Mineral content changes in bone associated with damage induced by the electron beam

Abstract

Summary:Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200× embedded, 200× unembedded, and 1000× embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200×, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200×, 1000× data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.

Ca/P concentration ratio at different sites of normal and osteoporotic rabbit bones evaluated by Auger and energy dispersive X-ray spectroscopy

“…As stated above, the physical origin of matrix effects in EDX, for multi-element samples, stems from differences in elastic and inelastic scattering and in the propagation of X-rays [29,55]. The experimental Ca/P error with this procedure was evaluated to 1-2%.…”

“…cm −2 [54]. In EDX experiments, the critical parameters are accelerating voltage, scan time, magnification, sample coating, and beam spot size [55,56]. Irradiating a specimen with an electron beam results in energy transfer to the sample in the form of heat, which can lead to specimen damage.…”

Relationship between the v2PO4/amide III ratio assessed by Raman spectroscopy and the calcium content measured by quantitative backscattered electron microscopy in healthy human osteonal bone

“…31 Other studies using energy dispersive x-ray analysis (EDX) measured Ca/P weight percent ratios between 2.1 and 2.2, which corresponds to the Ca/P ratio range mentioned above. 32,33 This variability is likely due to changes in the apatite composition due to type A and type B carbonate substitutions as well as Mg, Na, or K or even the presence of calcium ion lattice vacancies in the apatite crystal. [34][35][36][37][38] Of course, alterations in the mineral composition or phase change both Raman and qBEI outcomes.…”

Spectroscopic Assessment of Normal Cortical Bone: Differences in Relation to Bone Site and Sex

“…Then, the tissues were slightly polished, further dehydrated by freeze drying, and coated with a conductive carbon layer using a JEOL JEE-4X Vacuum Evaporator. The electron beam can induce significant damage on biological substances [11], so we performed excessive tests on operating conditions. Thus, the magnification was kept low (50 -110) and each spectrum was collected twice every 60 sec until a total collection time of 120 sec was reached.…”