Neuroprotective effects of low-concentration alpha-tocopherol Confocal laser microscopy observations

BACKGROUND： Alpha-tocopherol （ α-tocopherol） can effectively relieve neuronal damage induced by oxygen-centered free radicals. However, the effective dose remains controversial. OBJECTIVE： To evaluate the protective effects of low-concentration α-tocopherol on neuronal membranes. DESIGN, TIME AND SETTING： Contrast observation and in vitro study, performed at Laboratory of Neurosurgery, Tianjin Huanhu Hospital between April and September 2006. MATERIALS： Fetal cortical neurons were derived from two 14-day pregnant SD rats, and α-tocopherol was provided by Sigma, USA. METHODS： The neurons were randomly assigned to six groups： （1） normal： neurons were cultured under normal conditions; （2） oxidative damage： oxidative free radicals was damaged using the Fenton reaction; （3） α-tocopherol： neurons were cultured in different concentrations of -tocopherol 10, 20, 40, and 80 mg/L for 2 hours, respectively. MAIN OUTCOME MEASURES： Neuronal membrane damage was observed using a confocal laser microscope, and malonaldehyde production was detected using the thiobarbituric acid method. RESULTS： At normal, biological concentrations （10 mg/L）, α-tocopherol induced no change in the damaged neurons （P 〉 0.05）. However, at a concentration of 80 mg/L, the number of damaged neurons was significantly reduced, compared with the damage group （P 〈 0.05）. Malonaldehyde levels following 80 mg/L α-tocopherol treatment were less than the oxygen free radical damage group （P 〈 0.05）, but greater than the control group （P 〈 0.01）. CONCLUSION： A concentration of 80 mg/L α-tocopherol can effectively protect the neuronal cell membrane from oxidative damage

Dissolution behavior of Al_(2)O_(3)inclusions into CaO-MgO-SiO_(2)-Al_(2)O_(3)-TiO_(2)system ladle slags

To investigate the dissolution behaviors of Al_(2)O_(3)inclusions in CaO-5wt%MgO-SiO_(2)-30wt%Al_(2)O_(3)-TiO_(2)system ladle slags,confocal scanning laser microscopy was conducted on the slags with different TiO_(2)contents(0-10wt%),and scanning electron microscopy was performed to study the interfacial reaction between Al_(2)O_(3)and this slag system.The results disclose that the dissolution of Al_(2)O_(3)inclusions does not result in the formation of new phases at the boundary between the slag and the inclusions.In TiO_(2)-bearing and TiO_(2)-free ladle slags,there is no difference in the dissolution mechanism of Al_(2)O_(3)inclusions at steelmaking temperatures.Boundary layer diffusion is found as the controlling step of the dissolution of Al_(2)O_(3),and the diffusion coefficient is in the range of 4.18×10^(-10)to 2.18×10^(-9)m^(2)/s at 1450-1500℃.Compared with the solubility of Al_(2)O_(3)in the slags,slag viscosity and temperature play a more profound role in the dissolution of Al_(2)O_(3)inclusions.A lower viscosity and a lower melting point of the slags are beneficial for the dissolution.Suitable addition of TiO_(2)(e.g.,5wt%)in ladle slags can enhance the dissolution of Al_(2)O_(3)inclusions because of the low viscosity and melting point of the slags,while excessive addition of TiO_(2)(e.g.,10wt%)shows the opposite trend.

Effect of melatonin on the spatial and temporal changes of [Ca^(2+) ]i in single living cells of cortical neurons by laser scanning confocal microscopy

Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order to explore the possible antiaging mechanisms of melatonin (MT) Methods Using the highly fluorescent Ca 2+ sensitive indicator Fluo 3/AM, cortical neurons cultured in a 35?mm Tissue Culture Dish were in incubated for 45?min at room temperature with 5?μmol/L Fluo 3/AM, resulting in proper intracellular dye concentration to provide adequate signal strength for detection and excellent Laser Scanning Confocal Microscopy (LSCM) imaging of [Ca 2+ ]i while not disturbing normal intracellular physiology The changes in fluorescent intensity were monitored by LSCM Results Bay K8644 (10 6 ?mol/L), KCl (20 ?mmol/L), sodium L glutamate (Glu, 50?μmol/L) caused a rapid increase of [Ca 2+ ]i in cortical neurons, and this increase could be significantly attenuated by 10 6 and 10 7 mol/L MT Conclusions MT could antagonize the extracellular Ca 2+ influx, reduce Ca 2+ overload, and have a protective effect on neurons This may be one of the important antiaging mechanisms of MT

Single Particle-Based Confocal Laser Scanning Microscopy for Visual Detection of Copper Ions in Confined Space

Main observation and conclusion A single particle-based confocal laser scanning microscopy was developed for the visual detection of copper ions in confined space.A fluorescence microparticle,named AuNCs/ZIF-8,was synthesized by coating gold nanoclusters(AuNCs)onto the outer surface of zeolitic imidazolate framework-8(ZIF-8).

Laser confocal microscopy findings of Thygeson superficial punctate keratitis

Thygeson superficial punctate keratitis （TSPK） is a rare,chronic,inflammatory disorder,with adverse impact on the visual function and quality of life in patients.It was first described by Thygeson in 1950,1 as typically bilateral,corneal epithelial opacities without associated stromal involvement or corneal edema.Recurrent episodes of tearing,foreign body sensation,photophobia,and reduced vision are observed.To date,virus infection and immune factors have been considered as important risk factors for visual deterioration in the patients with TSPK,but the origin of the comeal opacities remains poorly understood.Keywords：Thygeson superficial punctate keratitis ; laser confocal microscopy

Large-field objective lens for multi-wavelength microscopy at mesoscale and submicron resolution

Conventional microscopes designed for submicron resolution in biological research are hindered by a limited field of view,typically around 1 mm.This restriction poses a challenge when attempting to simultaneously analyze various parts of a sample,such as different brain areas.In addition,conventional objective lenses struggle to perform consistently across the required range of wavelengths for brain imaging in vivo.Here we present a novel mesoscopic objective lens with an impressive field of view of 8 mm,a numerical aperture of 0.5,and a working wavelength range from 400 to 1000 nm.We achieved a resolution of 0.74μm in fluorescent beads imaging.The versatility of this lens was further demonstrated through high-quality images of mouse brain and kidney sections in a wide-field imaging system,a confocal laser scanning system,and a two-photon imaging system.This mesoscopic objective lens holds immense promise for advancing multi-wavelength imaging of large fields of view at high resolution.

IN SITU OBSERVATION OF GROWTH BEHAVIOR AND MORPHOLOGY OF DELTA-FERRITE AS FUNCTION OF SOLIDIFICATION RATE IN AN AISI304 STAINLESS STEEL

It was presented the in situ observation of growth behavior and morphology of delta-ferrite as a function of solidification rate in an AISI304 stainless steel. The specimens have been solidified and observed using confocal scanning laser microscopy (CSLM). The δ-phase always appears like cells on the sample surface when critical supercooling occurs, during which the L→δ transformation starts. The solid-liquid (S-L) interface is found to be finger shaped and has no faceted shape. γ phase appears among δ grains due to partitioning of Ni into the melt during solidification, when solidification rate is higher. The mergence of observed δ cells is possible for the steel sample cooled at 7.5℃/min. The formation of dendrites can be observed on the free surface of the steel sample cooled at 150℃/min. The size of solidified delta grains decreases from 120 to 20-80μm, and the volume fraction of solidified austenite increases with increase in solidification rate from 7.5 to 150℃/min. The relation between the tip radius of δ cell and its growth rate is deduced, and the results agree with the experimental values.

Changes of meibomian glands in patients with type 2diabetes mellitus

AIM： To investigate the morphological changes of meibomian glands in patients with type 2 diabetes mellitus （DM）.METHODS： Of 118 eyes （118 patients） with type 2 DM （DM group） and 100 eyes of 100 control subjects （control group） were enrolled. After completing an ocular surface disease index （OSDI） questionnaire, the non-invasive tear film break-up time （NI-BUT） and the structure of the meibomian glands （MGs, meibography） were assessed by the Keratograph 5M system. Partial or complete loss of MG was scored for each eyelid from grade 0 （no loss） to grade 3 （lost area was 〉2/3 of the total MG area）, which were also examined by laser scanning confocal microscopy （LSCM）. The primary outcomes were meibomian gland acinar unit density （MGAUD）, meibomian gland acinar longest diameter （MGALD） and meibomian gland acinar shortest diameter （MGASD）.RESULTS： Compared with control group, the OSDI was significantly higher in DM group （Z=-5.916; P〈0.001）, while the NI-BUT was significantly lower （Z=-7.765; P〈0.001）. Keratograph showed that there were more MGs dropout in DM group than that in control group. The meiboscore was significantly higher in DM group compared with control group （Z=-3.937; P〈0.001）. LSCM revealed that there were cytological alterations of MGs in DM group compared with control group, which included enlargement of MG acinar units and decreased in density of MG acinar units. Specifically, there were lower MGAUD, larger MGALD and MGASD in DM group than control group （Z=-10.120, -9.4442, -7.771; P〈0.001）.CONCLUSION： Compared with the normal control participants, the patients with type 2 DM had more unstable tear films and severe symptoms of dry eye. Using Keratograph 5M system and LSCM, we found that the patients with type 2 DM had more significant morphological and cytological changes and dysfunction in MGs.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Lead Can Inhibit NMDA-,K^+-,QA/KA-Induced Increases in Intracellular Free Ca^(2+) in Cultured Rat Hippocampal Neurons

Objective To examine the effects of Pb2+ on N-methyl-D-aspartate (NMDA)-, K+- and quisqualate(QA)/kainite(KA)-induced increases in intracellular free calcium concentration ([Ca2+],) in cultured fetal rat hippocampal neurons in order to explain the cognitive and learning deficits produced by this heavy metal. Methods Laser scanning confocal microscopy was used. Results The results clearly demonstrated that adding Pb2+ before or after NMDA/glycine stimulation selectively inhibited the stimulated increases in [Ca2+], in a concentration-dependent manner. In contrast, Pb2+ treatment did not markedly affect increases in [Ca2+], induced by an admixture of QA and KA. The minimal inhibitory effect of Pb2+ occurred at 1 μmol/L, and more than seventy percent abolition of the NMDA-stimulated increase in [Ca2+]; was observed at 100 Jμmoll/L Pb2+. Evaluation of Pb2+-induced increase in [Ca2+], response to elevating extracellular concentrations of NMDA, glycine or calcium revealed that Pb2+ was a noncompetitive antagonist of both NMDA and glycine, and a competitive antagonist of Ca2+ at NMDA receptor channels. In addition. Pb2+ inhibited depolarization-evoked increases in [Ca2+], mediated by K+ stimulation(30μmol/L). indicating that Pb2+ also depressed the voltage-dependent calcium channels. Also, the results showed that Pb2+ appeared to be able to elevate the resting levels of [Ca2+|, in cultured neurons, implying a reason for Pb2+-enhanced spontaneous release of several neurotransmitters reported in several previous studies. Conclusion Lead can inhibit NMDA-. K+-, QA/KA-jnduced increases in intracellular [Ca2+], in cultured hippocampal neurons.

Interference study on the free calcium in the human peripheral lymphocyte by acrylamide

The role of acrylamide on the human peripheral lymphocytes was studied by laser scanning confocal microscopy technique and fluo-3. The calibration value of the apparent dissociation constant （Kd） of the fluo-3-Ca^2＋ complex was obtained as 4.83 × 10^-7 moi/L. Acrylamide （〈54 μg/mL） evoked a rise in free intracellular calcium concentration [Ca^2＋]i, in a dosedependent manner. Acrylamide induced the increase of [Ca^2＋]i was discussed in detail.

Continuous live cell imaging of cellulose attachment by microbes under anaerobic and thermophilic conditions using confocal microscopy

Live cell imaging methods provide important insights into the dynamics of cellular processes that cannot be derived easily from population-averaged datasets.In the bioenergy field,much research is focused on fermentation of cellulosic biomass by thermophilic microbes to produce biofuels;however,little effort is dedicated to the development of imaging tools to monitor this dynamic biological process.This is,in part,due to the experimental challenges of imaging cells under both anaerobic and thermophilic conditions.Here an imaging system is described that integrates confocal microscopy,a flow cell device,and a lipophilic dye to visualize cells.Solutions to technical obstacles regarding suitable fluorescent markers,photodamage during imaging,and maintenance of environmental conditions during imaging are presented.This system was utilized to observe cellulose colonization by Clostridium thermocellum under anaerobic conditions at 60℃.This method enables live cell imaging of bacterial growth under anaerobic and thermophilic conditions and should be widely applicable to visualizing different cell types or processes in real time.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

COMPLEX OPTICAL CHARACTERIZATION OF MESHIMPLANTS AND ENCAPSULATION AREA

Complex investigation of mesh implants was per formed involving laser confocal microscopy,backscattered probing and OCT imaging methods.The growth of endomysium and fat tissue with microcirculation vessels was observed in the mesh encapsulation region.Confocal microscopy analysis shows that such pat hologies complications such as necrosis formation and microcavities were localized in the area near implant fibers with the size compatible with fiber diameter.And the number of such formnations increase with the increase of the size,number and density of microdefects on the implant surface.Results of mumerical sinulations show that it is possible to control implant installation up to the depth to 4mm with a help of bac kscattering probing.The applicability of OCT imaging for mesh implant control was demonstrated.Special two stage OCT image noise reduction algorithm,including empirical mode decomposition,was proposed for contrast increase and better abnormalities visualization by halving the signal-to-noise ratio.Joint usage of backscattered probing and OCT allows to accurately ascertain implant and surrounding tissue conditions,which reduces the risk of relapse probability.

Rice Protoplasts Isolated Directly from Mature-Embryo-Derived Calli

Mature-embryo-derived calli or japonica rice (Oryza sativa L)Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisractory yield of 5. 20×106～8. 96× 106 protoplasts/g FW(fresh weight).The remaining three callus lines Initiated from seeds of cryopreserved- callus-derived plants had rooty calli, resulting in low yield or protoplasts and a large number or isolated banana- shape intact cells. Viability or protoplasts ranged 87.46% ～94.15%.The average size or Protoplasts was 207. 49～379. 04μm2 in different callus lines.Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts.

Comparative Analysis of Bacillus thuringiensis Vip3Aa57 and Vip3Aa62 Insecticidal Activities

Bacillus thuringiensis(Bt)exhibits strong insecticidal activity and is harmless to non-target organisms such as human and animals.Bt becomes the most commonly used environment-friendly insecticidal microorganism.However,the insecticidal activities of different Bt strains variy significantly.Therefore,it is particularly important to compare the insecticidal activities of different strains and explore their insecticidal effector mechanisms to expand Bt insecticidal spectrum and enrich transgenic resources.Here,the insecticidal activities of Vip3Aa57 and Vip3Aa62 strains,carrying vegetative insecticidal protein-encoding genes that were inserted into the expression vector pET-21b and transformed into Escherichia coli Rosetta(DE3)strain,expressing 88 ku protein,were compared.Vip3Aa57 protein reportedly displayed body weight inhibition effect on Spodoptera exigua without affecting Heliothis armigera while Vip3Aa62 protein was known to have strong insecticidal activity against S.exigua(LC50=5.124 ng•mg^(-1)).A low H.armigera activity(LC50=870.1 ng•mg^(-1))was observed.The paraffin sectioning results showed that Vip3Aa57 protein affected S.exigua midgut cell morphology.The laser confocal microscopic imaging results showed that Vip3Aa57 bound to receptors in the midgut without damaging the midgut tissue morphology.This study would be conducive for making full use of Bt strains in the soil to compare the insecticidal activities of different Vip insecticidal genes.It could thus provide significant help in revealing the underlying insecticidal mechanisms of Vip3Aa insecticidal genes,developing new insecticidal proteins and delaying pest resistance problems.

Combined quantitative microscopy on the microstructure and phase evolution in Li1.3Al0.3Ti1.7(PO4)3 ceramics

Lithium aluminum titanium phosphate(LATP)is one of the materials under consideration as an electrolyte in future all-solid-state lithium-ion batteries.In ceramic processing,the presence of secondary phases and porosity play an important role.In a presence of more than one secondary phase and pores,image analysis must tackle the difficulties about distinguishing between these microstructural features.In this study,we study the phase evolution of LATP ceramics sintered at temperatures between 950 and 1100℃by image segmentation based on energy-dispersive X-ray spectroscopy(EDS)elemental maps combined with quantitative analysis of LATP grains.We found aluminum phosphate(AlPO4)and another phosphate phase((Lix)PyOz).The amount of these phases changes with sintering temperature.First,since the grains act as an aluminum source for AlPO4 formation,the aluminum content in the LATP grains decreases.Second,the amount of secondary phase changes from more(Lix)PyOz at 950℃to mainly AlPO4 at 1100℃sintering temperature.We also used scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM)to study the evolution of the LATP grains and AlPO4,and LATP grain size increases with sintering temperature.In addition,transmission electron microscopy(TEM)was used for the determination of grain boundary width and to identify the amorphous structure of AlPO4.

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice

Embryo sac abortion is one of the major reasons for sterility in indicaljaponica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

In situ observation and thermodynamic calculation of MnS in 49MnVS3 non-quenched and tempered steel

Based on thermodynamics calculation, the results of the formation temperature of MnS inclusions of non-quenched and tempered steel during heating process were dis- cussed. It is shown that while the solid fraction is 0.9, MnS inclusions began to precipitate in the final stage of solidification. The solidification process of 49MnVS3 non-quenched and tempered steel during heating has been observed in situ using a confocal scanning laser microscope （CSLM）, which agrees well with the thermody- namics calculation. MnS particles were coarsening during heating process, which would reduce the pinning effect on the austenite grain boundaries and bring about the ~11rtden ~r^wth ~f ,qnrn~ ~11,qtenif.~ ~r^ins in thi~ st~.g~.