The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the various organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.
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 OS04 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 frpm 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 althoAigh 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.The homeostasis of calcium within eukaryotic cells is the result of complex equilibria among multiple pools located in the cytosol as well as within the nucleus and various organelles, where the element is known to play roles of fundamental importance (1). The development of fluorescent indicators (2, 3) together with videoimaging techniques, and the use of recombinant photoproteins (4, 5), have recently provided powerful tools for the dynamic investigation of the fraction of these pools that is in the free, ionized Ca2+ state. In contrast, information about the distribution of total calcium in identified subcellular compartments is still fragmentary, due primarily to limitations of analytical electron microscopy techniques and/or inadequacy of preparative procedures for the maintenance in the specimens of the original element distribution (1, 6-11). So far conclusive results have been obtained only by the use of cryosections analyzed by electron probe x-ray microanalysis (EPMA) (7,8,12). However, even the latter approach requires the use of relatively thick, unstained preparations. Therefore, solid data have been obtained mainly on large or geometrically distributed structures: nuclei, mitochondria, clusters of vesicles or cisternae, or sarcoplasmic reticulum (SR) (6,(13)(14)(15)(16)(17)(18)).An alternative approach that can offer advantages in terms of spatial resolution and organelle identification is electron energy loss microanalysis (19-23), employed in the spectrum mode (electron energy loss spectroscopy; EELS) and/or in the image mode (electron spectroscopic imaging; ESI). With this technique, however, the need to use very thin speci...
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