Thermal effects of the electron beam and implications of surface damage in the analysis of bone tissue

Abstract

Summary: Electron beam interactions with specimens in the scanning electron microscope (SEM) can lead to increased surface temperatures and damage. These changes may have significant consequences in the analysis of bone tissue. An investigation was performed to measure the surface temperature changes associated with the electron beam on a thermocouple with systematic variations in operating conditions. Probe currents, magnifications, and accelerating voltages were incrementally adjusted to measure the temperature changes and to make assessments for determining optimal operating conditions for the SEM in future analyses of bone tissue. Results from this study suggest that thermal effects were minimal at lower accelerating voltages (< 20 kV), lower probe currents (< 10 nA), and lower magnifications, but surface damage may still occur during the analysis of bone tissue.

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

“…However, compared with scan mode, point mode could produce increased damage because of relatively greater energy absorbed when using the smaller analyzed volume and longer dwell time of the electron beam. We have observed similar differences in bone tissue damage between low magnification (e.g., 200×) and high magnification (e.g., 1000×), which might similarly be related to greater energy absorbed by the analyzed region at higher magnification (Holmes et al 2000).…”

“…This finding is important since the analysis of minute regions within bone microstructure, such as cement lines and interlamellar seams, require high magnification and small analysis regions. Holmes et al (2000) also suggested that probe currents below 2.0 nA help reduce "bleaching" artifact when point mode is required at high magnification. Based on this suggestion, Skedros et al (2005) rendered the "bleaching" artifact negligible in a study of cement line mineral content in 1500× images by using point mode with a probe current of 0.75 nA.…”

“…The EDX spectrum was subsequently collected (described above) from the region represented by the entire BSE image. Once the spectrum was complete, the probe current was checked and manually adjusted as needed; this ensured consistent image contrast (Holmes et al 2000, Vajda et al 1996. Four additional coupled BSE/EDX measurements were conducted at the same location.…”

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

“…The criterion must be peak resolution and signal-to-noise ratio. Temperature levels at the surface of bone specimens are also positively related to the degree of magnification [58], which in our experiments was kept to low values. Carbon coating prevents charging effects but also reduces the effects of beam damage [59] without compromising the validity of EDX results for calcified tissues [60] provided that it does not contribute to the signal as in the present case with Ca and P measurements.…”

Response to referee Adam Hitchcock

“…When an accelerated ion interacts with a material, it loses its kinetic energy either via electronic (inelastic scattering of electrons) or nuclear processes (elastic collisions). Both pathways are relevant in ion milling, but nuclear processes play the predominant role (Ishitani and Kaga, 1995;Prenitzer et al, 2003). Besides the sputtering of material, it is well-known that FIB milling also implants Ga + ions in a surface-near layer (Balcells et al, 2008;Cairney et al, 2000;Prenitzer et al, 1998;Rubanov and Munroe, 2001) and thus creates Ga-rich phases with Ga fractions of up to 20 wt % in the damage layer (Susnitzky and Johnson, 1998) and up to 30 wt % in the redeposition layer (Rajsiri et al, 2002), which can melt at low temperatures (Li and Liu, 2017).…”