Computational protocol: Validation of Growth Layer Group (GLG) depositional rate using daily incremental growth lines in the dentin of beluga (Delphinapterus leucas (Pallas, 1776)) teeth

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Protocol publication

[…] Photomicrographs for quantification of incremental line spacing were taken on a Zeiss Imager M2 compound microscope equipped with a high-resolution CMOS camera (monochrome) and motorized stage. Images of the daily short-period lines were taken with a 63X oil immersion objective as virtual stacks of around 20 images with a z-spacing of 0.2–0.3 μm, producing a stack of images with successive deeper focal planes. Short-period lines are more distinct in some focal planes than others, hence the z-stack improves our resolving abilities over having to pick a single plane of focus. Images of the Mayer’s hematoxylin stained sections were taken with a green filter in the light path to improve image contrast.The one ground section was imaged under differential interference contrast on a compound microscope (Olympus BX60) and camera (monochrome), using a 40X oil lens. A coverslip was temporarily attached using glycerin and regular immersion oil was used between the coverslip and the objective. The lower index of refraction of the glycerin improved the contrast over use of immersion oil between the cover glass and the tissue.The computer software Fiji [], a variant of NIH ImageJ, was used for image analysis and measurement. The Pairwise Stitching plugin [] written for Fiji was used to “stitch” adjacent image stacks together producing one stack covering the entire width of one GLG (typically 4–5 stacks). Measurements of incremental line spacing were made in Fiji. As different focal planes offered the best clarity for a given region it was impossible to measure all the lines in a single focal plane. Images figured in this paper were prepared using the 3D EDF plugin [] for Fiji. This plugin takes the in-focus areas from an image stack and merges them together to produces a composite image containing only the focused regions.Seven unique GLGs from three teeth were selected for the quantification of incremental line spacing based on the clarity of the incremental lines within a given GLG, and the certainty in which the boundaries of the GLG could be identified. GLGs that had ambiguous boundaries were avoided. Some incremental lines had indistinct boundaries, and we chose to measure the distance between two lines from the center of one dark line to the next, as the center of each line could be picked more precisely in the photomicrographs. Only one measurement was taken between any two incremental lines. Total GLG thickness was measured in the same image stack as the incremental line spacing. All measurements were taken following the direction of the tubules () which follow the direction of dentin growth [–].We tested the hypothesis that one GLG is deposited annually by predicting the spacing between adjacent incremental lines under two scenarios, one in which a GLG represents one year, and one in which two GLGs are deposited on a semi-annual basis (). The predicted mean incremental line thickness for one GLG per year is simply total GLG thickness divided by 365. The predicted spacing between daily incremental lines if two GLGs are deposited per year is GLG thickness divided by 182.5 (). The estimated number of days represented by each GLG in our sample was calculated as the thickness of the GLG divided by the mean spacing of daily incremental lines within that GLG (). Errors are given as three times the standard error to provide a more conservative estimate of error in estimation of the average incremental line thickness and the estimated days per GLG and are justified by our relatively small sample size. […]

Pipeline specifications

Software tools ImageJ, Stitching
Application Microscopic phenotype analysis
Organisms Homo sapiens, Huso huso