN, Forward KM, Morse LS, Telander DG (2013) The comparative histologic effects of subthreshold 532 nm and 810 nm diode micropulse laser on the retina. Invest Ophthalmol Vis Sci 54(3):2216–2224. doi: 10.1167/iovs.12-11382 PubMedCrossRefGoogle Scholar
  10. 10.
    Hoerster R, Muether PS, Vierkotten S, Schröder S, Kirchhof B, Fauser S (2012) In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice. Graefes Arch Clin Exp Ophthalmol 250:1579–1586PubMedCrossRefGoogle Scholar
  11. 11.
    Bahr GF, Bloom G, Friberg U (1957) Volume changes of tissues in physiological fluids during fixation in osmium tetroxide or formaldehyde and during subsequent treatment. Exp Cell Res 12:342–355PubMedCrossRefGoogle Scholar
  12. 12.
    Davies DJ, Brewer DB, Hardwicke J (1978) Urinary proteins and glomerular morphometry in protein overload proteinuria. Lab Invest 38:232–243PubMedGoogle Scholar
  13. 13.
    Weibel ER (1979) Morphometry of the human lung: the state of the art after two decades. Bull Eur Physiopathol Respir 15:999–1013PubMedGoogle Scholar
  14. 14.
    Hatton WJ, von Bartheld CS (1999) Analysis of cell death in the trochlear nucleus of the chick embryo: calibration of the optical disector counting method reveals systematic bias. J Comp Neurol 409:169–186PubMedCrossRefGoogle Scholar
  15. 15.
    Gardella D, Hatton WJ, Rind HB, Rosen GD, von Bartheld CS (2003) Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections. J Neurosci Meth 124:45–59CrossRefGoogle Scholar
  16. 16.
    Li Q, Onozato ML, Andrews PM, Chen CW, Paek A, Naphas R, Yuan S, Jiang J, Cable A, Chen Y (2009) Automated quantification of microstructural dimensions of the human kidney using optical coherence tomography (OCT). Opt Express 17:16000–16016PubMedCrossRefGoogle Scholar
  17. 17.
    Mujat M, Greco K, Galbally-Kinney KL, Hammer DX, Ferguson RD, Iftimia N, Mulhall P, Sharma P, Pikal MJ, Kessler WJ (2012) Optical coherence tomography-based freeze-drying microscopy. Biomed Opt Express 3:55–63PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Gambichler T, Jaedicke V, Terras S (2011) Optical coherence tomography in dermatology: technical and clinical aspects. Arch Dermatol Res 303:457–473PubMedCrossRefGoogle Scholar
  19. 19.
    Regar E, Ligthart J, Bruining N, van Soest G (2011) The diagnostic value of intracoronary optical coherence tomography. Herz 36:417–429PubMedCrossRefGoogle Scholar
  20. 20.
    Knott EJ, Sheets KG, Zhou Y, Gordon WC, Bazan NG (2011) Spatial correlation of mouse photoreceptor–RPE thickness between SD-OCT and histology. Exp Eye Res 92:155–160PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Anger EM, Unterhuber A, Hermann B, Sattmann H, Schubert C, Morgan JE, Cowey A, Ahnelt PK, Drexler W (2004) Ultrahigh resolution optical coherence tomography of the monkey fovea. Identification of retinal sublayers by correlation with semithin histology sections. Exp Eye Res 78:1117–1125PubMedCrossRefGoogle Scholar
  22. 22.
    Curcio CA, Messinger JD, Sloan KR, Mitra A, McGwin G, Spaide RF (2011) Human chorioretinal layer thicknesses measured in macula-wide, high-resolution histologic sections. Invest Ophthalmol Vis Sci 52:3943–3954PubMedCrossRefGoogle Scholar
  23. 23.
    Abbott CJ, McBrien NA, Grünert U, Pianta MJ (2009) Relationship of the optical coherence tomography signal to underlying retinal histology in the tree shrew (Tupaia belangeri). Invest Ophthalmol Vis Sci 50:414–423PubMedCrossRefGoogle Scholar
  24. 24.
    Baur R (1969) Error correction by sterologic measuring on human placenta. 2. Cutting compression. Experientia 25:1173PubMedCrossRefGoogle Scholar
  25. 25.
    Paulus YM, Jain A, Gariano RF, Stanzel BV, Marmor M, Blumenkranz MS, Palanker D (2008) Healing of retinal photocoagulation lesions. Invest Ophthalmol Vis Sci 49:5540–5545PubMedCrossRefGoogle Scholar
  26. 26.
    Birngruber R (1991) Choroidal circulation and heat convection at the fundus of the eye. In: Wolbarsht ML (ed) Laser applications in medicine and biology. Plenum Press, New York, pp 277–358CrossRefGoogle Scholar
  27. 27.
    Verolino M, Nastri G, Sellitti L, Costagliola C (1999) Axial length increase in lid-sutured rabbits. Surv Ophthalmol 44(Suppl 1):103–108CrossRefGoogle Scholar
  28. 28.
    Park HS, Kim JY, Shin JP, Choi YJ, Kim SY (2005) Effect of experimental scleral shortening on axial length of the rabbit eye. Korean J Ophthalmol 19:101–205PubMedCrossRefGoogle Scholar

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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