High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation.

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 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...

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

“…Dehydration, infiltration, and embedding in Araldite were done as described previously (Watanabe et al, 1983 (Torri Tarelli et al, 1985). Frozen specimens were then transferred into an ultra-highvacuum chamber (Cryofract 190) and freeze-dried under controlled conditions as described previously (Grohovaz et al, 1996). At the end of the drying cycle (3-4 d), when pressure and temperature were restored to room values, the specimens were removed from the vacuum apparatus and treated as described in the following sections.…”

“…The final calcium map (coded in pseudocolors) was then superimposed on the ultrastructural organization of the same field obtained at 240 eV, i.e., at an energy loss where most of the elements contribute to the image (Colliex, 1986). The nature of calcium signals in the different regions of the maps was confirmed by EELS spectra (for further details, see Grohovaz et al, 1996).…”

“…The adequacy of such an approach was confirmed by our results in the frog muscle where the high calcium content of the sarcoplasmic reticulum terminal cisternae, revealed over 20 years ago by subAbbreviations used: CRT, calreticulin; EELS, electron energy loss spectroscopy; ER, endoplasmic reticulum; ESI, electron spectroscopic imaging; GC, Golgi complex; MANII, mannosidase II; qf-fd, quick freezing/freeze drying; TGN, trans-Golgi network. cellular fractionation studies and then validated by X-ray microanalysis (see Pozzan et al 1994), was definitely confirmed at high resolution. Moreover, in the presynaptic compartment of the neuromuscular junction, calcium was shown to be concentrated not only within the ER cisternae but also within synaptic vesicles, where the level of the element previously had remained controversial (Grohovaz et al, 1996).…”

“…Most of the studies have in fact been carried out on samples first exposed to chemical treatments, with fixatives and/or dehydration agents, that are known to induce, or to be inadequate to prevent, artifactual changes of the cell native state (Echlin, 1992). In our previous work (Grohovaz et al 1996), fibers of a thin frog muscle were first quick frozen and slowly freeze-dried at low temperature [quick freezing-freeze drying (qf-fd)], after which fixation with OSO4 vapors was applied. The subsequent direct resin embedding of the tissue permitted the preparation of ultrathin (<25-nm) sections from which appropriate EELS-ESI signals could be recorded.…”

“…Recently, a protocol for high-resolution revelation of the cellular distribution of calcium has been developed in our laboratory (Grohovaz et al, 1996) using electron energy loss spectroscopy (EELS) employed in its imaging mode (ESI; Ottensmeyer and Andrew, 1980;Colliex, 1986;Leapman and Ornberg, 1988). In this microanalytical approach, the electrons that are scattered inelastically by the specimen are collected, and the information about their energy distribution is converted into maps of the local elemental composition.…”