Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae)

Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non‐target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide. Microsc. Res. Tech. 77:274–281, 2014. © 2014 Wiley Periodicals, Inc.     

Structural and ultrastructural features of the Malpighian tubules of Dolycoris baccarum (Linnaeus 1758), (Heteroptera: Pentatomidae)

The morphology and ultrastructure of the Malpighian tubules of Dolycoris baccarum were analyzed by scanning (SEM) and transmission (TEM) electron microscopy in order to determine their functional organization. The Malpighian tubules are compared with similar structures of other insects based on cell structure and functional organization. The Malpighian tubules of D. baccarum extend from the midgut–hindgut region of the digestive tract. The Malpighian tubules are divided into two regions: the proximal segment is short and flattened and the distal segment is long, stringy in shape and free in hemolymph. The tubules are generally long and narrow. There is a large number of trachea around the tubules. They consist of a single layer of epithelial cells. It is observed in the TEM observation that the epithelial cells have numerous microvilli at the apical side of the cells. At the basal side of the cells, there is a great number of membrane foldings and mitochondria among them. Besides some spherites, mitochondria, lysosome‐like bodies, and large or small granules can be distinguished in the cells. With this study, we aimed to demonstrate the ultrastructure of the Malpighian tubules of D. baccarum and differences or similarities with other species.     

Embryonic development of the alimentary canal of the scorpionfly Panorpa obtusa Cheng (Mecoptera: Panorpidae)

The embryonic origin of the alimentary canal, especially the midgut, is a controversial problem in insects, and it has not been satisfactorily resolved to date. The organogenesis of the digestive system in the embryonic development was observed in the scorpionfly Panorpa obtusa Cheng using light, transmission, and scanning electron microscopy. The embryonic development lasts about 150–160 h at 24°C. The stomodaeum is formed from an invagination in the medioposterior portion of the protocephalon mid‐ventrally posterior to the labral segment at 76 h after oviposition. The proctodaeum arises as an invagination from the caudal end of the abdomen at 78 h. Four anal forks are formed from within the opening of proctodaeum. Three pairs of proctodaeal evaginations are formed from the anterior part of the proctodaeum, and eventually developing into Malpighian tubules, thus are of ectodermal origin. The cardiac and pyloric valves develop from stomodaeum and proctodaeum, respectively, and also of ectodermal origin. The midgut epithelium originates from anterior and posterior midgut rudiments in blind ends of the stomodaeum and proctodaeum, and it is of endodermal origin. The two cell‐bands (rudiments) cover the yolk ventrally and then dorsally, elongate to each other, and eventually fuse to form the midgut. The midgut formation pattern is briefly discussed in different insects. Microsc. Res. Tech. 76:457–466, 2013. © 2013 Wiley Periodicals, Inc.     

Influence of the insecticide pyriproxyfen on the flight muscle differentiation of Apis mellifera (Hymenoptera, Apidae)

The Brazilian africanized Apis mellifera is currently considered as one of the most important pollinators threatened by the use of insecticides due to its frequent exposition to their toxic action while foraging in the crops it pollinated. Among the insecticides, the most used in the control of insect pragues has as active agent the pyriproxyfen, analogous to the juvenile hormone (JH). Unfortunately the insecticides used in agriculture affect not only the target insects but also beneficial nontarget ones as bees compromising therefore, the growth rate of their colonies at the boundaries of crop fields. Workers that forage for provisions in contaminated areas can introduce contaminated pollen or/and nectar inside the beehives. As analogous to JH the insecticide pyriproxyfen acts in the bee's larval growth and differentiation during pupation or metamorphosis timing. The flighty muscle is not present in the larvae wingless organisms, but differentiates during pupation/metamorphosis. This work aimed to investigate the effect of pyriproxyfen insecticide on differentiation of such musculature in workers of Brazilian africanized honey bees fed with artificial diet containing the pesticide. The results show that the bees fed with contaminated diet, independent of the insecticide concentration used, show a delay in flight muscle differentiation when compared to the control.     

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high‐pressure freezing unit

The high‐pressure freezing (HPF) technique is known to cryofix water‐containing materials with little ice‐crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light‐exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight‐exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.     

Bundles of hexagonally arranged tubules in timothy grass pollen: Detection of a novel pollen component using anhydrous fixation and image analysis techniques in transmission electron microscopy

Pollen from timothy grass (Phleum pratense L.) was subjected to various aqueous and non‐aqueous fixation and preparation protocols for transmission electron microscopy. Only in the cytoplasm of anhydrously prepared pollen grains were conspicuous inclusions observed that range in size from less than 1 μm up to 8 or 10 μm. These bodies have so far not been described in the literature. Higher magnifications show that these inclusions consist of bundles of hexagonally arranged small tubules. In order to obtain details of the ultrastructure of this novel pollen component, TEM micrographs of ultrathin sections of hexagonally arranged tubules were analyzed using Fourier transform techniques of image analysis. It was found that the tubules form groups with quasi‐periodic hexagonal arrangement, with an average centre‐to‐centre spacing between the neighbouring tubules of approximately 42 nm. Individual tubules are formed by 12 or 13 particles. The outer diameter of the tubules ranges between 22 and 24 nm. From our experiments, we conclude that the quasi‐periodic hexagonally arranged tubules forming conspicuous cytoplasmic inclusions in dry timothy grass pollen grains are structurally similar to microtubules.     

Localization of metals in cells of pterygote insects

Insects maintain the equilibrium of their internal milieu by diffusion mechanisms, but more frequently by a substantial storage of metals in the cells of numerous organs: digestive tract, Malpighian tubules, fat body, integument, and genital organs. This storage implies the precipitation, of numerous cations (Ca, Mg, K, Mn, Fe, Zn, Cu) in a structure called the spherocrystal, which originates from the endoplasmic reticulum-Golgi complex: elements precipitate on a glycosaminoglycan nucleus in thin peripheral strata. Some spherocrystals contain exclusively mineral compounds, frequently phosphates, whereas others may contain organic compounds such as urates. In some species mineralized lysosomes store Ca, Fe, Zn, and Cu. When fed additional metals found in the environment, insects such as cockroach and ant are able to stay alive and to trap the metals (Cd or Pb, for example) in the peripheral strata of spherocrystals; the cytoplasm is not altered. It seems that these insects are able to resist exposures to high levels of toxic metals. The lysosomes are able to retain toxic heavy metals (Cd or Hg, for example) within metallothionein-like proteins.     

水培油麦菜对吡虫啉的降解研究

吡虫啉是一种低毒新型烟碱类杀虫剂,具有触杀、渗透、胃毒和内吸等多种杀虫方式,与其他杀虫剂没有交互抗性。本实验利用水培油麦菜降解吡虫啉,并对降解规律进行了研究,吡虫啉含量用高效液相色谱法检测。实验结果表明:(1)在水培蔬菜生长的嫩芽期,吡虫啉能够被降解,但是降解非常缓慢;(2)在水培油麦菜的生长期和衰老期,吡虫啉的降解快速下降最后趋于平缓;(3)水培蔬菜生长的不同时期对吡虫啉降解的能力不同,水培油麦菜在生长期和衰老期对营养液中吡虫啉的降解能力要强于嫩芽期。     

Morphological characterization of the nymphs Rhipicephalus sanguineus ticks (Latreille, 1806) (Acari: Ixodidae). Description of the testes, integument, Malpighian tubules, and midgut on the detachment day

This study presents the morpho-histological and histochemical characterization of the testes, integument, Malpighian tubules, and midgut of engorged Rhipicephalus sanguineus nymphs on the detachment day, showing the morphological and physiological characteristics to this phase in the life cycle of these individuals. The testis is constituted by germinative cells (only spermatogonia) with large, round-shaped and strongly stained nuclei which are organized into cysts by a thin layer of somatic cells. The integument consists of a cuticle subdivided into epicuticle (lipoprotein) and procuticle (glycoproteic), and a layer of epithelial cells which present glycolipoprotein elements. The procuticle presents two distinct regions: the exocuticle (next to the epicuticle) and the endocuticle (next to the epithelial layer). The Malpighian tubules present a simple epithelium with small flat and/or cubic cells, which form its wall and delimitates a lumen full of lipoprotein material. The midgut consists of an epithelial wall formed by two types of digestive cells, spent cells and empty digest cells, and by generative cells supported by a basal lamina and a thin layer of muscular tissue. This study described the main organs of engorged nymphs of R. sanguineus, to generate information that can help researchers to better understand the biology of these ectoparasites; which is fundamental for the development of compounds that are less aggressive to the environment. In addition, if the immature stages of the ticks are controlled, the number of adult ticks able to cause damages to the animals--and to the man as well--is also under control.     

Quantitative elemental X-ray imaging of frozen-hydrated biological samples

It is shown that quantitative X-ray imaging of planed, frozen-hydrated, biological bulk samples that have not been etched is possible. X-ray imaging represents a better alternative to static beam (selected area) analysis of fractured frozen-hydrated samples. This procedure avoids the undesirable necessity of etching planed frozen-hydrated samples to provide an interpretable electron image. Qualitative oxygen and carbon X-ray images, which can be acquired in a short time, can be used for distinguishing morphological features and remove the requirement for electron images. In test samples of frozen-hydrated albumin, containing salts, analyses by X-ray images compared well with static beam (selected area) analyses from the same samples. An example of an analysis of frozen-hydrated insect Malpighian tubules is given in which the response to ouabain treatment was analysed. In this example X-ray imaging showed that ouabain resulted in a significant increase in cytoplasmic and luminal Na and a significant decrease in cytoplasmic and luminal K. X-ray imaging also showed that there was a significant increase in cellular water content. The presence of a potassium gradient in soybean root nodules was also demonstrated. The use of standard deviation images for processing low count images increases analytical precision but results in underestimates of the true concentrations.     

Toxicity of trifluralin herbicide in a representative of the edaphic fauna: Histopathology of the midgut of Rhinocricus padbergi (Diplopoda)

The aim of this study was to use the diplopod Rhinocricus padbergi as test organism to verify the possible toxic effects of the herbicide trifluralin in a representative of the edaphic fauna. Histological and histochemical techniques were applied in the midgut of the animals after exposure to different concentrations of trifluralin for 7 and 90 days (acute and subchronic exposure, respectively). The individuals from the control group did not present alterations to any of the exposures. In the acute exposure, the group exposed to a quarter and half of the field dose presented an accumulation of cytoplasmic granules in the hepatic cells; the field dose group presented an increase in the rate of epithelial renewal and the group exposed to double the field concentration presented an increase in the release of secretory vesicles. The subchronic response was obtained with the field dose and double field dose because the animals from a quarter and half groups did not survive until the end of the experiment. In both groups, the animals presented disruption of the epithelium and higher occurrence of hemocytes among the hepatic cells. The group exposed to double the field dose presented sites of cytoplasmic vacuolization, dilatation of the intercellular space, increase in the epithelial renewal, and release of secretory vesicles. The results alert for a careful use of this herbicide, since it promoted alterations in the midgut cells of the test organism used. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Cytogenetical and ultrastructural effects of copper on root meristem cells of <i>Allium sativum</i> L.

Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.     

Immunolocalization of estrogen and progesterone receptors in ewe mammary glands

Immunopresence of estrogen receptor α (ERα), β (ERβ) and progesterone receptor (PR) were examined in the ewe mammary gland from prepubertal stage to involution. Immunolocalization of ERα revealed a strong positive staining in nuclei of cells composing terminal ductular units (TDUs) in prepubertal ewes. A mild immunoreactivity was identified in early lactating gland. During late lactation immunoreactive product to ERα was observed in the cytoplasm of glandular epithelial cells in all alveoli. In mammary glands at involution ERα positivity was clearly nuclear, with weak to moderate cytoplasmic staining. Cytoplasmic strong immunostaining for ERβ was detected in cells of TDUs, whereas some stromal cells exhibited nuclear staining. A nuclear ERβ immunostaining was observed at early lactation, instead during late lactation, the positivity for ERβ showed only a moderate cytoplasmic distribution. At involution, ERβ positivity was very moderate and detected just in the cytoplasm of shrunken alveoli. Scattered nuclear staining of PR was observed just in mammary glands at early lactation. These results showed that in the mammary glands of sheep both estrogen receptor isoforms were displayed during lactation cycle and that PR appeared just at early lactation, reflecting their regulatory role in alveolar cells. Microsc. Res. Tech. 76:955–962, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.     

Toxicity assessment of nano‐TiO2 in Apis mellifera L., 1758: histological and immunohistochemical assays

The aim of this study was to evaluate the toxicity of titanium dioxide nanoparticles (TiO₂NPs) by short‐term toxicity tests in Apis mellifera, considered an excellent bioindicator organism mainly due to its sensitivity. Bees have been exposed to several concentrations of TiO₂NPs (1 × 10^?3, 1 × 10^?4, 1 × 10^?5, 1 × 10^?6 mg/10 ml) for 10 days. Morphostructural and histological assays were done on gut and honey sac. The research of exposure biomarkers like metallothioneins 1 (MT1) and Heat Shock Protein 70 (HSP70) was performed to verify if a detoxification mechanism has been activated in the exposed animals. No histological alteration on the epithelium of the gut and honey sac were observed in exposed samples. A significant positivity for anti‐MT1 antibody was observed only in the honey sac cells. A weak positivity for HSP70 was observed in both structures analyzed. In several studies have shown the non‐toxicity of TiO₂NPs on other model organisms, in our study, titanium dioxide nanoparticles was proven to be highly toxic at the highest concentration tested (100% of lethality to 1 × 10^?3 mg/10 ml) and moderately toxic at lower concentrations. Honey bees proved to be excellent models for study of NPs toxicity and for monitoring environment.     

Histochemical and ultrahistochemical localization of heavy metals in calf organs

Cadmium, zinc, selenium, and copper were administered, singly or in combination, orally or subcutaneously. Experiment I included 32 calves of both sexes; six received Cd (two groups), Zn, Cd, and Zn, and Cd and Se (two groups) and one group was a control. In Experiment II (21 bulls), three were given Cd, Cd, and Cu, and Cd and Zn, respectively, and one group was a control. For light microscopy, in Experiment I the highest amounts of silver granules were present in the samples of liver, small intestine, and vesicular gland of all the exposed groups; in Experiment II the most affected organs were liver, kidney, and small intestine. For electron microscopy, in Experiment I, after administration of Cd and Zn, the highest amounts of granules were seen in the cytoplasm of hepatocytes and cells of the proximal and distal renal tubules and the lowest amounts were found in glandular cells of the pancreas. Administration of Cd and Se resulted in the presence of large numbers of granules in the nuclei and nucleoli of spermatogonies. In Experiment II, ingestion of Cd and Zn in feed led to the appearance of highest amounts of granules in the nucleoli, nuclei, and cytoplasm of cells in testes, kidneys, and pancreas. Following Cd intake, the highest accumulation of granules was observed in the nucleoli of hepatocytes and cells of the proximal and distal renal tubules. Combined Cd and Cu produced the highest number of granules in cells of the proximal and distal renal tubules and in the nucleoli and nuclei of germinal epithelium.     

Degenerative process and cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged female exposed to the acaricide permethrin

Ticks are ectoparasites of great medical and veterinary importance around the world and synthetic chemicals such as permethrin have been used for their control. This study provides a cytochemistry analysis of both degenerative and cell death processes in salivary glands of the brown dog tick Rhipicephalus sanguineus semi-engorged females exposed to 206, 1,031, and 2,062 ppm of permethrin. The results presented herein demonstrate that permethrin is a potent chemical acaricide that would act on the glandular tissue's morphophysiology in this tick species by eliciting severe changes in the acinus shape, intense vacuolation of the acinar cells' cytoplasm, marked glandular tissue disorganization, culminating in an advanced degenerative stage with consequent formation of many apoptotic bodies (cell death). In addition, permethrin induced major changes in the acinar cells' nucleus, such as a change both in its shape and size, chromatin marginalization, nuclear fragmentation, and appearance of picnotic nuclei, especially when the highest concentrations of the product were used. Thus, permethrin induced early degeneration of this tissue characterized by significant changes in the structure of acinar cells and production of enzymes related to the cell death process, in addition to interfering directly in the genetic material of these cells.     

The bioactive compound carvacrol as a potential acaricide: An assessment of its effects on the integument of female Rhipicephalus sanguineus sensu lato ticks

Studies seeking control methods for infestation of Riphicephalus sanguineus sensu lato (s.l.) ticks (dog ticks) have been carried out in order to minimize damage to both the tick's hosts and the environment, the latter due to the misuse of acaricide products. In this regard, carvacrol has been used as a natural alternative against ticks as it displays several properties including acaricidal. In this context, the present study aimed to evaluate the ultramorphology and morphohistochemistry of the integument of semi-engorged R. sanguineus s.l. females exposed to different carvacrol concentrations. The findings indicate that the integument surface of females exposed to the highest carvacrol concentration (25 μl/ml) became wrinkled, suggesting dehydration or the result of integument cuticular and epithelial layer disorganization in response to the toxic product. Morphohistochemical integument layer alterations were more significant and intense in females exposed to the highest carvacrol concentration (25 μl/ml), confirming dose-dependent carvacrol action. Among other cell and tissue alterations, changes in epithelial cell shape, size, and arrangement (epidermal layer) were noted, alongside altered and pyknotic-shaped nuclei, suggesting a death process for these cells. This epithelium changed from simple cubic to stratified, also in response to the presence of the evaluated bioactive compound. Thus, the findings reported herein demonstrate that carvacrol may be an alternative for an efficient and more sustainable tick control in the near future.     

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers

A Nd‐YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament‐dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR‐laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 µm but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament‐mediated processes.     

Ultrastructural preservation of nuclei and chromatin: improvement with low-temperature methods

The ultrastructure of chromatin has been examined in nuclei prepared by a variety of low-temperature methods. Embedding glutaraldehyde (GA)-fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature-dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0°C or in conventional epoxy resin at 60°C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross-linking of nuclear proteins. In acetone, however, cross-linking by GA occurs at — 45°C, or at lower temperatures if the water content of the acetone-based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive-free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low-temperature embedding.