A cryostat approach to ultrathin ‘dry’ frozen sections for electron microscopy: a morphological and X‐ray analytical study

Appleton, T. C.

doi:10.1111/j.1365-2818.1974.tb03913.x

Cited by 118 publications

(62 citation statements)

References 11 publications

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“…(2) Effects of non-uniform distribution of Na in the oocyte. Crystallization of NaCl during freezing or freeze-drying is not a likely cause of significant nonuniformity since the work of Appleton (1974) suggests that the gaps between such crystals would not exceed 2 Asm so that they would not much affect measurements made in a 3 x 3 4um square, or at least they should affect cell and standard equally; in any case the crystallization of KCI, which presumably occurs, does not appear to affect measurements of K. On the other hand sequestration of non-Li-replaceable Na in the cytoplasm (Dick et al 1970) could give rise to significant non-uniformity in the distribution of at least a part of the oocyte Na. If, for example, 16 m-mole/l.…”

Section: Comparison Of Na Assay By Microprobe Analysis and Flame Photmentioning

confidence: 99%

The distribution of sodium, potassium and chloride in the nucleus and cytoplasm of Bufo bufo oocytes measured by electron microprobe analysis.

Dick¹

1978

The Journal of Physiology

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Section: Comparison Of Na Assay By Microprobe Analysis and Flame Photmentioning

confidence: 99%

The distribution of sodium, potassium and chloride in the nucleus and cytoplasm of Bufo bufo oocytes measured by electron microprobe analysis.

Dick¹

1978

The Journal of Physiology

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Section: Tissue Freezing and Cryoultramicrotomymentioning

confidence: 99%

“…The supercooled Freon 22 was found to yield superior freezing to a liquid N2 slush produced by cooling liquid N2 through vacuum pumping a surrounding container of liquid N2 (boiling point, -196~ freezing point, -210~ as judged by the number of ice crystal-free sections obtained. Preservation of ice crystal-free structures at the electron microscope level requires freezing rates of > 5,000 ~ 10,000~ s. (5,19,23,42,71,77).The LKB cryoultramicrotome (LKB Produkter, Bromma, Sweden) was modified so that the ambient temperature in the cryo chamber was -130~ A similar modification was reported while this work was in progress (30). The liquid N2 normally pumped into the knife holder was directed into a large foil trough of about 7 x 3 inches at one end of the chamber.…”

mentioning

confidence: 99%

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

Somlyo¹,

Shuman²,

Somlyo³

1977

The Journal of Cell Biology

264

172

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A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusible ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A. V. Somlyo, and A. P. . Ultramicros. 1:317-339.) of freeze-dried ultrathin cryo sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of C1 in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition of striated muscle.There was no evidence of sequestered C1 in the terminal cisternae of resting muscles, although calcium (66 mmol/kg dry wt -+ 4.6 SE) was detected. The values of [C1]l determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles and over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial C1 ratio of 2.4 -+ 0.88 SD.The elemental concentrations (mmol/kg dry wt -+ SD) of muscle fibers measured with 0.5-9-/zm diameter electron probes in normal frog striated muscle were: P, 302 -4.3; S, 189 -+ 2.9; C1, 24 -1.1; K, 404 +-4.3, and Mg, 39 +-2.1. It is concluded that: (a) in normal muscle the "excess Cl" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic Cl in low [K]o solutions exceeds that predicted by a passive electrochemical distribution.Hypertonic 2.2 x NaC1, 2.5 • sucrose, or 2.2 • Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P -~(6 Ca + 1 Mg)/6 P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules. 828THe JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977" pages 828-857 on May 11, 2018 jcb.rupress.org Downloaded from http://doi.org/10. 1083/jcb.74.3.828 Published Online: 1 September, 1977 KEY WORDS electron probe analysis 9 cryoultramicrotomy 9 striated muscle hypertonicity 9 sarcoplasmic reticulum The distribution of diffusible ions in, respectively, the extracellular space, cytoplasm, and cellular organelles, represents one of the most general problems of cell function. The main techniques available for determining the concentrations of ions in different compartments are the measurement of radio isotope ion fluxes in intact tissues and organeUe fractionation. These techniques are, however, subject to the respective uncertainties of the ultrast...

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“…Electron probe analysis combined with cryoultramicrotomy permits the analysis of cell constituents (Z 2 11) with a spatial resolution and sensitivity unavailable through conventional techniques (1,2). It has been shown that it is feasible to detect with commercially available instruments calcium and other cations in single (50-80 nm diameter) mitochondrial granules (3,4).…”

mentioning

confidence: 99%

Electron probe x-ray analysis of single ferritin molecules.

Shuman¹,

Somlyo²

1976

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

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Single molecules and groups of two or three ferritin molecules were subjected to electron probe x-ray microanalysis in a transmission electron microscope equipped with a liquid nitrogen cooled stage. Significant Fe Ka peaks were generated during 100-sec counts when single ferritin molecules were excited with a probe current of 0.35 nA/60 nm spot, less than the maximal current available in a thermionic gun. There was a linear relationship between the number of ferritin molecules analyzed and count rates. The experimental results are compared to the theoretically calculated Fe KI yields and to the results of Isaacson and Johnson [(1975) To realize the optimal minimal detectable mass attainable with this system it is essential to reduce to a minimum the background radiation (x-ray continuum) generated by extraneous sources. Contamination, the major source of extraneous continuum with small diameter (50 nm or less) probes in our system, can be eliminated or at least substantially reduced by operating at low temperatures (10). Therefore, the suspensions of ferritin molecules placed on thin (about 4 nm) carbon films were analyzed at -110°, using a Philips liquid nitrogen cooled specimen stage modified to permit the exit of generated x-rays towards the detector. Analysis at even lower temperatures was not feasible due to icing of the specimen. The stage drift of 1.5 nm/sec (specified by the manufacturer) is evident in the electron micrographs obtained and required continuous vigilance by the operator for maintaining the ferritin target in the center of the probe. RESULTS Electron micrographs of a single ferritin molecule before and after electron probe x-ray microanalysis with 60 nm diameter probe for 100 sec and of a group of three molecules are shown in Fig. 1. The hole formed in the carbon support is evidence of the continuous mass loss under the electron beam even at this low temperature. It may be possible to reduce or eliminate mass loss, at comparable current densities, by operating at liquid He temperatures (11) or by using lower current densities (ref. 12; Shuman and Somlyo, unpublished observations). The x-ray spectra obtained from a single ferritin molecule and from a group of three are shown in Fig. 2, and the results of analyses of 1, 2, and 3 ferritin molecules are shown in Table 1. The latter shows the linear increase in Fe Kaf count rate with the number of ferritin molecules analyzed.The theoretical count rate can be estimated from the x-ray production cross section, absolute detector efficiency, and measured probe current. The data in Table 1 were obtained with a measured probe current of 0.35 nA in a 60 nm diameter probe. The average current density is J = 6.2 A/cm2 and, assuming a gaussian probe, the current density at the center of the probe is Jo = 12.5 A/cm2. The x-ray production cross section is composed of two factors, the ionization cross section q and the x-ray yield w. For Fe K x-rays, w = 0.347 and q = 3.95 X 10-22 cm2 from Durup and Platzman's expression (7, 13) for ionization cros...

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A cryostat approach to ultrathin ‘dry’ frozen sections for electron microscopy: a morphological and X‐ray analytical study

Cited by 118 publications

References 11 publications

The distribution of sodium, potassium and chloride in the nucleus and cytoplasm of Bufo bufo oocytes measured by electron microprobe analysis.

The distribution of sodium, potassium and chloride in the nucleus and cytoplasm of Bufo bufo oocytes measured by electron microprobe analysis.

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

Electron probe x-ray analysis of single ferritin molecules.

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