Freeze-substitution of plant and animal tissue for the localization of water-soluble compounds by electron probe microanalysis

Zyl, Johann van; Forrest, Q.G.; Hocking, Colin; Pallaghy, Charles K.

doi:10.1016/0047-7206(76)90005-4

Cited by 18 publications

(7 citation statements)

References 20 publications

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“…Cryomethods can be considered as the most satisfactory sample preparation technique for ion analysis and imaging (Edelmann, 1994;Grignon et al, 1997;Palsgard et al, 1996;Quintana and Bonnet, 1994;Sitte et al, 1994). However, even with freeze-substitution, it cannot be excluded that ions are either lost or relocated during the ice/organic-solvent exchange or at other steps of this process (Edelmann, 1991;Van Zyl et al, 1976). With plant samples this risk must be particularly taken into account because of the presence of large aqueous vacuoles, which often span more than 90% of the cell volume.…”

Section: Introductionmentioning

confidence: 96%

Dynamic‐SIMS imaging and quantification of inorganic ions in frozen‐hydrated plant samples

Dérue

Gibouin

Demarty

et al. 2006

Microscopy Res & Technique

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We present here SIMS images of the distribution of inorganic cations (Na, K, Mg and Ca) in frozen-hydrated samples of three plant species, ivy, camomile, and flax. The samples were cryofixed using fast plunge-freezing. Stigmatic images were obtained, at 100 K, under dynamic SIMS conditions by fast atom bombarding (FAB). Even though the images obtained with the frozen-hydrated plant samples are still not of upper quality, they show that the method used to prepare these samples preserves existing ionic gradients between the outer and the inner part of the cells, between adjacent cells, including cells with the same type of differentiation, and between tissues. We also describe the quantification of the relative proportions of the ions in the vacuoles of flax. The reasonable accuracy achieved for quantification of the vacuole ion ratios permitted to show (i) that radial gradients of ion ratios in hypocotyls change when the plant is becoming older and (ii) that large differences may exist between adjacent cortical cells of the same type. The role of these substantial differences in vacuole ion balance ratios is a largely unexplored issue in plant physiology.

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Section: Introductionmentioning

confidence: 96%

Dynamic‐SIMS imaging and quantification of inorganic ions in frozen‐hydrated plant samples

Dérue

Gibouin

Demarty

et al. 2006

Microscopy Res & Technique

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“…As a substitution medium we routinely use a freshly prepared and quickly cooled solution of 2% osmium tetroxide in analytical grade acetone (water content <0.1%). For comparison we also used pure acetone, pure methanol, acetone with up to 3% glutaraldehyde (Hobot et al, 1987), methanol containing 1% osmium tetroxide, 0.5% uranyl acetate, 3% glutaraldehyde, 3% water (Muller et al, 1980), or diethyl ether with 20% acrolein (Van Zyl et al, 1976). To the latter, and also to the medium containing glutaraldehyde in acetone, molecular sieve (Linde 0.3 nm, Serva, Heidel-berg, Germany) was added.…”

Section: Introductionmentioning

confidence: 99%

Freeze‐Substitution for morphological and immunocytochemical studies in insects

Steinbrecht

1993

Microscopy Res & Technique

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Methods of plunge freezing and freeze-substitution (FS) for insect antennae and similar body appendages are described. In these more or less cylindrical specimens, usually a layer below the cuticular surface of 10-15 microns thickness is well preserved without freezing damage, further inwards ice-crystal ghosts of increasing size are encountered, but in the very centre of antennal branches (diameter approximately 80 microns) of the silkmoth, Bombyx mori, freezing damage is usually reduced again. The frost-hardy species, Poecilocampa populi and Boreus hiemalis, exhibit regions free from freezing damage up to 40 microns below the cuticular surface. Secondary freezing damage in silkmoth sensory hairs is observed only after deliberately warming the specimens to -43 degrees C for >> 10 min before FS. Secondary artefacts due to the substitution process are investigated by comparison with freeze-etching and by comparing different FS media and protocols. Methanol is not recommended as a substitution medium for insect specimens. Structures particularly liable to substitution damage are the stimulus-conducting pore tubules of olfactory sensilla and the receptor cell membrane. Extraction of soluble components is more likely with pure organic solvents without added chemical fixing agents and with prolonged substitution at elevated temperatures. Such extraction may also be a possible artefact with soluble antigens in immunocytochemical studies. A review is given of the major achievements attained with these techniques is insect functional morphology and immunocytochemistry.

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“…); by the latter method also sugars, amino acids and other organic compounds can be detected. Van Zyl et al (1976) found no significant losses of 22Na, 86Rb and 36Cl after FS in diethylether containing 20% acrolein, in contrast to considerable losses after FS with polar solvents. Van Zyl et al (1976) found no significant losses of 22Na, 86Rb and 36Cl after FS in diethylether containing 20% acrolein, in contrast to considerable losses after FS with polar solvents.…”

Section: Effects On Diffusible Specimen Componentsmentioning

confidence: 54%

Cryotechniques in Biological Electron Microscopy

1987

121

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Freeze-substitution of plant and animal tissue for the localization of water-soluble compounds by electron probe microanalysis

Cited by 18 publications

References 20 publications

Dynamic‐SIMS imaging and quantification of inorganic ions in frozen‐hydrated plant samples

Dynamic‐SIMS imaging and quantification of inorganic ions in frozen‐hydrated plant samples

Freeze‐Substitution for morphological and immunocytochemical studies in insects

Cryotechniques in Biological Electron Microscopy

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