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

Abstract

BackgroundFor effective investigation of the developing structure and chemistry of bone, comprehensive studies including compositional analysis can be achieved through the gradual observation from the micro- to nanometer scale via correlative light and electron microscopy (CLEM). This technique is particularly useful considering the complex hierarchical arrangement of bioapatite and collagen fibrils which may vary according to specific bone tissue types (i.e., lamellar bone and woven bone) and different growth stages. Scanning electron microscopy (SEM) accompanied with the attachment of the scanning transmission electron microscopy (STEM) detector, referred to as the STEM-in-SEM can be utilized to produce high contrast images from materials composed of light elements, and efficiently allows the selection of suitable accelerating voltage for energy-dispersive spectroscopy (EDS). This study aims to emphasize the efficacy of CLEM techniques through applying STEM-in-SEM and EDS analyses, and its application to comparative murine bone investigation in differing ontogenetic stages.FindingsWe have designed a new grid-holder which can be used for both light and electron microscopy, and we presented an imaging technique for TEM specimens via reflective light microscopy (RLM). For performing CLEM, ultra-thin-sections (UTS) prepared from the femoral bones of 1- to 16-week old of Sprague-Dawley (SD) rats provided light and electron micrographs that can be correlated based on the regions of interest (ROIs). STEM-in-SEM micrographs revealed information not attainable by secondary electron (SE) and back-scattered electron (BSE) micrographs. In addition, for analyzing chemical variation according to growth and development of femoral bones from 1- to 16-week-old rats, comparative chemical analysis was performed through STEM-in-SEM EDS with two reference materials.ConclusionHerein, from femoral bones of SD rats, we have confirmed the rapid chemical and structural variations within the first 8 weeks after birth. STEM-in-SEM micrographs revealed the bone development process of the early stage porous bone matrix subsequently being filled with collagen fibrils and bioapatite. In addition, chemical analysis for carbon and oxygen showed the ratios of inorganic to organic phases according to growth and progress in bone mineralization. As a result, we were able to postulate the growth mechanism of murine femoral bone in the neonatal stages of development. We also anticipate that our CLEM techniques can be further utilized for more thorough investigation of bone structure and chemistry in diverse scales.