Development of SEM and STEM-in-SEM grid holders for EDS analysis and their applications to apatite phases

Abstract

Background: With the application of the scanning transmission electron microscopy (STEM) detector, transmitted electron images similar to transmission electron microscopy (TEM) can be obtained from scanning electron microscopy (SEM), which is referred to STEM-in-SEM imaging. Compared to TEM-energy dispersive spectroscopy (EDS), SEM-EDS is expected to be more efficient and reliable for chemical analysis of TEM specimens due to the larger sample space in SEM and the higher takeoff angle of the SEM-EDS detector. Unfortunately, this advantage is not well applied to TEM specimens in practice, mainly because of fault signals generated from the commercial grid holders used in SEM and STEM-in-SEM. This study is designed to solve this problem by modifying the commercial holders and to test them through EDS analysis of apatite phases. Findings: We first changed the way of assembling parts of the commercial multi-grid holder for SEM. This new assembly was capable of producing a thinner upper cover part, resulting in the reduction of the EDS fault signals generated from the holder. Furthermore, the thin upper part allowed us to get images in shorter working distances, that is, at higher magnifications. This design concept was also applied to the commercial single-grid holder for STEM-in-SEM, producing a new multi-grid holder to be used for loading multiple samples. Conclusion: We confirmed that the new holders produced reliable chemical data from apatite phases. In case of oxygen analysis, despite of low electron brightness from the tungsten source, 5 kV provided more stable acquisition and signal yields than 15 kV. We expect that these modified holders facilitate more efficient EDS analysis for multiple samples under the same analytical conditions in SEM and STEM-in-SEM.

Effective investigation of murine femoral bone development utilizing correlative light and electron microscopy (CLEM)

“…Although there are a variety of manufactured TEM gridholders for SEM instruments, we have previously developed a multi-grid-holder suitable for EDS analysis in a table-top SEM designed to minimize EDS noise signals from the holder itself (Kwon et al 2019). In this study, a new multi-grid-holder called as the 'CLEM holder' has been designed to broaden its utilization for CLEM of biological samples, which is capable of loading nine TEM grids and can be used to reliably perform EDS analysis as shown in Fig.…”

“…Because the thin plates themselves were insufficient to firmly hold TEM grids, the plates were fixed using the plate supporters and side screws. The newly applied assembly of the holder mitigated the limitations of the previous holder which was only capable of loading four grids due to the bulky lower support part (Kwon et al 2019). Furthermore, with the increased holder capability up to nine TEM grids, the new holder was much more efficient to investigate multiple biological samples under consistent analytical conditions.…”

“…We have utilized ultra-thin-sections (UTS) prepared from the femoral bones of SD rats via an ultramicrotome, which allowed us to observe the transverse cross section of the sampled bones via CLEM including RLM and STEM-in-SEM. By utilizing STEM-in-SEM EDS analysis, we have carried out comparative chemical analysis on seven UTS specimens of SD rats with two reference materials, synthetic apatite ((Ca 5 (PO 4 ) 3 (OH)) and precipitated calcium carbonate (CaCO 3 ), all within the same analytical conditions (Kwon et al 2019). In order to interpret the changes of inorganic (Ca, P and O) and organic (C and O) elements according to the ages of the SD rats, we have particularly focused on carbon analysis with consideration of potential carbon sources that may originate from the analytical environment.…”