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

Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca2+]i) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca2+]i. Cryomethods, quick-freezing and freeze-drying, and OsO4 vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.