Tunable glass reference materials for quantitative backscattered electron imaging of mineralized tissues

Abstract

Backscattered electron microscopy provides gray-level contrast resulting from variations in atomic composition. Through the use of reference materials, quantitative backscattered electron (qBSE) imaging can be used to measure the mineral content of mineralized tissues at submicron resolution. We have developed novel tunable reference materials that can be adjusted for analysis of an individual tissue or a wide range of tissues with variable atomic density. As an alternative to conventional metallic reference materials, these amorphous materials maintain long-term stability and possess no long-range order that may induce channeling contrast. Using these reference materials, we characterized the mineral content of a broad range of mineralized tissues from immature mouse femur to whale bulla. Mineral volume fraction correlated to more traditional measurements of mineral content with microcomputed tomography and ashing techniques. Further, we demonstrate the advantage of location-matched measurements of nanomechanical properties and qBSE mineral content.

Moderate chronic kidney disease impairs bone quality in C57Bl/6J mice

“…Following nanoindentation, samples were sputter coated with carbon in preparation for scanning electron microscopy using backscattered electron imaging (qBSE, JEOL JASM6490 LV, 20 kV, aperture 60 μm, 14-mm working distance) [Boyde et al, 1999, Campbell et al, 2012]. Images were acquired at 300x to include the nanoindentation array using both backscatter compositional and shadow modes.…”

“…Images were acquired at 300x to include the nanoindentation array using both backscatter compositional and shadow modes. Raw grey levels of compositional images were calibrated using borosilicate glass reference standards [Campbell et al, 2012]. Custom Matlab code was employed to measure the mean grey level in circular regions of interest (ROI) situated over the nanoindentation indents, and convert mean grey level to corresponding mineral volume fraction (minVf) based on a previously published calibration curve [Campbell et al, 2012].…”

“…Raw grey levels of compositional images were calibrated using borosilicate glass reference standards [Campbell et al, 2012]. Custom Matlab code was employed to measure the mean grey level in circular regions of interest (ROI) situated over the nanoindentation indents, and convert mean grey level to corresponding mineral volume fraction (minVf) based on a previously published calibration curve [Campbell et al, 2012]. The ROI radius was selected to be 2.5 times the indent contact radius ( R ) after performing a sensitivity analysis with ROI ranging from 1-3 ; minVf was observed to stabilize at and beyond 2.5 R .…”

Lacunar-canalicular bone remodeling: Impacts on bone quality and tools for assessment

“…When measurement conditions are the same, a linear relationship is expected between grey level and backscattered coefficient 113 , thus measured grey level from BSEM can be related to the effective Z for the bone tissue composite.…”

“…35,80,115 Other reference standards, such as custom-made lithium borosilicate glasses, are less frequently used but can closely bracket the expected elemental density range of bone. 113,116 qBEI and qBSE employ somewhat different approaches for measuring bone composition. In qBEI, the grey level of the sample as well as calibration standards are measured.…”