共查询到20条相似文献,搜索用时 15 毫秒
1.
Yeshayahu Talmon 《Journal of microscopy》1982,125(2):227-237
Although low temperature fixation seems to be superior to chemical fixation with respect to structure preservation, complex interactions between the electron beam and frozen hydrated specimens can limit its applications. A review is given of the present knowledge of beam heating and radiation damage with emphasis on recent developments in the field. Beam heating is found not to be a major limitation to cold stage microscopy. But radiolysis of ice and the formation of free radicals that subsequently attack organic matter in contact with ice can severely alter the specimen structure before a useful image is recorded. Use of low dose techniques may help in some cases, but the problem cannot be overcome in X-ray microanalysis of very small regions, when high beam doses must be used. The use of liquid helium stages operating near 4 K may reduce damage also in frozen hydrated specimens. 相似文献
2.
The analytic solution for the model of electron beam heating a moderately thick STEM/SEM specimen was used to calculate the steady state temperature profiles developed in the bulk of the specimen. For thin specimens one maximum in the temperature profile was found near the surface. The location of this maximum shifted away from the surface with increasing sample thickness. For specimens of thickness approaching the electron range two maxima were found: one close to the surface (as in thin samples) due to a ‘self-insulating’ effect, and another maximum near the sample-substrate interface—a result of very rapid increase in the energy loss by the electrons near their penetration range. These results are of particular interest for X-ray microanalysis where high beam currents are used, resulting in potentially large temperature rises in the bulk of the specimen. 相似文献
3.
Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples 总被引:3,自引:0,他引:3
M. MARKO C. HSIEH W. MOBERLYCHAN C. A. MANNELLA & J. FRANK† 《Journal of microscopy》2006,222(1):42-47
The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30‐keV Ga+ beam, and cryo‐transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200–150 nm, changes characteristic of heat‐induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that ‘cryo‐FIB thinning’ of bulk frozen‐hydratred material will be capable of producing specimens for TEM cryo‐tomography with much greater efficiency than cryo‐ultramicrotomy, and without the specimen distortions and handling difficulties of the latter. 相似文献
4.
A new SEM technique for imaging uncoated non-conducting specimens at high beam voltages is described which employs a high-pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation. A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy. 相似文献
5.
We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability. 相似文献
6.
A series of modifications have been devised which allow the peak to background ratio X-ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen-hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X-rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back-scattered imaging system is described, which helps to overcome the problem of spurious X-ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X-ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X-ray interactive volume to electron penetration and X-ray emission in both frozen hydrated and frozen dried cells. The problems of X-ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves. 相似文献
7.
Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit. 相似文献
8.
Jacob Bastacky Christian Goodman Thomas L. Hayes 《Microscopy research and technique》1990,14(1):83-84
A miniature vise built into a copper stub is described that holds bulk, pre-frozen, hydrated biological specimens during examination under the electron beam of the scanning electron microscope. 相似文献
9.
Quantitative x‐ray microanalysis of model biological samples in the SEM using remote standards and the XPP analytical model
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ALAN T. MARSHALL 《Journal of microscopy》2017,266(3):231-238
It is shown that accurate x‐ray microanalysis of frozen‐hydrated and dry organic compounds, such as model biological samples, is possible with a silicon drift detector in combination with XPP (exponential model of Pouchou and Pichoir matrix correction) software using ‘remote standards’. This type of analysis is also referred to as ‘standardless analysis’. Analyses from selected areas or elemental images (maps) were identical. Improvements in x‐ray microanalytical hardware and software, together with developments in cryotechniques, have made the quantitative analysis of cryoplaned frozen‐hydrated biological samples in the scanning electron microscope a much simpler procedure. The increased effectiveness of pulse pile‐up rejection renders the analysis of Na, with ultrathin window detectors, in the presence of very high concentrations of O, from ice, more accurate. The accurate analysis of Ca (2 mmol kg?1) in the presence of high concentrations of K is possible. Careful sublimation of surface frost from frozen‐hydrated samples resulted in a small increase in analysed elemental concentrations. A more prolonged sublimation from the same resurfaced sample and other similar samples resulted in higher element concentrations. 相似文献
10.
Frozen hydrated specimens of various latex spheres were used as well-defined systems for the study of electron beam radiation damage to organic inclusions in vitreous, cubic and hexagonal ice. We found that radiolysis of organic material is modified by the presence of ice and that radiolysis in vitreous ice is different from that in crystalline ice. The pattern of damage depends also on the nature of the irradiated polymer, e.g., damage to poly(vinylchloride) is quite different from damage to polyacrylates, although in both polymers the main radiolytic process is chain scission. Some polymers such as polyacrylates were found to be much more stable in vitreous ice than in crystalline ice. The experimental results indicate that free radicals formed at the ice–organic matter interface play an important role in the radiolysis process which affects both the ice and embedded organic particles. Ice may play also a physical role in the process by limiting the diffusion of free radicals away from the interface. Although net mass loss is not much affected by ice, massive structural changes including repolymerization take place in its presence. 相似文献
11.
The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts. 相似文献
12.
Electron-beam-induced amorphization of ice III or IX obtained by high-pressure freezing 总被引:1,自引:0,他引:1
Cryo-electron microscopy of vitrified specimens makes it possible to observe fully hydrated biological samples unimpaired by chemical fixation, staining and dehydration. High-pressure freezing represents important progress since it allows a 10-fold increase in the vitrification depth. High-pressure freezing can also induce the formation of undesirable high-pressure forms of ice. We show that ice III or IX is amorphized under the electron beam at a dose of about 2400 electronsnm−2 and that the resulting amorphous ice is similar to the vitreous water obtained by high-pressure freezing. 相似文献
13.
Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. 总被引:4,自引:0,他引:4
When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures. 相似文献
14.
Karl Zierold 《Microscopy research and technique》1988,9(1):65-82
The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples. 相似文献
15.
《Nuclear Instruments and Methods in Physics Research》1983,191(1-3):375-382
SIMS microanalysis is readily achieved by the adoption of the liquid metal field ion source for formation of the primary ion probe. These sources exhibit high brightness and small source size and permit the formation of high intensity sub-micron ion beams at low energies (⩽10 KeV). Operation is simple, UHV compatible and well suited to mulit-technique surface analysis systems, as well as dedicated SIMS instrumentation.Beam characteristics for a gallium probe allow image resolutions down to 1000 Å with probe currents ranging from 50 pA at high resolution, to maximum currents in excess of 100 nA. Image recording times range from 10 s to 10 min, typically.A gallium source has been in routine operation for over one year on a quadrupole based SIMS instrument, and a wide range of experience gained on various specimens. These include metals, semiconductors, insulators, polymers and biological materials.Some comparisons have been made between the use of argona and gallium beams with respect to spectral content and elemental sensitivities. In general the sensitivities are similar, while the main difference in spectral content is the presence of secondary ions of gallium and gallium complexes.Insulating specimens which have proved very difficult in Auger analysis such as optical fibres, or powders, have been studied more readily with the gallium probe in SIMS analysis. In this case charge neutralization is effected with an accurately positioned 500 eV electron beam. 相似文献
16.
Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample. 相似文献
17.
Wolfgang Fuchs Bernd Lindemann Jobst D. Brombach Walter Trsch 《Journal of microscopy》1978,112(1):75-87
A cooling chain for the handling of frozen hydrated bulk specimens is described. Use of this method permits the specimen to be kept fully hydrated. After quench freezing, the specimen is transferred to a freeze etch apparatus, freeze fractured, carbon coated and transferred onto the precooled cold stage of the SEM by means of an airlock. The specimen is examined in the secondary electron mode and analysed using an energy dispersive X-ray analyser. The midgut of Chironomus thummi larvae and frog skin epithelium were used to test the performance. 相似文献
18.
Diane M. Hopkins Alan D. Jackson Kenneth Oates 《Microscopy research and technique》1991,18(2):176-182
The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X-ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was an inverse relationship between coating thickness and elemental X-ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together. 相似文献
19.
Energetic beams of electrons and ions are widely used to probe the microscopic properties of materials. Irradiation with charged beams in scanning electron microscopes (SEM) and focused ion beam (FIB) systems may result in the trapping of charge at irradiation induced or pre-existing defects within the implanted microvolume of the dielectric material. The significant perturbing influence on dielectric materials of both electron and (Ga(+)) ion beam irradiation is assessed using scanning probe microscopy (SPM) techniques. Kelvin Probe Microscopy (KPM) is an advanced SPM technique in which long-range Coulomb forces between a conductive atomic force probe and the silicon dioxide specimen enable the potential at the specimen surface to be characterized with high spatial resolution. KPM reveals characteristic significant localized potentials in both electron and ion implanted dielectrics. The potentials are observed despite charge mitigation strategies including prior coating of the dielectric specimen with a layer of thin grounded conductive material. Both electron- and ion-induced charging effects are influenced by a delicate balance of a number of different dynamic processes including charge-trapping and secondary electron emission. In the case of ion beam induced charging, the additional influence of ion implantation and nonstoichiometric sputtering from compounds is also important. The presence of a localized potential will result in the electromigration of mobile charged defect species within the irradiated volume of the dielectric specimen. This electromigration may result in local modification of the chemical composition of the irradiated dielectric. The implications of charging induced effects must be considered during the microanalysis and processing of dielectric materials using electron and ion beam techniques. 相似文献
20.
Patrick Echlin 《Journal of microscopy》1991,161(1):159-170
There are several limitations to the low-temperature techniques which are currently being used for the preparation, examination and analysis of biological and organic samples by means of high-energy beam instrumentation. The low thermal conductivity of samples and the inadequacy of rapid cooling techniques means that, with the exception of thin-film suspensions and the surface of impact-cooled bulk specimens which may be vitrified, ice crystals of varying sizes will be present in nearly all samples which are quench cooled. Data are presented which indicate the depth to which adequate cryo-fixation may be achieved for both morphological and analytical studies. Although dynamic processes may be time resolved in the outer parts of quench-cooled samples, the decreased freezing rate below the surface makes resolution of these processes much less certain. The quality of information which may be obtained from quench-cooled samples is limited by radiation damage. Low-dose microscopy of vitrified thin-film suspensions of macromolecules continues to provide valid structural information at the molecular level. The increased doses needed for X-ray microanalysis present serious problems with the high spatial resolution analysis of thin frozen-hydrated sections although much less damage is observed in dried samples. A case is presented for using the outer fracture faces of frozen-hydrated bulk samples for low-resolution analysis of cells and tissues. 相似文献