Combined use of confocal laser scanning microscopy and transmission electron microscopy for visualisation of identical cells processed by cryotechniques

Summary. Successive visualisation of identical plant cells by light and electron microscopy is reported. For this purpose segments of pea and barley leaves were prepared by high-pressure freezing, freeze-substitution, and low-temperature embedding. The use of Safranin O during low-temperature dehydration allowed, on one hand, staining of all cellular components as investigated by confocal laser scanning microscopy and, on the other hand, excellent ultrastructural and antigenic preservation. A newly constructed specimen holder enabled precise relocation of the target cells for electron microscopic investigations. Transmission electron microscopy and immunohistochemistry revealed that during the whole procedure the ultrastructure of the cells as well as the antigenicity of cell constituents were preserved.Correspondence and reprints: Central Microscopy, Center of Biology, University of Kiel, Am Botanischen Garten 5, 24098 Kiel, Federal Republic of Germany.     

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4’,6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.     

A differential staining method to identify lignified and unlignified tissues

We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.     

Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.     

紫花含笑（♀）、灰岩含笑（♂）及其杂种F1代花粉萌发试验研究

对紫花含笑(Michelia crassipes)、灰岩含笑(M.calcicola)及其杂种F1代花粉生活力进行了研究,为基于紫花含笑和灰岩含笑杂种F1代的含笑属观赏植物新品种培育与种质创新提供科学数据及研究资料.研究发现,亲本(紫花含笑和灰岩含笑)新鲜花粉萌发率均可达90％以上,杂种F1代花粉萌发率从38％到79％不等,平均为57.7％,低于双亲.亲本及其杂种F1代花粉萌发的最适温度为25℃,温度过高花粉管的伸长受到抑制,并导致花粉管顶端破裂.亲本及多数杂种F1代的新鲜花粉在100 g/L和150 g/L的蔗糖浓度下萌发率都较高;经-20℃贮藏后的花粉对蔗糖浓度的敏感性要高于新鲜花粉.杂种F1代及其亲本的花粉在离体培养中均会出现双萌发管现象.番红染料对液体培养基中的花粉有致死和染色作用,有利于统计杂种F1代及其亲本的花粉萌发率.     

Silver nanoparticles biosynthesis using Saussurea costus root aqueous extract and catalytic degradation efficacy of safranin dye

Nanobiotechnology is a fast growing field in which instruments are created by nano size particles of approximately 1 to 100 nm (1 to 100 nm) of the scale of nanometers. Nanoparticles today have potential implications for life sciences and human health applications. In this research, silver nanoparticles (AgNPs) were successfully synthesized using Saussurea costus root aqueous extract and AgNPs have been characterized by the use of UV–Vis, Scanning Electron Microscopes (SEM), and Electromicroscopy of transmission (TEM) and Energy Dispersive X-ray Spectroscopy (EDXs). The highest number of particles are in the 5 to 15 nm range. AgNPs have been added in saffron dye solution for degradation dye biosynthesizing, and product analysis using UV/vision spectrophotometer, FTIR and HPLC has been performed. Green-summed AgNPs effectively degraded the color, with UV/VIS spectrophotometers, around 84.6 percent at 72 h of exposure time. The decrease in tested dye and presence of multiple new highs in the samples treated with different retention times (Rt) 2.30, 6.10 and 12.24 min, is positive for the biodegradation compared to the untreated dye with single high at 10.31 min, respectively. This green chemistry is very advantageous for AgNPs biosynthesis, for example, cost-effectiveness and usability for medicinal, pharmaceutical and extensive industrial applications. Furthermore, the bio-recovery unit for plant extracts provides a greater ease of handling, compared to micro-organisms.     

A differential staining method to identify lignified and unlignified tissues

We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.