Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging

Abstract

Subcellular localization of total calcium requires tissue processing that preserves the chemical composition of the samples and a highly sensitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ultrastructural preservation was good in the superficial sections: osteoblasts near the bone mineral had clearly defined plasma and nuclear membranes, dense mitochondria, and numerous ribosomes. Electron energy loss spectroscopy allowed high-resolution calcium-sensitive images to be obtained using ionization edge loss electrons. In biological samples, the Ca-L2,3 signal is superimposed on the carbon edge and artifacts may result from thickness and scattering effects. Therefore the relative thickness was established for each area analyzed (t/lambda <0.5). Background was subtracted using the three-images method, allowing high resolution calcium-sensitive images of intramitochondrial granules and of intracellular compartments, and semiquantitative data from the granules to be obtained. Calcium maps were confirmed by spectra collected on defined areas of the images and the shape of the net Ca-L2,3 edges was compared to the characteristic Ca-L2,3 edge of bone crystals. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distribution of other elements.

Distribution of iron oxide nanoparticles in rat lymph nodes studied using electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)

“…Details of data acquisition and computer processing have already been reported (17). Briefly, the EFTEM Zeiss EM 902 was equipped with a monochrome CCD TV camera and an electron detector connected to a PC.…”

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

“…Conventional processing of cells may lead to the loss of ions and/or to their translocation (Grignon et al 1997). We have previously shown in fetal bone cells that high-pressure freezing (Leica HPF) and freeze substitution allows to obtain definite intracellular calcium maps (Bordat et al 1998), but the quality of the images was limited. We have then tested the newly designed high-pressure freezing machine (Leica EMPACT), but the small size of the containers was inadequate for our samples.…”

Fluid Dynamics of the Excretory Flow of Zymogenic and Mucin Contents in Rat Gastric Gland Processed by High-pressure Freezing/Freeze Substitution

“…Thus far, many publications state that HPF processing is much superior to conventional processing in terms of excellent preservation of some protein epitopes, such as the distinct protein components of cuticlin of nematodes, CUT-1 and CUT-2 (Favre et al 1995), the major cell cycle regulator complex cyclin B in the yeast Schizosaccharomyces pombe (Audit et al 1996), the worm cuticular collagen and the vertebrate Type I fish scale collagen (Nicolas et al 1997), the organic matrix components in the adult echinoid skeleton and tooth (Ameye et al 1999) and filaggrin of human epidermis (Pfeiffer et al 2000). In addition, Bordat et al (1998) succeeded in demonstrating a precise calcium distribution in HPF bone cells by electron energy-loss spectroscopy and electron spectroscopic imaging. The application of HPF/freeze substitution to glycoconjugate histochemistry has provided excellent retention of some carbohydrate molecules of not only the epithelial cells of rat gastric glands but also the intraluminal exocytosed contents, suggesting the formation of a seromucous interface between the exocytosed zymogenic and mucin contents without diffusion (Suganuma et al 1998(Suganuma et al ,2001.…”