Scanning Microscopy


Pure metal standards have been used to calibrate the operating environment in quantitative backscattered electron (BSE) imaging of mineralized tissue, allowing comparisons to be made between various mineralization states of bone at the microscopic level. It has not previously been documented that calibration procedures produce consistent, reliable results over multiple imaging sessions. In this study, BSE images were obtained from bones, pure metals, and a naturally occurring mineral in multiple imaging sessions over a six day period. The graylevel histogram profile (GHP) from each specimen was analyzed for changes in the shape and relative placement on the graylevel spectrum. Computer controlled calibration and a retrospective calibration method using pure aluminum and pure magnesium-aluminum-zinc demonstrated consistency between imaging sessions. Calibrated weighted mean gray levels (WMGLs) for biological materials had an average standard deviation of 5.9 graylevels (2.4% variation) during the course of the study. WMGLs for inorganic materials had an average standard deviation of 0.9 graylevels (0.4% variation). A trend towards increased image brightness, due to specimen and/or embedding media degradation, was observed in the biological tissues. No increase in brightness was observed for the inorganic specimens. Kurtosis and skewness tests revealed a slight deviation from normality in all specimens, which remained consistent between multiple imaging sessions. These results demonstrate that BSE image analysis of bones and mineral can be calibrated with negligible precision error allowing comparisons between data within and between multiple imaging sessions.

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