Conventional Chemical Fixation Methods Research Articles

Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development.

The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as 'amorphous,' lacking in ultrastructural features, and is often observed as a thin (~80-100nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

Immunocytochemical identification of interstitial cells of Cajal in the murine fundus using a live-labelling technique

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.

Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

Development of peltate glandular trichomes of peppermint.

Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha x piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.

Ultrastructure of the vitelline coat in the ascidians Phallusia mammillata , Ascidia mentula and Ciona intestinalis : new aspects revealed by freeze-substitution and deep-etching

The microstructure of the vitelline coat (VC) of the three ascidian species Phallusia mammillata, Ascidia mentula and Ciona intestinalis was analyzed by transmission electron microscopy (TEM) using freeze-substitution and deep-etching. In addition, eggs were examined by light microscopy and prepared by conventional chemical fixation methods for TEM in order to illustrate the advantages of cryopreparation for the preservation of the VC ultrastructure. In conventionally fixed eggs of P. mammillata and A. mentula the VC appeared as a tripartite structure, consisting of a thin outer fibrous layer (OFL), a central dense layer (CDL) and a relatively wide inner fibrous layer (IFL). In C. intestinalis a sparse OFL is made up by fine tufts of filaments but an IFL is lacking. In all three species the test cells were located in the area between the inner VC boundary and the egg cell membrane. Using our modified freeze-substitution method a clear improvement in preservation of the VC matrix was obtained. In all three species the OFL as well as the IFL in P. mammillata and A. mentula appeared as extended fibrous meshworks. Furthermore, the area of the perivitelline space, empty after conventional TEM preparation, was found to be filled by an additional matrix, designated here as perivitelline fibrous matrix (PVFM). Quick-freeze/deep-etch/rotary-shadowing enabled us to confirm the overall architecture of the egg matrix and allowed a further detailed investigation of the VC fine structure in P. mammillata. Accordingly, the vitelline coat of P. mammillata and A. mentula is a multilayered matrix composed of four distinct subunits (OFL, CDL, IFL, PVFM), whereas in C. intestinalis an IFL is lacking.

Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds.

The barbel taste buds of catfish are widely used as a model system for investigating the structure and function of vertebrate taste buds. We have examined the ultrastructure of the taste buds of the channel catfish, Ictalurus punctatus, as part of a comparative study of the morphology of taste buds in various mammalian and non-mammalian vertebrates. Since conventional chemical fixation methods have limited usefulness for certain kinds of ultrastructural studies (i.e., localization of diffusible substances or labeling techniques requiring retention of biological activity), we have developed methods for fixing catfish barbel taste buds by high-pressure freezing followed by freeze-substitution (HPF/FS) and have compared the ultrastructure of taste buds fixed by this technique and by chemical fixation procedures. The morphological details of the cells within taste buds are significantly affected by the method of fixation employed. In general, membrane contours are smoother and intracellular organelles more regular in shape in HPF/FS samples as compared with the chemically fixed specimens. Absolute and relative electron-densities of various tissue components are also affected by the fixation method employed. Certain ultrastructural features are more clearly visualized by one or the other of the fixation procedures. Fixation of barbel taste buds by HPF/FS not only provides an alternative view of the ultrastructure of taste bud cells but also offers a method of tissue preparation that may prove to be preferable to chemical methods for ultrastructural investigations involving procedures such as immunohistochemical labeling.

Comparison of high‐pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds

The barbel taste buds of catfish are widely used as a model system for investigating the structure and function of vertebrate taste buds. We have examined the ultrastructure of the taste buds of the channel catfish, Ictalurus punctatus, as part of a comparative study of the morphology of taste buds in various mammalian and non-mammalian vertebrates. Since conventional chemical fixation methods have limited usefulness for certain kinds of ultrastructural studies (i.e., localization of diffusible substances or labeling techniques requiring retention of biological activity), we have developed methods for fixing catfish barbel taste buds by high-pressure freezing followed by freeze-substitution (HPF/FS) and have compared the ultrastructure of taste buds fixed by this technique and by chemical fixation procedures. The morphological details of the cells within taste buds are significantly affected by the method of fixation employed. In general, membrane contours are smoother and intracellular organelles more regular in shape in HPF/FS samples as compared with the chemically fixed specimens. Absolute and relative electron-densities of various tissue components are also affected by the fixation method employed. Certain ultrastructural features are more clearly visualized by one or the other of the fixation procedures. Fixation of barbel taste buds by HPF/FS not only provides an alternative view of the ultrastructure of taste bud cells but also offers a method of tissue preparation that may prove to be preferable to chemical methods for ultrastructural investigations involving procedures such as immunohistochemical labeling. © 1996 Wiley-Liss, Inc.

Ultrastructure and X-ray microanalysis of epiphyseal growth cartilage of femoral head processed by rapid-freezing and freeze-substitution.

Epiphyseal growth cartilage of the femoral head obtained from Wistar rats was investigated after fixation by a rapid-freezing and freeze-substitution. Liquid helium was used in order to achieve a fast cooling rate without ice-crystal damage during the rapid freezing. Use of the rapid-freezing and freeze-substitution procedure provided better ultrastructural preservation of the chondrocyte than conventional chemical fixation methods. This procedure allowed a more reliable approach to electron probe analysis. X-ray microanalysis of the specimens confirmed that calcium is not detected in the initial matrix vesicles as a result of the freezing process. The results suggest that calcium release from precipitates occurs in the free state without any detectable formation of hydroxyapatite at the initial stage of calcification and that calcium is not tightly bound to the matrix vesicles.

Morphometric study of glomerular slit diaphragms fixed by rapid-freezing and freeze-substitution

The widths of slit diaphragms in rat glomeruli were quantitatively measured on electron micrographs obtained by the rapid-freezing and freeze-substitution (FS) fixation method and compared to those prepared by conventional chemical fixation procedures, such as tannic acid, glutaraldehyde and osmium tetroxide (TGO) or glutaraldehyde and osmium tetroxide (GO) fixation. When fixed by FS using 2% osmium tetroxide, glomerular slit diaphragms had widths of 33.8 +/- 3.6 nm, but when fixed by TGO or GO they had widths of 47.0 +/- 4.3 and 48.7 +/- 5.5 nm, respectively. The width of slit diaphragms was significantly narrower after cryofixation than after conventional TGO or GO fixation (P less than 0.001). Similarly, the distance between neighboring foot processes at 50 nm from slit diaphragms was significantly narrower after cryofixation (P less than 0.001). The widths were 98.6 +/- 17.1 nm after TGO fixation, 122.0 +/- 29.3 nm after GO fixation and only 54.8 +/- 18.7 nm after FS fixation. These foot processes were significantly broader after FS fixation. The foot processes were 179.2 +/- 59.4 nm in breadth after TGO fixation (P less than 0.001), 188.8 +/- 78.2 nm after GO fixation (P less than 0.01) and 235.1 +/- 87.2 nm after FS fixation. These results suggest that the cytoplasm of foot processes shrinks when the tissue is prepared by conventional chemical fixation methods, and that the broadening of slit diaphragms is an artifact separation of the margins of adjacent foot processes due to shrinkage. We propose that TGO fixation alters the structure of slit diaphragms by causing them to shrink.

Elemental composition of polyphosphate bodies in aluminum-treated Pisolithus tinctorius: Comparison of chemical and freeze-substitution methods

The toxic effects of aluminum (Al) ions on plants growing in acidic soils may be ameliorated by mycorrhizal (M) fungi, which form symbiotic associations with roots of many forest trees. Pisolithus tinctorius, a M fungus found in many acid soils, is tolerant of high levels of Al. It is not known, however, if Al is excluded from the hyphae or compartmentalized within specific organelles or areas of the hyphae. Many M fungi have been shown to accumulate divalent cations, especially Ca, with P in polyphosphate (polyP) bodies. Whether Al ions might also be localized in polyP bodies is unclear. Previously, most ion localization studies in M fungi have been performed using conventional chemical fixation (CF) methods. However, ion movement due to aqueous fixation has been demonstrated. The inhibition of ion movement during fixation is a superior feature of specimen preparation by rapid freezing followed by freeze substitution (FS). The purpose of this study was to determine by energy dispersive X-ray microanalysis (EDS) of ultrathin sections the elemental composition of polyP bodies and other hyphal areas in P. tinctorius grown at different Al levels and prepared by CF or FS methods.

Ultrastructure of the Septal Pore Apparatus and Early Septum Initiation in Auricularia auricula-judae

The septal pore apparatus and septum initiation in vegetative hyphae of Auricularia auricula-judae were studied by light and electron microscopy for phylogenetic comparisons. Both freeze substitution and conventional chemical fixation methods were employed for electron microscopic studies. Septa developed at the base of the clamp connection and in the main hypha near the clamp where the nuclear divisions had occurred. Septum formation lasted about 4 min. Septal swellings were much larger in chemically fixed than in freeze-substituted hyphae. Fine filaments at the adseptal side of the septal pore cap appeared to be attached to the orifice area of the septal pore and to the cap via intermediate structures. Broader filaments, 7.6 to 9.4 nm in diam, usually connected the plasma membrane bordering the cross wall with endoplasmic reticulum which was continuous with the pore cap. There was a central plate in the septal pore. Ribosomes were absent between the plate and the pore cap. Septum initiation and development involved microvesicles, apparently from the Golgi apparatus, and a third type of filament, 10.6-13.2 nm diam. Septal structure supports the relationship of Auricularia to Tremella and functional analysis suggests a general absence of transport of larger organelles between cells, unlike the situation in homobasidiomycetes.