Wide-range calibration of corneal backscatter analysis by in vivo confocal microscopy.
OBJECTIVE
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.
Rotterdam Ophthalmic Institute, Rotterdam, The Netherlands. t.hillenaar@oogziekenhuis.nl
Toine
T
Hillenaar
Victor Arni D P
VA
Sicam
Koenraad A
KA
Vermeer
Boy
B
Braaf
Lies
L
Remeijer
Roger H H
RH
Cals
Johannes F
JF
de Boer
D002138:Calibration; D003315:Cornea / Q000737:chemistry*; D006801:Humans; D008027:Light; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000592:standards*; D019904:Polymethyl Methacrylate / Q000737:chemistry; D012015:Reference Standards; D012542:Scattering, Radiation; D020058:Styrene / Q000737:chemistry; D014753:Vinyl Compounds / Q000737:chemistry
D014753:Vinyl Compounds; D020058:Styrene; D019904:Polymethyl Methacrylate; C004985:divinyl benzene