Multiple least-squares fitting for quantitative electron energy-loss spectroscopy — an experimental investigation using standard specimens

Door, R.; Gängler, Dietmar

doi:10.1016/0304-3991(94)00198-v

Cited by 19 publications

(12 citation statements)

References 34 publications

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“…Although in absolute terms the elemental detection is higher for ESI/EELS than EPMA because of the requested thinness of the analyzed specimens and the large contribution of the carbon peak, only relatively high calcium levels can be detected. While the detection sensitivity of EELS has been analyzed in nonbiological standards (21,40) (30) to be used for experimental tasks other than elemental analysis. Two aspects of our procedure are worth emphasizing.…”

Section: Discussionmentioning

confidence: 99%

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

Grohovaz

Bossi

Pezzati

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Discussionmentioning

confidence: 99%

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

Grohovaz

Bossi

Pezzati

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…3). Additionally, where sulphur or phosphorus edges are detected, an original spectrum of the specimen with its background subtracted by multiple least-squares fitting (Leapman & Swyt, 1988;Door & Gängler, 1995) is shown together with a micrograph of the component. …”

Section: Resultsmentioning

confidence: 99%

“…Carbon films were prepared by evaporation of spectroscopically clean carbon in a Balzers EVM052 evaporator as described by Door & Gängler (1995). The relative thickness t/l of the carbon films was measured by EELS (Door et al, 1991).…”

Section: I D E O D E N S I T O M E T Ry F O R M I C Roa Na Lys I Smentioning

confidence: 99%

Videodensitometric analysis of electron spectroscopic micrographs — a tool for detection of biologically relevant elements with high resolution

Door¹,

Breitig

Martin³

1997

Journal of Microscopy

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SummaryElectron energy-loss spectroscopic imaging (ESI) yields highresolution, element-sensitive images. However, ESI suffers from difficulties in distinguishing element-specific and background contributions. New methods have therefore been introduced which use grey-level measurements in micrographic images for a more accurate detection of element distributions. A videodensitometric method allowed the detection of low phosphorus levels in axoplasmic neurofilaments of squid giant axons. Here we further verify these results by investigating the relationship of videodensitometry and electron energy-loss spectroscopy (EELS), particularly considering the peculiarities of these methods in terms of automatic background correction and representation of the results. Six biological specimens and two nonbiological specimens were examined both by EELS and by videodensitometry. In all cases comparable results were obtained. The overlapping P L2,3 and S L2,3 ionization edges could clearly be recognized individually by both methods, and controls showed that mass density variations within the specimens did not impair elemental analysis. Additional evidence supporting the detection of phosphorylation sites in squid neurofilaments was obtained in both EELS and videodensitometric measurements of neurofilament-enriched pellets and of aggregated axoplasmic particles. Thus, videodensitometry appears to be a useful tool for an improved exploration of the full imaging capabilities of energy filtering electron microscopy.

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“…The fitting of the low-loss part of these spectra has been performed following the method described previously. The intensity I(E,03B8c) of spectra above the first K-edge after background removal can be written in direct space [17,34] :…”

Section: Calculation Proceduresmentioning

confidence: 99%

Quantitative EELS by Spectrum Parametrization

Aitouchen

Kihn

Zanchi

1997

Microsc. Microanal. Microstruct.

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Multiple least-squares fitting for quantitative electron energy-loss spectroscopy — an experimental investigation using standard specimens

Cited by 19 publications

References 34 publications

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

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

Videodensitometric analysis of electron spectroscopic micrographs — a tool for detection of biologically relevant elements with high resolution

Quantitative EELS by Spectrum Parametrization

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