Quantitative analysis of knock-on and thermal damage of biological specimens by electron irradiation in transmission electron microscopy

High-energy electrons are able to transfer momentum to nuclei, which results in displacement on to the interstitial lattice sites with a maximum transferred energy of 4 · 10⁴ eV for carbon at 100 keV. Moreover, most of the energy dissipated in energy losses is converted into heat, which results in melting and evaporation.The specimen temperature rise was calculated by the heat conduction theory and confirmed by the specimen drift due to the thermal damage. The damage can be reduced by a small area of illumination, the use of a metal-coated microgrid and small area scanning.A further displacement due to the knock-on collision and the resulting etching rate of biological specimens was measured. The damage is proportional to the current density in c cm^-2 at the specimen. The allowable maximum dose was obtained from the measurement of an etching rate with the weight loss and dry density of the specimens.It was found that the images affected by the electron irradiation, in which -H, C-H, C-N bonds break molecules in proteinaceous biological specimens are removed, and the remaining molecules are changed to stable carbon-rich molecules by deposition, polymerization and contamination. In addition, defect images were observed in high contrast, when compared with unaffected images taken with a small area scanning method.     

Interaction of an ion beam with the target in electron microscope applications

When based on the power-potential law of Lindhard et al. (Mat. Fys. Dan. Vid. Selsk, 33: 1–42, 1963) for ionic impact phenomena on the surfaces of a target, the universal curves of nuclear and electronic energy loss-energy, their resulting yield-energy relationships of sputtering and secondary electron emission yield-energy and range-energy have consistently been derived.According to the results obtained from the above experimental data, a diffusion model of an ion beam penetrating a target is proposed, which takes place throughout a hemisphere with its centre located at half the diffusion depth, and which is found to agree well with the empirical data of ion beam penetration, energy-dissipation profiles and the backscattering coefficient as a function of the reduced depth.Owing to the diffusion model's data, the total secondary electron emission yield due to both primary and backscattering ions is obtained. More importantly, radiation damage in ion beam applications is consistently evaluated as a function of the reduced energy ratio.     

Study of the permeability of tomato pericarp etched by low-energy ion beams based on α-particles Irradiation

Low-energy ion beam bio-technology has been applied in the biological field and has gained remarkable success in crop and microbe breeding. However, to understand how low-energy ion beams interact with biological materials remains a challenge for researchers who work for the development of ion-beam bio-technology. In this work, tomato pericarp was used as the target sample to study the effect of ion beams on the permeability of biological objects. A series of experiments were conducted via irradiating tomato pericarp samples with low-energy (10 keV ～ 25 keV) ion beams followed by measuring the pericarp’s permeability using transmissive α particles. The transmissive spectra of α particles and the measurement of the tip number in CR39 gave a quantitative evaluation of the sputtering effect caused by low-energy ions. Meanwhile, natural red dye was used to examine the permeability of irradiated tomato pericarp samples. It was found that the sputtering effect is not only proportional to the ion energy and dose, but dependent on the ion type as well. The damage caused by Ar ions due to sputtering was much more severe than that caused by N ions sputtering with the same dose. Therefore, this study not only demonstrates the permeability difference of biological membranes before and after ion irradiation, but also provides the information on how to optimize the experimental conditions for application of the low-energy ion beam in biology.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

ELECTRON MICROSCOPIC OBSERVATION OF SPECIMENS UNDER CONTROLLED GAS PRESSURE

A technique for encasing specimens in a thin gas layer during their observation in the Siemens Elmiskop I is described. All gases can be employed at pressures up to one atmosphere. Destruction of specimens can occur in the beam; all organic specimens are particularly liable to decompose. The conditions under which this can be avoided are given. A useful application of the technique allows one to prevent specimens from drying out, as they normally do in vacuum. A further application uses the controlled removal of carbon for thinning organic layers and for selective etching of organic materials.     

Observations on charging effects of non-conductive and uncoated materials by ion beam pre-bombardment in scanning electron microscopy

The specimen charging defects of non-conductive materials in scanning electron microscopy are discussed with reference to the surface electric field generated by the illuminating electron beam dose. If the charge density depends on the relaxation time constant as defined by a product of the permittivity and resistivity when known or available, the electric field can be evaluated by the incident dose stored when illuminated by an electron scanning beam.It was found by observation that uncoated or non-conductive materials pre-bombarded by a positive ion beam, which contributes to the generated negative field, together with the charging effects, could be eliminated at the optimum time of neutralization.In the normal process of double fixation and staining of biological specimens, the local electric field produces increased contrast due to polarization effects. The dark and bright images of secondary and backscattered electrons, respectively, can be analysed by taking into account local polarization, in addition to voltage contrast.     

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.     

DNA complex lesions induced by protons and ⋅-particles: track structure characteristics determining linear energy transfer and particle type dependence

The yield of DNA double-strand breaks (dsb) and DNA complex lesions induced by protons and α-particles of various energies was simulated using a Monte Carlo track structure code (MOCA15) and a simple model of the DNA molecule. DNA breaks of different complexity were analysed. The linear energy transfer (LET) and particle-type dependence of lesions of higher complexity seems to confirm the importance of clustered damage in DNA as a relevant step leading to biological endpoints such as cell inactivation. The detailed structure of proton and α-particle tracks was analysed to identify the main characteristics possibly responsible for such a dependence. The role of the primary ion and of its secondary electrons in inducing dsb and complex lesions is described, showing that the relative contribution of secondary electron tracks alone in inducing clustered lesions is almost negligible at high LET, but tends to dominate below ≈10 keV/μm. This is consistent with the observed similar effectiveness of low-LET fast particle radiation and sparsely ionizing radiation such as x-rays. The dependence on LET and particle type is mainly due to energy deposition events of the primary ion together with short range electrons surrounding the ion track; the yield of complex lesions due to secondary electron tracks alone is substantially LET independent. The radial distributions of the energy contributing to the induction of complex lesions were analyzed and compared with the radial distributions of energy deposition of the full tracks. The results suggest that the stochastic behaviour (i.e. cluster properties) of the energy deposition pattern within a radius of a few nanometers around the ion track plays a relevant role in determining the biological radiation effectiveness. Received: 20 December 1996 / Accepted in revised form: 5 March 1997     

Experimental study of the dynamics of neutron emission from the GOL-3 multimirror trap

In experiments on the plasma heating and confinement in the GOL-3 multimirror trap, a deuterium plasma with a density of ～10¹⁵ cm^?3 and an ion temperature of 1–2 keV is confined for more than 1 ms. The plasma is heated by a relativistic electron beam. The ion temperature, which was measured by independent methods, reached 1.5–2 keV after the beginning of the beam injection. Since such a fast ion heating cannot be explained by the classical energy transfer from electrons to ions through binary collisions, a theoretical model of collective energy transfer was proposed. In order to verify this model, a new diagnostics was designed to study the dynamics of neutron emission from an individual mirror cell of the multimirror trap during electron beam injection. Intense neutron bursts predicted by this model were detected experimentally. Periodic neutron flux modulation caused by the macroscopic plasma flow along the solenoid was observed. The revealed mechanism of fast ion heating can be used to achieve fusion temperatures in the multimirror trap.     

Removing Contamination-Induced Reconstruction Artifacts from Cryo-electron Tomograms

Imaging of fully hydrated, vitrified biological samples by electron tomography yields structural information about cellular protein complexes in situ. Here we present a computational procedure that removes artifacts of three-dimensional reconstruction caused by contamination present in samples during imaging by electron microscopy. Applying the procedure to phantom data and electron tomograms of cellular samples significantly improved the resolution and the interpretability of tomograms. Artifacts caused by surface contamination associated with thinning by focused ion beam, as well as those arising from gold fiducial markers and from common, lower contrast contamination, could be removed. Our procedure is widely applicable and is especially suited for applications that strive to reach a higher resolution and involve the use of recently developed, state-of-the-art instrumentation.     

The ups and downs of beam damage,contrast and noise in biological electron microscopy

A personal account of the early problems associated with contrast in images obtained by electron microscopy from biological specimens is presented, together with the effects of electron beam damage. The author's experiences with different types of electron microscope as well as problems of contrast enhancement is described. A short account is given of the physical effects occuring during specimen preparation and their relation to structural preservation when attempting to achieve atomic resolution. Recent developments in biological electron microscopy are also discussed with a view to future trends.     

Dosimetric characteristics of a MOSFET dosimeter for clinical electron beams

The fundamental dosimetric characteristics of commercially available metal oxide semiconductor field effect transistor (MOSFET) detectors were studied for clinical electron beam irradiations. MOSFET showed excellent linearity against doses measured using an ion chamber in the dose range of 20–630 cGy. MOSFET reproducibility is better at high doses compared to low doses. The output factors measured with the MOSFET were within ±3% when compared with those measured with a parallel plate chamber. From 4 to 12 MeV, MOSFETs showed a large angular dependence in the tilt directions and less in the axial directions. MOSFETs do not show any dose-rate dependence between 100 and 600 MU/min. However, MOSFETs have shown under-response when the dose per pulse of the beam is decreased. No measurable effect in MOSFET response was observed in the temperature range of 23–40 °C. The energy dependence of a MOSFET dosimeter was within ±3.0% for 6–18 MeV electron beams and 5.5% for 4 MeV ones. This study shows that MOSFET detectors are suitable for dosimetry of electron beams in the energy range of 4–18 MeV.     

玻璃化法对离子注入拟南芥幼苗过程中的冻害防护效应

离子注入过程中活体材料往往由于真空造成的冻害而不能存活.文章以拟南芥幼苗为对象,尝试了玻璃化法对注入过程中冻害的防护效应.结果显示,玻璃化处理的2 d龄幼苗经液氮冻融处理后存活率最高,达80%以上.2 d龄幼苗经玻璃化处理后进行离子注入,在一定剂量范围内成活率在90%以上;而未经玻璃化处理的幼苗离子注入后均不能成活.玻璃化后注入的幼苗成活率随注入剂量的增加而呈下降趋势,但受注入的离子能量的影响不大.显示玻璃化法对离子注入过程中活体材料遭受的冻害有较好的防护效果,在应用上有一定的意义.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

K-shell radiation from double-shell argon plasma liners

Experiments on the generation of argon K-shell radiation during the implosion of double-shell plasma liners are described. The optimum liner length with respect to the maximum K-shell radiation yield is determined. At a liner current of ～1.4 MA, the conversion efficiency of the generator electric energy into the K-shell radiation energy attains 8–9%. The spectrum of the argon K-shell radiation is measured by a set of photoeission X-ray diodes with different filters (including an argon gas filter). Based on the measurements of the emission power in different spectral intervals and calculations by the collision-radiative model, the ion density and electron temperature of the pinch plasma are estimated.     

电子束辐射唐菖蒲球茎对M1叶片的影响初探

为了探讨电子束对唐菖蒲诱变育种的可行性和不同剂量的电子束对叶片的影响;用能量为3MeV的不同剂量电子束辐照唐菖蒲“超级”球茎,对其M1代的叶片进行了研究。 光合作用率,蒸腾速率,细胞间隙CO₂浓度和气孔导度等光合指标在低剂量时变化均不明显,随着剂量的增价,辐照对蒸腾速率和气孔导度起到明显的刺激作用并达到0.05的显著水平;通过对电子束处理后唐菖蒲叶片叶绿素含量的测定表明,叶绿素含量及叶绿素a/b比值均发生了变化,并且叶绿素a含量在240Gy处理后达到最大值,且达到了0.05的显著水平;扫描电镜观察发现,高剂量处理的M1代植株表皮毛与气孔结构均发生改变,叶表面组织特征明显排列杂乱不规则。由此表明,电子束辐照对唐菖蒲叶片的形态与生理均能产生明显的影响。     

低能离子束生物技术的应用

自从我国科学家发现离子注入生物学效应后,低能离子束生物技术的研究就在我国率先兴起。随后,越来越多的科学家基于低能离子与生物体之间存在的能量沉积、动量传递、质量沉积及电荷中和与交换的相互作用,对生物体内的遗传物质进行加工、修饰、重组,开辟了农作物和微生物等遗传改良及转基因的新方法。本文简要介绍了低能离子束生物技术产生的背景、低能离子束与生物体之间相互作用的机理和特点以及目前低能离子束在诱变育种和转基因等生物技术领域的研究进展,并展望了离子束技术在藻类基因工程方面的发展潜力。     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation

In order to obtain an industrial strain with higher chitosanase yield, the wild strain Bacillus sp. S65 cells were mutated by a novel mutagen, nitrogen ion beam, with energy of 15 keV and dose ranging from 2.6 × 10¹⁴ to 5.2 × 10¹⁵ ions/cm². One mutant, s65F5 with high yield of chitosanase was isolated. Results showed that the production of chitosanase of s65F5 was dramatically increased from 4.1 U/ml in s65 to 25 U/ml by ion beam implantation, while the fermentation time was shortened from 72 to 56 h, both of which greatly increased efficiency and reduced the cost of industrial production. Besides, the mutagenic effects of low-energy ion beam on survival rate showed characteristic down–up–down pattern, which was different from the traditional mutagens such as UV and γ-ray and the possible mutation mechanism was discussed.     

Kinetics Studies on the Fluorescence Quencher in Isolated Chloroplasts

The chlorophyll fluorescence yield in isolated chloroplasts without an added electron acceptor is increased by actinic illumination. The decline in the fluorescence yield when the actinic illumination is extinguished can be accurately represented by three, independent, exponential decays with half-times of approximately 0.8, 5, and 30 sec. These results have been interpreted using Duysens' theory of fluorescence quenching by a compound (Q) on the reducing side of photosystem II. This theory states that changes in fluorescence yield are indicative of electron flow through Q. The most rapid decay is eliminated by an EDTA washing of the chloroplasts and the half-time is increased by uncoupling with ammonia and by added electron acceptors in suboptimal concentrations. Thus, this decay may represent electron flow from Q to intermediates on the oxidizing side of photosystem I. The decay with a half-time of 5 sec is affected in the same manner as the decay with the shortest half-time by the same procedures. However, electron donors to photosystem II lengthen the half-time of the 5 sec decay while eliminating the most rapid decay. This 5 sec decay can be interpreted as electron flow from Q to intermediates either on the reducing side of photosystem II or on the oxidizing side of photosystem I. The decay with the longest half-time is affected only by pH and electron donors to photosystem II. Therefore, this decay may indicate electron flow from Q to intermediates on the oxidizing side of photosystem II which may be connected to the regeneration of the oxygen burst.