Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping

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

“…This makes the use of cryosections problematical (see ref. 24 and the Introduction). Although the use of ESI instrumentation with higher accelerating voltage should, in principle, allow the use of thicker sections, this approach has not been widely exploited yet.…”

“…As a consequence, until now only a few attempts were made to perform microanalyses on samples where diffusion of the element had been minimized. In particular, the use of cryosections proved problematical, because computational analyses of spectra from multiple areas were necessary to sort out results (24), which turned out to be averages rather than maps of calcium in individual cells. Most other studies were carried out on samples conventionally fixed and embedded in resin, where calcium was identified in precipitates generated by treatment with various anions (see, for example, refs.…”

Drug Targeting and Metabolic Investigations of Cryoprepared Tumor Cells with Analytical Electron Energy Loss Spectroscopy

“…Nev ertheless, in contrast to conventional methods to follow up the uptake and distribution of molecules in cells, the most important advan tage of analytical EELS is the achievable cor relation of ultrastructural and chemical data on small specimens, but horribile dictu, due to the high vacuum in the electron microscope no continuous acquisition of data which re flect dynamic cellular processes is possible. In the case of the calcium metabolism, for in stance, the physiological state of the cell is not only reflected by the total amount of calcium in a compartment, but also by the dynamic propagation of Ca2+ in the cell which gives important information about molecular and cellular events with respect to neoplastic growth [21,22,41], In the past, many authors have reported interesting and critical data about method ological problems in analytical EELS [1,6,9, 10,51, 52], To appreciate the potential useful ness and the practical limitations of biological EELS, valuable information is provided in a publication from Leapman et al [35], All these papers clearly show that EELS is in prin ciple a very powerful and stimulating method, but in practice, there exist even today many technical and physical problems, beginning with a suitable mode for spectra acquisition and ending with the complicated procedure of spectral processing.…”

“…The data presented above show changes in element concentrations after stimulation of the cells which are sometimes extremely low. Detec tion of such low concentrations and variations in the chemical composition in the biological specimens is a great challenge in microanaly sis [35], Large ultrathin cryosections of noncryoprotected material (as they are ideally re quired for analytical investigations on histopathological specimens) are almost impossi ble to produce. The material is too brittle and the sectioning properties of common ultrami crotomes arc not suited for this application.…”

“…For this purpose, we have developed a rapid and economical preparation line also for sampling and freezing of biological materi al using cryoprocedures [30][31][32] which in clude an ultracryomicrotome with a reliable section thickness performance [33,34], One of the most important limitations of EELS in biomolecular analysis are radiation damage [3, 4. 18] and the relatively low con centration of biologically important elements such as calcium, sulfur, chlorine and phos phorus in cryosections in relation to carbon [10,35], When investigating the uptake and distribution of xenobiotics with inherent trac er elements or molecules with labelled com pounds (e.g. fluorine, iodine, boron), the ac tual concentrations of such substances are ex tremely low compared to the dominant car bon peak [11,17].…”