Registration of Confocal Scanning Laser Microscopy and Quantitative Backscattered Electron Images for the Temporospatial Quantification of Mineralization Density in 18-Month Old Thoroughbred Racehorse Articular Calcified Cartilage

M. Doube, E. C. Firth,* A. Boyde

Biophysics, Barts and The London School of Medicine and Dentistry, Centre for Oral Growth and Development, Queen Mary, University of London, U.K.; *Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand

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Summary: Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (D_m) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine D_m, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and D_m data. The repeatability of the registration and measurement protocols was determined. D_m profiles between calcein labels were explored for an association with time. D_m was 119.7 ± 24.5 (mean ± standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 µm/day, respectively. Coefficients of variation (CV) for D_m were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between D_m and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.

Key words: confocal scanning laser microscopy, quantitative backscattered electron scanning electron microscopy, image registration, articular calcified cartilage, equine

PACS: 07.05.Pj, 42.30.Va, 68.37.Hk