Low temperature techniques for X‐ray microanalysis in pathology: Alternatives to cryoultramicrotomy

A comparative study of some aspects of freeze-substitution as a preparative method for X-ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X-ray microanalysis of thin sections of mouse pancreas were used. Freeze-substitution for 3 weeks in diethyl ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze-substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze-substitution in acetone resulted in noticeable loss and redistribution of ions and freeze-substitution in methanol gave very poor results.

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“…Freeze-substitution may be preferred to cryosectioning in special circumstances (Wroblewski et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Comparative study of freeze‐substitution techniques for x‐ray microanalysis of biological tissue

Påsgård

Lindh

Roomans

1994

Microscopy Res & Technique

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“…The dry cut section of freeze-substituted low temperature embedded materials was used in former XRMA studies (Wroblewski and Wroblewski 1984;Wroblewski et al, 1988). However, the concentration of diffusible elements is generally lower than that obtained with thin freeze-dried cryosections.…”

Section: Cryomethods For Xrma Applicationsmentioning

confidence: 99%

X‐ray microanalysis of cell nuclei

Quintana

1991

J. Elec. Microsc. Tech.

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The principal component analysis, a multivariate statistical analysis of data, has been used to process X-ray microanalytical data from cell nuclei. Sixty-seven measurements from different areas of chromatin, nucleoli of rat follicular cells, and nucleoli of rat oocyte cells in their antral stage have been studied. The variables are the X-ray characteristic signals for P, S, Al, Fe, Cu, and Zn. This method demonstrates four distinct groups, the chromatin area, which is associated with a higher concentration of P; the compact mass of oocyte nucleolus which possesses the highest content in S, Al, and Zn, and two groups of nucleolar areas. The fibrillar component is richer in S, Al, and Zn than the granular component. The high degree of correlation between these three elements proves the chemical affinity of metals for the proteins (S being the signature for proteins). Cryoembedding in Lowicryl resin at even lower temperatures (213 degrees K in K11M) after quick cryofixation and cryosubstitution in the absence of chemical fixatives gives good ultrastructural preservation and the possibility of simultaneously performing X-ray microanalysis and immunocytochemistry.

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“…However, the extracellular fluid, which is essential for maintaining the cells during fura-2 measurements, had to be eliminated to analyze cells without contamination. Any attempt to wash away the extracellular fluid was suspect as potentially artifactual (7,8). This obstacle was avoided by using a sandwich-freeze-fracture freeze-drying methodology recently developed in our laboratory (5 (9).…”

Section: Methodsmentioning

confidence: 99%

Quantitative imaging of free and total intracellular calcium in cultured cells.

Chandra

Gross

Ling

et al. 1989

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

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Techniques of fluorescence and ion microscopies were combined to study the free [Ca2+] and total Ca in NIH 3T3 fibroblast and L6 rat myoblast cells. Free Ca2+ measurements with the Ca2+ indicator fura-2 and digital imaging reveal an inhomogeneous distribution of free cytoplasmic Ca + in both cell lines. Fura-2 also reveals a difference in free Ca2+ activity between the nucleus and cytoplasm of cells. Ion microscopic observations on sister cells show that total Ca in the cytoplasm is also inhomogeneously distributed and that mean cytoplasmic levels of total Ca are higher than levels in the MATERIALS AND METHODSCombining Fluorescence and Ion Microscopy. Combining these two entirely different techniques required the modification of sample preparation procedures of each technique to accommodate the needs of the other. Subcellular free Ca2" measurements using fura-2 are made on live cells, while the measurements for total Ca with ion microscopy must be made on fixed cells grown on electrically conducting substrate since the cell sample is biased at 4500 V in the high vacuum chamber (-10-8 torr; 1 torr = 133.3 Pa) of the ion microscope. Semiconductor grade silicon wafer pieces (=1 cm2) polished to submicron flatness serve well as a cell growth substrate for measurements by ion microscopy. This substrate is nontoxic and allows comparable cell growth rates and morphology as compared to cells grown on glass coverslips. In addition, cells cultured on silicon can also be examined by fluorescence microscopy, as demonstrated by previous DNA staining work (5). Quenching of fluorescence for fluorphores near a conducting silicon surface occurs efficiently over distances of =50 nm (6). Thus, quenching of cytoplasmic fura-2 fluorescence by this mechanism is negligible.After the free cytoplasmic Ca2' distributions within several cells were measured with fura-2 by fluorescence microscopy, cells on the same silicon wafer or on another bearing sister cells maintained under identical conditions were processed for ion microscopic measurements. At this stage in the procedure, the cells were fixed in their native state for ion microscopic analysis. However, the extracellular fluid, which is essential for maintaining the cells during fura-2 measurements, had to be eliminated to analyze cells without contamination. Any attempt to wash away the extracellular fluid was suspect as potentially artifactual (7,8

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Low temperature techniques for X‐ray microanalysis in pathology: Alternatives to cryoultramicrotomy

Cited by 15 publications

References 21 publications

Comparative study of freeze‐substitution techniques for x‐ray microanalysis of biological tissue

Comparative study of freeze‐substitution techniques for x‐ray microanalysis of biological tissue

X‐ray microanalysis of cell nuclei

Quantitative imaging of free and total intracellular calcium in cultured cells.

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