Environmental scanning electron microscope
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In this paper we introduce new methodology for the observation of living biological samples in an environmental scanning electron microscope (ESEM). The methodology is based on an unconventional initiation procedure for ESEM chamber pumping, free from purge-flood cycles, and on the ability to control thermodynamic processes close to the sample. The gradual and gentle change of the working environment from air to water vapor enables the study of not only living samples in dynamic in situ experiments and their manifestation of life (sample walking) but also its experimentally stimulated physiological reactions. Moreover, Monte Carlo simulations of primary electron beam energy losses in a water layer on the sample surface were studied; consequently, the influence of the water thickness on radiation, temperature, or chemical damage of the sample was considered.
The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella.
Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.
- MeSH
- aktivní transport MeSH
- arsen farmakokinetika toxicita MeSH
- biotechnologie MeSH
- buněčné linie MeSH
- fyziologický stres MeSH
- klíčení účinky léků MeSH
- látky znečišťující životní prostředí farmakokinetika toxicita MeSH
- měď farmakokinetika toxicita MeSH
- mikroskopie elektronová rastrovací MeSH
- smrk účinky léků embryologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The paper discusses the real-time monitoring of the changing sample morphology during the entire lyophilization (freeze-drying) and vacuum-drying processes of model biopharmaceutical solutions by using an environmental scanning electron microscope (ESEM); the device's micromanipulators were used to study the interior of the samples in-situ without exposing the samples to atmospheric water vapor. The individual collapse temperatures (Tc) of the formulations, pure bovine serum albumin (BSA) and BSA/sucrose mixtures, ranged from -5 to -29 °C. We evaluated the impact of the freezing method (spontaneous freezing, controlled ice nucleation, and spray freezing) on the morphologies of the lyophiles at the constant drying temperature of -20 °C. The formulations with Tc above -20 °C resulted in the lyophiles' morphologies significantly dependent on the freezing method. We interpret the observations as an interplay of the freezing rates and directionalities, both of which markedly influence the morphologies of the frozen formulations, and, subsequently, the drying process and the mechanical stability of the freeze-dried cake. The formulation with Tc below -20 °C yielded a collapsed cake with features independent of the freezing method. The vacuum-drying produced a material with a smooth and pore-free surface, where deep cracks developed at the end of the process.
This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts.
- MeSH
- elektronová kryomikroskopie metody MeSH
- konfokální mikroskopie metody MeSH
- mikroskopie elektronová rastrovací metody MeSH
- nízká teplota MeSH
- semena rostlinná ultrastruktura MeSH
- smrk * embryologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Norsko MeSH
In this study three types of scanning electron microscopes were used for the size determination of spermatozoa of sterlet Acipenser ruthenus - high vacuum scanning electron microscope (SEM, JEOL 6300), environmental scanning electron microscope (ESEM, Quanta 200 FEG), field emission scanning electron microscope (FESEM, JEOL 7401F) with cryoattachment Alto 2500 (Gatan) and transmission electron microscope (TEM, JEOL 1010). The use of particular microscopes was tied with different specimen preparation techniques. The aim of this study was to evaluate to what degree the type of used electron microscope can influence the size of different parts of spermatozoa. For high vacuum SEM the specimen was prepared using two slightly different procedures. After chemical fixation with 2.5% glutaraldehyde in 0.1M phosphate buffer and post-fixation by 1% osmium tetroxide, the specimen was dehydrated by acetone series and dried either by critical point method or by means of t-butylalcohol. For ESEM fresh, unfixed material was used, which was dropped on microscopic copper grids. In FESEM working in cryo-mode the specimen was observed in a frozen state. Ultrathin sections from chemically fixed and Epon embedded specimens were prepared for TEM observation. Distinct parts of sterlet spermatozoa were measured in each microscope and the data obtained was statistically processed. Results confirmed that the classical chemical procedure of specimen preparation for SEM including critical point drying method led to a significant contraction of all measured values, which could deviate up to 30% in comparison with values measured on the fresh chemically untreated specimen in ESEM. Surprisingly sperm dimensions determinated on ultrathin sections by TEM are comparable with values obtained in ESEM or FESEM.
Methods for additive hydration are presented that enable longtime observation of very wet biological specimens in an environmental scanning electron microscope. The changes of structure due to dehydration for specimens placed on a Peltier-cooled holder, put on a special agar base or embedded in it or blown over by water vapor are compared. The degree of dehydration damage of the observed specimen structures is evaluated and compared with the structure of a nondestructively dried specimen, prepared by the critical point drying method.
In this study a non-conductive biological sample is observed free of charging artefacts when placed on a cooled Peltier stage in the specimen chamber of an alternative form of the environmental scanning electron microscope, equipped with a specially designed hydration system. This system was used to create dynamically changing surrounding conditions leading to controlled dehydration of the sample enabling us to visualize the topographical structure of a rat tongue in the transition region between the liquid and the gas state of water in the microscope specimen chamber.
- MeSH
- jazyk chemie ultrastruktura MeSH
- krysa rodu rattus MeSH
- mikroskopie elektronová rastrovací metody přístrojové vybavení MeSH
- teplota MeSH
- tlak MeSH
- voda chemie MeSH
- vysoušení metody přístrojové vybavení MeSH
- životní prostředí MeSH
- změna skupenství MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The Extended Low Temperature Method (ELTM) for the in-situ preparation of plant samples in an environmental scanning electron microscope enables carrying out repetitive topographical and material analysis at a higher resolution in the vacuum conditions of a scanning electron microscope or in the low gas pressure conditions of an environmental scanning electron microscope. The method does not require any chemical intervention and is thus suitable for imaging delicate structures rarely observable with common treatment methods. The method enables both sample stabilization as close to their native state as possible, as well as the transfer of the same sample from a low vacuum to an atmospheric condition for sample storage or later study. It is impossible for wet samples in the environmental scanning electron microscope. Our studies illustrate the high applicability of the ELTM for different types of plant tissue, from imaging of plant waxes at higher resolution, the morphological study of highly susceptible early somatic embryos to the elemental microanalysis of root cells. The method established here provides a very fast, universal and inexpensive solution for plant sample treatment usable in a commercial environmental scanning electron microscope equipped with a cooling Peltier stage.
A macroscopic, microscopic and scanning electron microscope study was performed on the pathological bone changes of the mandibles of wild red deer (n = 61) exhibiting severe dental fluorosis. The animals originated from a highly fluoride polluted area in Central Europe (Ore mountains and their southern foreland, Czech-German border region) and constituted 11.2 % of the studied red deer sample (n = 545) from this area. Pathologically increased wear and fracture of fluorosed teeth caused a variety of mandibular bone alterations, including periodontal breakdown, periostitis, osteitis and chronic osteomyelitis. As a further consequence of severe dental attrition, opening of the pulp chamber and formation of periapical abscesses were occasionally observed. In case of severe periodontal breakdown, loss of teeth from the mandibles was found. In addition to the inflammatory bone changes, the occurrence of osteofluorotic alterations was also diagnosed in the specimens with the highest bone fluoride concentrations (> 4000 mg F-/kg dry wt). These changes comprised extended apposition of periosteal bone onto the mandibular cortex as well as deformation of the mandibular body, which was attributed to a fluoride-induced osteomalacia. The present study provided circumstantial evidence that, in addition to fluoride induced dental lesions, the occurrence of marked periodontal disease and tooth loss is an important factor responsible for a reduction of life expectancy in severely fluorotic wild red deer.
- MeSH
- dentální fluoróza patologie veterinární MeSH
- fluoridy * MeSH
- mandibula patologie ultrastruktura MeSH
- mikroskopie elektronová rastrovací MeSH
- vysoká zvěř anatomie a histologie MeSH
- znečištění životního prostředí * MeSH
- ztráta zubů chemicky indukované MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Německo MeSH
