Wide-range calibration of corneal backscatter analysis by in vivo confocal microscopy

Toine Hillenaar, Victor Arni D P Sicam, Koenraad A Vermeer, Boy Braaf, Lies Remeijer, Roger H H Cals, Johannes F de Boer

Research output: Contribution to journalArticleResearchpeer-review


PURPOSE: To report intra- and interinstrument calibration methods for corneal backscatter analysis by in vivo confocal microscopy.

METHODS: Applicability of two reference standards was evaluated for corneal backscatter calibration. Repeated measurements of four concentrations of AMCO Clear (GFS Chemicals, Inc., Powell, OH) suspension and three transparencies (26%, 49%, and 65%) of polymethylmethacrylate (PMMA) slabs were performed to assess image intensity acquisition in a wide backscatter range. Intra- and intersession repeatability and lot-to-lot variation were determined for both standards. The effect of light intensity (LI) variation on image intensity acquisition was evaluated by examination of PMMA slabs with nonreference (60% and 80%) and reference (72%) LIs. Both reference standards were implemented in the protocol. Intrainstrument calibration was verified by measuring three normal corneas with 60%, 72%, and 80% LIs. Interinstrument calibration was tested by measuring PMMA slabs on a second, similar confocal microscope.

RESULTS: AMCO Clear was used to express image intensity in absolute scatter units (SU), whereas the 49% transparent PMMA slab showed best repeatability, without image saturation, to adjust for LI variation. Intrainstrument calibration for LI variation reduced mean differences from -38.3% to 1.7% (60% LI) and from 33.9% to -0.6% (80% LI). The mean difference between similar microscopes decreased from 18.4% to 1.2%, after calibration of the second microscope.

CONCLUSIONS: Large interinstrument differences necessitate calibration of corneal backscatter measurements. With AMCO Clear suspension and PMMA slabs, standardization was achieved in a wide backscatter range corresponding to normal and opaque corneas. These methods can easily be applied in ophthalmic practice.

Original languageEnglish
Pages (from-to)2136-46
Number of pages11
JournalInvestigative ophthalmology & visual science
Issue number5
Publication statusPublished - 5 Apr 2011


  • Calibration
  • Cornea/chemistry
  • Humans
  • Light
  • Microscopy, Confocal/instrumentation
  • Polymethyl Methacrylate/chemistry
  • Reference Standards
  • Scattering, Radiation
  • Styrene/chemistry
  • Vinyl Compounds/chemistry


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