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.

The research combines backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) to study tissue mineralisation in 18-month old thoroughbred horses. The paper finds that these methods in combination are a reliable tool to understand the mineralisation of equine articular calcified cartilage.

Methodology

• The researchers injected Thoroughbred horses with calcein, a fluorescent dye, 19 and 8 days before euthanasia. This served to label areas of active mineralisation.
• They used CSLM to image the articular calcified cartilage (ACC) from the horses’ distal third metacarpal condyles, providing data on the linear accretion rate (LAR).
• They employed BSE SEM, specifically quantitative BSE (qBSE), on the same areas to determine the mineralisation density (Dm).
• An in-house software program was used to register the CSLM images to the qBSE images. Registering means the images were superimposed and matched for exact alignment.
• The researchers used ImageJ and Sync Windows for simultaneous data collection of LAR and Dm.

Findings

• Measurements indicate that Dm was roughly 119.7 (in gray levels), while modal and maximum LAR was 0.45 and 3.45 microm/day, respectively.
• The researchers ran the registration protocol multiple times to check its repeatability. It showed low variation, with coefficients of variation (CV) for Dm scoring at 0.70% with repeat registration and 0.77% without.
• Similarly, CVs for LAR were 1.90% with repeat registration and 2.26% without, demonstrating low variation and high repeatability.
• The research found no discernible relationship between Dm and time within the 11-day interlabel interval.

Conclusion

• The research concludes that the registration of CSLM to qBSE images provides a reliable and repeatable methodology for quantitative studies on equine ACC mineralization.
• This combined method allows researchers to accurately map mineralization within tissues, even though they did not find a relationship between Dm and time.