title: Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm.
abstract: Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.
authors.affiliation: Department of Biological Sciences, Clemson University; vbschan@clemson.edu.
authors.forename: Vera B S
authors.initials: VB
authors.lastname: Chan
authors.affiliation: Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
authors.forename: Takashi
authors.initials: T
authors.lastname: Toyofuku
authors.affiliation: Advanced Material Research Laboratory (AMRL), Clemson University.
authors.forename: George
authors.initials: G
authors.lastname: Wetzel
authors.affiliation: Advanced Material Research Laboratory (AMRL), Clemson University.
authors.forename: Laxmikant
authors.initials: L
authors.lastname: Saraf
authors.affiliation: Swire Institute of Marine Sciences and School of Biological Sciences, The University of Hong Kong.
authors.forename: Vengatesen
authors.initials: V
authors.lastname: Thiyagarajan
authors.affiliation: Department of Biological Sciences, Clemson University; mount@clemson.edu.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Mount
mesh_terms: D000818:Animals; D002114:Calcinosis / Q000000981:diagnostic imaging*; D007814:Larva / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D013052:Spectrometry, X-Ray Emission