Resolution and Reproducibility of Measures of the Diameter of Small Collagen Fibrils by Transmission Electron Microscopy--application to the Rabbit Corneal Stroma

Aim: To assess the impact of measurements of different numbers of small collagen fibrils at different final magnification values on the resultant mean values for fibril diameter (FD).

Methods: Very high magnification (33,000x) electron micrographs were taken of the posterior-central zone of the corneal stroma from young adult rabbits (2 kg), printed at 46,000 or 50,000x, scanned at 400 d.p.i. and 35 mm slides prepared. These were projected to give final magnifications between 150,000x and 450,000x. An overlay of fibril outlines was prepared from the projected images and the fibril diameters (FD's) measured to within 0.5 mm.

Results: On the overlays, at different projection magnifications, the average FD's ranged from 4 to 13.5 mm to allow measures to be made at a real resolution of between 3.5 and 1 nm. Using a fixed sized region of interest (ROI) on the overlays, the average values of FD's ranged between 24.9 and 31.7 nm, and could vary (for any particular micrograph) by up to 3.6 nm according to the number of FD's measured (n=34-384/ROI). Using a fixed number of FD measures (n=100) at different projection magnifications yielded average FD values from different corneas of between 25.1 and 35.2 nm, which could vary by up to 4.3 nm depending on the magnification used.

Conclusions: The results indicate that different average values for measures of fibril diameter of small collagen fibrils can easily be obtained according to the final magnification used and the number of fibrils measured, and that the overall averages can depend on whether the data sets are averaged or pooled. These aspects of the morphometry of small collagen fibrils therefore need to be carefully specified.