High resolution ultrastructural mapping of total calcium

Electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation

Fabio Grohovaz, Mario Bossi, Roberta Pezzati, Jacopo Meldolesi, Francesca Torri Tarelli

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze- drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.

Original languageEnglish
Pages (from-to)4799-4803
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number10
DOIs
Publication statusPublished - May 14 1996

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Energy-Filtering Transmission Electron Microscopy
Electron Energy-Loss Spectroscopy
Neuromuscular Junction
Calcium
Mitochondria
Smooth Endoplasmic Reticulum
Freeze Drying
Synaptic Vesicles
Sarcoplasmic Reticulum
Anura
Organelles
Freezing
X-Rays
Muscles
Skin

Keywords

  • mitochondria
  • sarcoplasmic reticulum
  • smooth endoplasmic reticulum
  • synaptic vesicles

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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abstract = "We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze- drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.",
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