Experimental setup for studying the effects of alpha particles on zebrafish embryos

In the present work, we have studied the feasibility to use an experimental setup based on polyallyldiglycol-carbonate (PADC) films to study effects of alpha particles on dechorionated zebrafish embryos. Thin PADC films with a thickness of 16 μm were prepared from commercially available CR-39 films by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 4 h post fertilization (hpf) with absorbed doses up to 2.3 mGy. Images of the embryos at 48 hpf were examined for identification of morphologic abnormalities. The preliminary results showed that absorbed doses corresponding to the abnormally developed embryos ranged from 0.41 to 2.3 mGy, which was equivalent to 0.21-1.2 mGy in human.     

Designing experimental setup and procedures for studying alpha-particle-induced adaptive response in zebrafish embryos in vivo

The present work was devoted to designing the experimental setup and the associated procedures for alpha-particle-induced adaptive response in zebrafish embryos in vivo. Thin PADC films with a thickness of 16 μm were fabricated and employed as support substrates for holding dechorionated zebrafish embryos for alpha-particle irradiation from the bottom through the films. Embryos were collected within 15 min when the light photoperiod began, which were then incubated and dechorionated at 4 h post fertilization (hpf). They were then irradiated at 5 hpf by alpha particles using a planar ²⁴¹Am source with an activity of 0.1151 μCi for 24 s (priming dose), and subsequently at 10 hpf using the same source for 240 s (challenging dose). The levels of apoptosis in irradiated zebrafish embryos at 24 hpf were quantified through staining with the vital dye acridine orange, followed by counting the stained cells under a florescent microscope. The results revealed the presence of the adaptive response in zebrafish embryos in vivo, and demonstrated the feasibility of the adopted experimental setup and procedures.     

电离辐射引起的DNA链断裂损伤及其修复

DNA链断裂是电离辐射的主要生物学效应之一,单链断裂由一次击中造成,所以与辐射剂量成线性关系,双链断裂可以一次击中造成,也可由两个位置相关的单链断裂迭加而成,所以与辐射剂量成线性——平方关系。已有各种理化方法能测定活细胞内的单链断裂和双链断裂数。细胞具有修复DNA链断裂的能力,但其机制远不如切除修复机制那么清楚。未修复的链断裂是致死的,被修复的单链断裂完全恢复DNA的正常结构与功能,被修复的双链断裂的生物学效应如何还不清楚。剂量率对辐射的生物学效应有重要影响。     

Surface Etching and DNA Damage Induced by Low-Energy Ion Irradiation in Yeast

Bio-effects of survival and etching damage on cell surface and DNA strand breaks were investigated in the yeast saccharomyces cerevisiae after exposure by nitrogen ion with an en- ergy below 40 keV. The result showed that 16% of trehalose provided definite protection for cells against vacuum stress compared with glycerol. In contrast to vacuum control, significant morpho- logical damage and DNA strand breaks were observed, in yeast cells bombarded with low-energy nitrogen, by scanning electron microscopy (SEM) and terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) immunofluorescence assays. Moreover, PI (propidium iodide)fluorescent staining indicated that cell integrity could be destroyed by ion irradiation. Cell damage eventually affected cell viability and free radicals were involved in cell damage as shown by DMSO (dimethyl sulfoxide) rescue experiment. Our primary experiments demonstrated that yeast cells can be used as an optional experimental model to study the biological effects of low energy ions and be applied to further investigate the mechanism(s) underlying the bio-effectsof eukaryotic cells.     

Measurements of the W values in argon,nitrogen, and methane for 0.93 to 5.3 MeV alpha particles

The W values in argon, nitrogen and methane have been measured for alpha particles with energies from 0.93 MeV to 5.3 MeV by gridded ionization chambers. The energies of alpha particles emitted from ²¹⁰Po (5.3 MeV) were reduced by passage through Al foils and Mylar films of various thicknesses. The mean energies of these “reduced” alpha particles were determined using a silicon detector calibrated with five natural alpha sources. The W values for the gases and energies studied are obtained by assuming a W value of 26.31 eV in pure argon for 5.3 MeV alpha particles. The W value in argon is found to be slightly dependent on the energy of alpha particles. In nitrogen and methane, the W values for an alpha energy of 0.93 MeV are about 5 and 8% larger, respectively, than for an alpha energy of 5.3 MeV.     

Study of substrate topographical effects on epithelial cell behavior using etched alpha-particle tracks on PADC films

Micrometer-size pits on the surface of a polymer (polyallyldiglycol carbonate or PADC) substrate created by alpha-particle irradiation and subsequent chemical etching were used to study the topographical effects alone on cell behavior. Vinculin, the cell adhesion and membrane protrusion protein, was used as an indicator of cytoskeletonal reorganization on the substrate and localization of vinculin was used to demonstrate the presence of focal adhesions. In our experiments, vinculin expressed in epithelial HeLa cells cultured on PADC films with track-etch pits, but not in cells cultured on the raw or chemically etched blank films. In other words, vinculin expression was induced by the topography of track-etch pits, while etching of the substrate alone (without alpha-particle irradiation) did not cause up-regulation of vinculin protein expression. HeLa cells cultured on PADC films with track-etch pits also showed changes in cell proliferation, cell area and cell circularity, and were largely contained by the pits. In other words, the cell membrane edges tended to be in contact with the pits. By comparing the correlation between the positions of HeLa cells and the pits, and that between the positions of cells and computer-simulated pits, the tendency for membrane edges of HeLa cells to be in contact with the pits was recognized. This could be explained by inhibition of membrane protrusion at the pits. In conclusion, substrate track-etch pits were an important determinant of epithelial cell behaviors.     

Characterisation of a pre-cell hit detector to be used in single cell irradiation experiments at the Lund Nuclear Microprobe

This paper describes the characterisation of an ultra-thin silicon semiconductor ΔE detector to be used as a pre-cell ion hit detector in single ion experiments on individual, living cells. The characteristics of interest for this specific application are the hit detection efficiency, which has to be close to 100% to enable bombardment with either a single ion or a counted number of ions, the beam spreading, which should be as small as possible to maintain the targeting accuracy, and the vacuum tightness, since the detector is intended, if possible, to be used simultaneously as vacuum window. The hit detection efficiency was shown to be above 99% when detecting alpha particles or 2 MeV protons, the increase in beam size was about 1 μm and the vacuum tightness was comparable to that of the Si₃N₄ wafer which is normally used as vacuum window, thus the ΔE detector fulfils the main criteria to function properly as a single ion hit detector.     

用微孔滤膜法检测γ射线引起的哺乳动物细胞DNA双链断裂及其重接

本文改进了Bradley和Kohn测定DNA双链断裂的微孔滤膜法,并用于检测γ射线引起的中国仓鼠卵巢细胞和小鼠骨髓细胞DNA的双链断裂,得到较好的剂量曲线,证明此法重复性好,灵敏度远较中性蔗糖密度梯度离心法高。同时也证明在上述细胞中辐射所致的DNA双链断裂是能够重接的。为DNA双链断裂能够重接的论点提供了又一个实验证据。     

细胞质受照射的遗传损伤效应

通过使用精确单粒子微束装置照射人与中国仓鼠杂交AL细胞细胞质,显示α粒子照射细胞质只有轻微的致死率,照射16个α粒子后仍有76％的细胞可以存活。诱变试验结果发现,尽管单个α粒子照射细胞质在CD59基因位点诱变频率较低,但8个或多于8个α粒子可诱导出高于未受照射对照3倍的诱变频率聚合酶链式反应（PCR）分析发现,照射细胞质诱导的CD59^-基因突变子其11号染色体上Wilms肿瘤（WT）、过氧化氢酶（CAT）、（H－rasRAS)、甲状旁腺激素（PTH）、（脱脂蛋白－A1 APO－A1）等基因缺失率很小,其突变谱类似于自发突变子的突变谱。DMSO或BSO处理细胞的研究显示,自由基清除剂DMSO可降低照射细胞质所诱发的CD59基因突变频率;与此相反,能与细胞内的谷胱甘肽结合的BSO可增加突变频率。结果表明,自由基很可能是照射细胞质诱发突变的主要原因之一。此外,用IF7单克隆抗体进行的8－羟基脱氧鸟苷免疫过氧化物酶染色反应发现照射细胞质可在核内引起DNA的氧化损伤。     

Optical appearance of alpha particle tracks in CR-39 SSNTD

A ray tracing method based on geometrical optics was used to study the tracks from alpha particles with different energies and with an incident angle of 50°. The transmission operation mode of the microscope is simulated. Considering the distribution of light intensities from the tracks, the mean and the 80% percentile gray levels from real experiments are proposed as quantitative variables to differentiate among tracks. The gray level properties for the same track for different exposures can vary to large extents. We introduce three variables, κ, λ and ε, to make empirical corrections. It is interesting to see that these coefficients are very consistent for the same alpha particle track despite the very different gray level properties. Gray level results have been obtained for tracks from alpha particles with 50° incident angle and different incident energies. However, the track depths cannot be predicted by any one of the coefficients κ, λ and ε. Multivariate analyses can help separate the tracks corresponding to different alpha energies. By using discriminant analysis with κ, λ and ε as independents, effectively all alpha energies can be determined with an accuracy of ±0.5 MeV.     

小鼠乳癌细胞辐射敏感突变株SX-9DNA双链断裂及其重接修复研究

本研究采用一种新的细胞DNA双链断裂检测方法——脉冲电场电泳法,检测了两株来源相同而辐射敏感性不同的细胞SR-1和SX-9的X线照射所致DNA双链断裂的产生和修复。结果表明两株细胞的DNA双链断裂产生没有差别,而辐射敏感细胞SX-9的DNA双链断裂重接修复能力明显低于SR-1细胞,本文对此结果与细胞辐射敏感性的关系进行了讨论。     

Double Differential Proton and Alpha Emission Cross Sections for Structural Fusion Materials <Superscript>94,95,96</Superscript>Mo

In this study, double differential alpha and proton emission cross sections, which is necessary in determination of heating and damages in structural fusion material research, of ^94,95,96Mo target nuclei have been theoretically calculated by the TALYS 1.8 code at 14.8 MeV neutron incident energy and also compared with available experimental data in EXFOR library. The compound nucleus formation process and pre-equilibrium contribution were found as dominant in the emission of proton and alpha particles, the direct reaction contribution also was seen as dominant in higher particle emission energies.     

The live cell irradiation and observation setup at SNAKE

We describe a new setup at the ion microprobe SNAKE (Superconducting Nanoscope for Applied nuclear (Kern-) physics Experiments) at the Munich 14 MV Tandem accelerator that facilitates both living cell irradiation with sub micrometer resolution and online optical imaging of the cells before and after irradiation by state of the art phase contrast and fluorescence microscopy. The cells are kept at standard cell growth conditions at 37 °C in cell culture medium. After irradiation it is possible to switch from single ion irradiation conditions to cell observation within 0.5 s. First experiments were performed targeting substructures of a cell nucleus that were tagged by TexasRed labeled nucleotides incorporated in the cellular DNA by 55 MeV single carbon ion irradiation. In addition we show first online sequences of short time kinetics of Mdc1 protein accumulation in the vicinity of double strand breaks after carbon ion irradiation.     

Development of Cell Chip Based on Track Detector for Examination of Biological Damage by Alpha Particles

A cell chip was developed for the examination of biological damage of cells irradiated by high-energy alpha particles. A CR-39 track detector was employed as a chip substrate to identify high-energy charged particles traversing target cells. Moreover, the patterning of a photopolymer layer spatially controlled the cellular adhesiveness on the chip substrate. HeLa cancer cells were cultured on a micropatterned photopolymer layer. In this way, all the cells on the chip were individually addressed through the block number in the photopolymer pattern. The biocompatibility of the cell chip was examined through a viability test with fluophor reagent and measurement of the cell proliferation rate. HeLa cancer cells on the chip were irradiated with alpha particles and stained with a fluorescent probe molecule for DNA damage detection. The CR-39 substrate was etched by means of an alkali solution during cell incubation. The HeLa cells and alpha tracks were successfully observed by microscopy at once. It was confirmed that fluorescent spots corresponding to DNA damage were located in the direction of the major axis of oval alpha tracks.     

Simple preparation of thin CR-39 detectors for alpha-particle radiobiological experiments

Alpha-particle radiobiological experiments involve irradiating cells with alpha particles and require accurate positions where the alpha particles hit the cells. In the present work, we prepared thin CR-39 detectors from commercially available CR-39 SSNTDs with a thickness of 100 μm by etching them in 1 N NaOH/ethanol at 40 °C to below 20 μm. The desired final thickness was achieved within ∼8 h. Such etching conditions can provide relatively small roughness of the detector as revealed by atomic force microscope, and thus provide transparent detectors for radiobiological experiments. UV radiation was employed to shorten track formation time on these thin CR-39 detectors. After exposure to UV light (UVA + B radiation) for 2-3 h with doses from 259 to 389 W/cm², 5 MeV alpha-particle tracks can be seen to develop on these CR-39 detectors clearly under the optical microscope within 2 h in 14 N KOH at 37 °C. As an example for practical use, custom-made petri dishes, with a hole drilled at the bottom and covered with a thin CR-39 detector, were used for culturing HeLa cells. The feasibility of using these thin CR-39 detectors is demonstrated by taking photographs of the cells and alpha-particle tracks together under the optical microscope, which can allow the hit positions on the cells by the alpha particles to be determined accurately.     

14MeV单能中子致DNA链断裂及相应防护研究

利用中国原子能科学研究院600kV高压倍加器产生的14MeV的单能中子,以不同剂量对不含和含有甘露醇或香兰素衍生物VND3207的质粒DNA进行了照射,使用凝胶电泳技术观测了DNA分子的链断裂。结果表明,中子可诱发DNA分子发生单链和双链断裂,随剂量的升高,链断裂产额逐渐增加,具有明显的剂量响应。在含有甘露醇和VND3207的研究体系中,也存在一定的剂量响应,但相比不含两者的体系而言,剂量响应不明显,且在相同剂量下DNA分子的链断裂产额极低。此结果反映出研究中所选用浓度的甘露醇与VND3207可有效降低中子辐射所诱发的DNA单链和双链断裂的产生,对DNA分子起到极好的保护作用,具有一定的中子辐射防护能力。     

Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

Yeast strain Saccharomyces cerevisiae was irradiated with different doses of 85 MeV/u 20Ne10+ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T→G and T→C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.     

BaF2闪烁体探测器信号数字化方法研究

应用数字化方法对BaF₂闪烁体探测器脉冲信号进行了研究,信号的测量通过FADC和计算机系统实现,并对所采集的脉冲波形进行了离线分析。通过计算脉冲波形的面积提取了射线的能量信息,重建了能谱,并与传统电子学模拟信号测量方法得到的能谱进行了比较;应用过阈定时方法提取了脉冲的时间信息,测量并给出了4πBaF₂探测器系统的时间分布谱;应用脉冲形状甄别法对α粒子和γ射线进行了鉴别研究,根据脉冲信号快/慢成分比的差别清楚地将α粒子从γ射线中鉴别开来。本工作为在γ全吸收型4πBaF₂探测器系统中应用FADC进行多通道、多参数、大数据量数字化数据获取积累了经验。     

羧甲基-β-1,3-葡聚糖对人B淋巴母细胞的辐射防护作用

利用X射线辐照经羧甲基-β-1,3-葡聚糖预处理的人B淋巴母细胞(Human B lymphoblasts,HMy2.CIR),探究羧甲基-β-1,3-葡聚糖对辐射损伤的防护作用。采用CCK-8法检测细胞存活率,并筛选出最佳给药浓度和孵育时间(0.1μg/mL,72 h);流式细胞仪检测羧甲基-β-1,3-葡聚糖对辐照后HMy2.CIR细胞凋亡的影响;微核实验检测羧甲基-β-1,3-葡聚糖对辐照后HMy2.CIR细胞微核形成的影响;彗星实验检测对DNA损伤程度和修复的影响。结果表明,羧甲基-β-1,3-葡聚糖对HMy2.CIR无细胞毒性并具有增殖促进作用;羧甲基-β-1,3-葡聚糖能够抑制辐射造成的凋亡率和微核率的增加,降低DNA损伤程度,加快损伤DNA的修复。羧甲基-β-1,3-葡聚糖对HMy2.CIR细胞辐射损伤有防护作用,其机制可能与抑制辐射诱导的细胞凋亡和DNA损伤有关。     

Stopping power and ranges of electrons, protons and alpha particles in liquid water using the Geant4-DNA package

This paper presents stopping power and ranges of electrons, protons, and alpha particles in liquid water, calculated using the latest Geant4-DNA processes implemented in the Geant4 Monte Carlo simulation toolkit. Inelastic cross sections are obtained using the first Born approximation and semi-empirical formulas like Rudd’s model for ionisation and the Miller and Green formula for excitation. Elastic collisions and vibrational excitations are considered for tracking electrons until complete thermalisation (0.025 eV). A speed scaling procedure with an effective charge screening term was used to compute alpha particle and heavy ion cross sections. Geant4-DNA simulations were carried out using thin liquid water volumes to determine the linear energy loss (dE/dX), while larger volumes were used to obtain the particle range. While results converge for highly energetic particles, differences are observed for low energies when the applied theoretical models begin to diverge from each other. Results show a good agreement between the analytical calculations obtained from the models, the Geant4-DNA Monte Carlo simulation predictions and the data published in the ICRU reports. Geant4-DNA processes apply to the following energy ranges: 0.025 eV-1 MeV for electrons, 100 eV-100 MeV for protons and 1 keV-400 MeV for alpha particles in liquid water, however since experimental data for very low energies is scarce and very difficult to obtain these processes could not be thoroughly validated so they are recommended for energies above 1 eV for electrons, 1 keV for protons and 10 keV for alpha particles. Relativistic, highly charged ions were implemented in our own “house” version of the code and will be available in future releases of Geant4.