Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.     

New details on the fine structure of the rhoptry of Toxoplasma gondii

Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii: the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron‐dense neck, an amorphous and less electron‐dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen‐fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo‐vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze‐fractured replicas. Cryo‐fixed, deep‐etched samples showed several pore‐like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Use of high-pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the ³¹P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T_m) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T_m. Above T_m, when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.     

Dynamic study of intramembranous particles in human fresh erythrocytes using an "in vitro cryotechnique"

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (-193 degrees C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes.     

Direct observation of liquid crystals using cryo‐TEM: Specimen preparation and low‐dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.     

New insights into the cellular makeup and progenitor potential of palatal connective tissues

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco‐gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de‐epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de‐epithelialised MSCs (deMSCs) and re‐entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony‐forming unit‐ fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA‐DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA?, CD31?) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,—in the pericapillary regions and in remote regions of the lamina propria‐ and pericytes—surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision‐making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.     

Light and electron microscopy study of the spermatheca of Eupholidoptera chabrieri bimucronata (Ramme, 1927) and Uromenus brevicollis trinacriae La Greca 1964 (Orthoptera: Tettigoniidae)

A study by both optical and electron microscopy has been carried out on the spermatheca of Eupholidoptera chabrieri bimucronata and Uromenus brevicollis trinacriae (Orthoptera: Tettigoniidae). In both the examined species, the spermatheca consists of a sac/kidney‐shaped seminal receptacle and a more or less tortuous spermathecal duct that opens into the common oviduct. The wall of both the organs consists of a pseudostratified epithelium surmounted by a cuticular intima; the latter is made up of a thicker endocuticle and an epicuticle. The epithelium shows two different cell types, irregularly arranged and with well differentiated functions: cuticle‐forming and gland cells. In both the species, the cuticle‐forming cells perform other functions, in addition to producing the cuticular intima. The gland cells never come in contact with the cuticular intima, have inside the reservoir a secretion whose appearance can diversify also in contiguous zones of the seminal receptacle. Based on our findings in both the species, the functions of the seminal receptacle would differ from those of the spermathecal duct. In the latter, some areas of the wall of the connecting tract show an activity of lysis, by contiguous epithelial cells, that could play a role in control and selection of spermatozoa. As for the feather‐shaped spermatodesms, similar in both the species, freeze‐fracture observations have shown that the acrosome of each spermatozoon regularly covers three‐quarters of the extension of the acrosome of the following spermatozoon. Finally, the significance of our findings, compared with what is known in literature, is discussed. Microsc. Res. Tech. 78:577–586, 2015. © 2015 Wiley Periodicals, Inc.     

Application of atomic force microscopy on observing the ultra-fine structure of the neoproterozoic microfossils from China

Phosphatic microfossils from the Doushantuo Formation, Guizhou, China, have been reported with preserved cellular structure or even sub-cellular structure in micron scale. However, more details in sub-micro scale have not been reported as having been found. The Fossil embryos from the acid residue of the phosphorite rocks of the Neoproterozoic Doushantuo Formation in south China have been studied with a scanning electron microscope (SEM) and an atomic force microscope (AFM). Some ultra-structures in sub-micro scale have been found by AFM on the surface of the fossil embryos. There are four types of structures found on the surface of the selected fossil embryos, the sizes of which vary from 30 to 645 nm in diameter under our AFM. One of the structures is composed of several big sub-units integrated with each other, and the size of the big sub-units is from 250 to 645 nm. Meanwhile, we also found an ultra-layer structure on the surface of the big sub-units, the thickness of which was about 10 nm. Thus we speculate that it could most probably be of biological origin. Therefore, AFM provides a new method for direct observation of the ultra-structure of the Doushantuo fossils in the sub-micro scale.     

Towards more precise definition of conditions for satisfactory deep etching

Experiments were carried out to determine whether propane-jet freezing could be as satisfactory as impact freezing in deep etch work. The material used was the intestinal brush border, previously studied by Heuser and coworkers where the 5–6 nm decoration of actin rootlet filaments, and the fine network of filaments linking these rootlets, provide good criteria by which to judge the quality of the preparation, as regards ice crystal growth and surface contamination. Propane jet freezing was indeed found satisfactory provided appropriate conditions were met (viz thin specimen, fracture near the surface). Variable results were obtained until it was realized that with a Balzers freeze-etch unit fitted with a rotating specimen table there is a 10–15 min delay (when specimen temperature is reset) between the time the recording thermocouple shows a given temperature to have been obtained and the time the specimen block actually reaches this temperature. Appropriate allowance must be made for this lag to achieve satisfactory deep etch replicas.     

New insights into wear of Ti6Al4V by ultra-high molecular weight polyethylene under water lubricated conditions

Laboratory studies indicate that sliding Ti6Al4V against soft ultra-high molecular weight polyethylene (UHMWPE) pins produces severe damage to the titanium and the lubricant (water) changes colour suggesting chemical change. Blackening of periprosthetic tissues associated with titanium wear debris was also observed in clinical investigations. To increase scientific understanding of the mechanism involved, systematic characterisation work has been conducted employing grow discharge spectrometry (composition), scanning electron microscopy (wear morphology) and cross-sectional transmission electron microscopy (phase identification). Experimental results show that hydrogen may play an important role in promoting the formation of abrasive particles in the Ti6Al4V/UHMWPE tribosystem under water lubricated conditions. The observed abnormal wear of Ti6Al4V by soft UHMWPE can be to a large extent attributed to hydrogen evolution and formation of titanium hydride. Based on experimental results and discussion, a hydrogen-assisted wear mechanism is proposed.     

起动机动力涡轮叶片断裂故障研究与改进

系统归纳了动力涡轮叶片断裂故障的基本特征,分析了叶片断裂的根本原因在于叶片的一阶弯曲共振。在弯曲共振无法避免的情况下,从提高动力涡轮叶片的疲劳抗力入手,采用细晶＋方向性凝固＋热等静压的复合成型工艺,成功地解决了这一故障。     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

Comparative fine structure of the epididymal spermatozoa from three Korean shrews with considerations on their phylogenetic relationships.

This study examined the fine structures of epididymal spermatozoa on the lesser white-toothed shrew (Crocidura suaveolens), the Japanese white-toothed shrew (C. dsinezumi) and the big white-toothed shrew (C. lasiura) belonging to the subfamily Crocidurinae living in Korea. In the spermatozoa of C. suaveolens, the head has a large acrosome, a smooth inner acrosomal membrane and a wavy, finger-like, electron-dense apical body. The neck has a solid proximal centriole that is filled with electron-dense material. These results showed the spermatozoa of C. suaveolens possess the characteristics of both Crocidurinae and Soricinae. In C. dsinezumi and C. lasiura, the head has a large acrosome, a serrated inner acrosomal membrane and a common apical body. The neck has a fistulous proximal centriole with slightly dense electron granules. These results showed the typical characteristics of Crocidurinae. Although C. suaveolens belongs to the subfamily Crocidurinae, the spermatozoan morphology is different from C. dsinezumi and C. lasiurai because it has conserved characteristics of the subfamily Soricinae.     

Raman microscopy of freeze-dried mouse eyeball-slice in conjunction with the "in vivo cryotechnique"

The wavelength of Raman-scattered light depends on the molecular composition of the substance. This is the first attempt to acquire Raman spectra of a mouse eyeball removed from a living mouse, in which the eyeball was preserved using the "in vivo cryotechnique" followed by freeze-drying. Eyeballs were cryofixed using a rapid freezing cryotechnique, and then sliced in the cryostat machine. The slices were sandwiched between glass slides, freeze-dried, and analyzed with confocal Raman microscopy. Important areas including various eyeball tissue layers were selected using bright-field microscopy, and then the Raman spectra were obtained at 240 locations. Four typical patterns of Raman spectra were electronically mapped on the specimen images obtained by the bright-field microscopy. Tissue organization was confirmed by embedding the same eyeball slice used for Raman spectra into epoxy resin and the thick sections were prepared with the inverted capsule method. Each Raman spectral pattern represents a different histological layer in the eyeball which was mapped by comparing the images of toluidine blue staining and Raman mapping with different colors. In the choroid and pigment cell layer, the Raman spectrum had two peaks, corresponding to melanin. Some of the peaks of the Raman spectra obtained from the blood vessels in sclera and the photoreceptor layer were similar to those obtained from the purified hemoglobin and rhodopsin proteins, respectively. Our experimental protocol can distinguish different tissue components with Raman microscopy; therefore, this method can be very useful for examining the distribution of a biological structures and/or chemical components in rapidly frozen freeze-dried tissue.     

New developments in electron energy loss spectroscopy

In the electron microscope, spectroscopic signals such as the characteristic X-rays or the energy loss of the incident beam can provide an analysis of the local composition or electronic structure. Recent improvements in the energy resolution and sensitivity of electron spectrometers have improved the quality of spectra that can be obtained. Concurrently, the calculations used to simulate and interpret spectra have made major advances. These developments will be briefly reviewed. In recent years, the focus of analytical electron microscopy has moved away from single spectrum acquisition to mapping and imaging. In particular, the use of spectrum imaging (SI), where a full spectrum is acquired and stored at each pixel in the image is becoming widespread. A challenge for the application of spectrum imaging is the processing of such large datasets in order to extract the significant information. When we go beyond the mapping of composition and look to map bonding and electronic structure this becomes both more important and more difficult. Approaches to processing spectrum imaging data sets acquired using electron energy loss spectroscopy (EELS) will be explored in this paper.     

脉冲电流对ZA-27合金凝固组织的影响

研究了脉冲电流对ZA-27合金凝固组织的影响。结果表明，在金属液凝固过程中对其施加脉冲电流，能够使合金的凝固组织得到改善，晶粒得到细化，枝晶间距减小。其主要原因是脉冲电流产生的脉冲压力对晶核的形成和长大产生了影响。     

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.     

具有精细结构的圆对称爱里光束的传输特性研究

为了研究具有精细结构的圆对称爱里光束的传输特性,通过减小与光斑宽度相关的参数w,使初始光场具有与波长相近的精细结构。在此特定情况下,用瑞利-索末菲衍射理论对圆对称爱里光束的传输特性进行数值计算。研究结果表明:随着参数w的减小,圆对称爱里光束的精细结构增加,使得代表高频成分的离轴光场影响逐渐增大并参与聚焦行为,焦点光强峰值增大,焦点主光斑的半高全宽减小,突然自聚焦能力得到显著提升;当w减小到某一阈值后,由于持续增加的高频分量进入到倏逝波,焦点光强峰值减小,焦点主光斑的半高全宽增大,突然自聚焦性能下降。     

新型林木生物质细粉碎机的设计

介绍了一种新型林木生物质细粉碎机的基本结构和工作原理,并对主要部件的技术参数和动力参数进行了设计。振筛结构采取与主轴同方向布置振动电机的创新设计,产生轴向振动力,有利于物料过筛,提高筛分能力。在保证粉碎粒度的前提下,同时满足了高效产业化的生产需求。     

Ultrastructural modifications of cell membranes induced by "electroporation" on melanoma xenografts

Electroporation (EP) has been widely employed in the past years as a safe and effective technique to drive drugs and DNA plasmids into target cells both for experimental and therapeutic purposes. Despite the large bulk of literature on this topic, often describing successful outcomes, there is a lack of knowledge about the intimate mechanism(s) controlling this phenomenon. In this paper, we describe a number of ultrastructural alterations in the cellular membranes following the exposure of orthotopic melanomas and red blood cells to trains of biphasic pulses. Specifically, melanoma xenografts grown in nude mice were subject to trains of eight biphasic pulses using an electric field of 1250 or 2450 V/cm, excised after 5 min and processed for electron microscopy. The freeze-fracturing analysis of both cell types evidenced defects in the dynamic assembly of lipids and proteins, which generate "areas with rough structure" and intensive clustering of intramembrane proteins. Such modifications could be the hallmarks of lipid and protein alterations, of protein cohesion reduction, and of changes in lipid orientation inside cell membranes, as postulated in several mathematical models applied to electroporation, and warrant further investigations.