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).

Evaluation of the intracellular trafficking of siRNAs in A375 cells by confocal laser scanning microscopy

Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy （CLSM） method to evaluate a novel delivery system of 3＇-peptide-siRNA therapeutic, which was named 3＇-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3＇-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3＇-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.

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.

Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.

Revealing the F_actin Networks in Interphase Nuclei of Garlic Clove Cells by Confocal Fluorescence Microscopy

The interphase nuclei of parenchyma cells and epidermal cells of garlic ( Allium sativum L.) clove were labelled with rabbit anti_actin antibody and FITC_conjugated goat anti_rabbit IgG antibody. The authors observed results with fluorescence microscopy and confocal laser scanning microscopy. The nuclei showed prominent green_yellow fluorescence, indicating the presence of actin in the nuclei. Fluorescence examination with TRITC_phalloidin showed distinctive red fluorescence in the nuclei, indicating that F_actin is present in the nuclei. Confocal laser scanning microscopy indicated the presence of F_actin containing network structures in the nuclei, but the network structures were absent and the nuclei still showed red fluorescence when the cells were treated with cytochalasin D before fixation; the red fluorescence in the nuclei was hard to be observed when the cells were treated with unlabelled phalloidin before the cells were stained with TRITC_phalloidin. These results indicate that F_actin is in the nuclei and forms network structures in the nuclei of garlic cells.

Changes in physicochemical characteristics of wheat flour and quality of fresh wet noodles induced by microwave treatment

Fresh wet noodles(FWN) are popular staple foods due to its unique chewy texture and favorable taste. However,the development of FWN is limited by its short shelf life and high browning rate. It has been found that the quantity of original microorganisms in wheat flour produced by traditional method is relatively high, which is detrimental to the processing quality and storage stability of FWN. Consequently, it becomes imperative to decrease microorganisms in wheat flour. Microwave treatment has been regarded as a promising method in the food industry due to its potential in inhibiting microbial growth and inactivating enzymes without causing adverse effect on the food quality. This study aims to investigate the effects of microwave treatment of wheat kernels under different powers(1, 2, 3, 4, 5 kW) on the physicochemical properties of wheat flour and the quality of FWN. The results revealed that microwave treatment had a significant effect on microbial inhibition and enzyme inactivation, wherein the total plate count(TPC) and yeast and mold counts(YMC) decreased by 0.87 lg(CFU/g) and 1.13 lg(CFU/g) respectively, and PPO activity decreased from 11.40 U to 6.31 U. The dough quality properties, such as stability, extensibility, and starch viscosity, improved significantly under different microwave conditions. Confocal laser scanning microscopy(CLSM) images indicated that starch and proteins aggregated gradually in treated flour, altering rheological properties of dough. From the results of scanning electron microscopy(SEM), microwave treatment led to the appearance of disrupted structure in the gluten proteins, but the secondary structure of proteins altered slightly. Rheological properties of dough confirmed that the microwave treatment greatly affected processing characteristics of wheat flour products, with significant advantageous consequences on product quality, especially for textural properties of FWN. Furthermore, FWN darkening could be inhibited noticeably after microwave treatment, thereby prolonging its shelf life. Therefore, microwave treatment could thus be an effective, practical technology to produce low-bacterial flour and thereby enhance its product quality.

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

Colonization Pattern of Azospirillum brasilense Yu62 on Maize Roots

Plasmid pVK1001 which carried the gfp gene of GFPmut2, a mutant of GFP, was introduced into Azospirillum brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled baeteria and grown gnotobiotically in flask with semi-solid agar medium. Observations were performed with confocal laser scanning microscopy (CLSM) and electron microscopy, respectively, at 8 d and 12 d after inoculation. Confocal laser scanning microscopy showed that A. brasilense Yu62 could penetrate into the cortex tissue, colonizing in the intercellular spaces of the parenchyma cells of the cortex tissue. Transmission and scanning electron microscopy (TEM) showed that the majority of the bacteria colonized on the root surface and only a minority of them resided in the root interior.

Evidence of Ultrastructure and Physiology of F-actin as Component of Plasmodesmata

The characters and ultrastructure of the intercellular connection were revealed in the outer epidermis of the garlic clove sheath by means of fluorescent probe TRITC_Phalloidin (TRITC_Ph) labeling combined with confocal laser scanning microscopy (CLSM), immuno_gold labeling and transmission electron microscopy. These results show that transcellular channel is a complex of rod_like cytoplasm channel and grouped plasmodesmata (PDs) in pit. The former remains a portion of the cell protoplast. The diameter of PD is normally 60-70 nm. The PDs are the real intercellular symplasmic connections of the cells. The transcellular fibers labeled with the TRITC_Ph obviously become narrow in the primary pit fields, which is the same as the characters observed under the electron microscope. The bright fluorescent spot in the primary wall reflects the grouped PDs in pit, and hence the presence of F_actin in the PDs can be confirmed. In immuno_gold labeling experiment, a lot of gold particles were massively distributed in the rod_like cytoplasm channel and grouped PDs. The result provides effective support that these fluorescent filaments possibly are the existing form of F_actin.

Changes of the Microtubule Arrays During Mitosis in Prothallus Cells of Dryopteris crassirhizoma

Microtubule arrays in prothalli large-vacuolated and meristematic dividing cells of the fern Dryopteris crassirhizoma Nakai were studied using Steedman's wax, indirect immunofluorescence labelling and confocal laser scanning microscopy. Results showed that the use of high paraformaldehyde concentration (8%) allowed good fixation of prothallus cells, which are characterized by numerous (meristematic cells) and big (large-vacuolated cells) vacuoles. Results also plead for the efficiency of Steedman's wax embedding method in: (1) avoiding excessive use of enzyme for digesting cell wall in the process of the microtubule cytoskeleton labelling, (2) minimizing the autofluorescence effect in cells through utilization of alcohol in sample dehydration, and (3) permitting a clear visualization of microtubule patterns during the cell mitosis. Steedman's wax, coupled with immunofluorescence labelling and confocal laser scanning microscopy techniques, allows a good investigation of cell division process in plants by using simple multicellular organisms such as fern prothalli.

Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ 11 dextranase mouthwash

We adopted the response surface methodology using single factor and orthogonal experiments to optimize four types of antimicrobial agents that could inhibit biofilm formation by Streptococcus mutans, which is commonly found in the human oral cavity and causes tooth decay. The objective was to improve the function of marine Arthrobacter oxydans KQll dextranase mouthwash （designed and developed by our laboratory）. The experiment was conducted in a three-level, four-variable central composite design to determine the best combination of ZnSO4, lysozyme, citric acid and chitosan. The optimized antibacterial agents were 2.16 g/L ZnSO4, 14 g/L lysozyme, 4.5 g/L citric acid and 5 g/L chitosan. The biofilm formation inhibition reached 84.49%. In addition, microscopic observation of the biofilm was performed using scanning electron microscopy and confocal laser scanning microscopy. The optimized formula was tested in marine dextranase Arthrobacter oxydans KQ11 mouthwash and enhanced the inhibition of S. mutans. This work may be promoted for the design and development of future marine dextranase oral care products.

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.

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.

Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl-2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there’s a change of intracellular calcium distribution, moving from cytoplast especially Golgi’s apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi’s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi’s apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspase-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

Effect of solanine on the membrane potential of mitochondria in HepG_2 cells and [Ca^(2+)]i in the cells

AIM： To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca^2＋]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.METHODS： HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy （LCSM）. HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca^2＋]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca^2＋]i in the cells were observed using LCSM.RESULTS： Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in the cells at the same time as it lowered the membrane potential of mitochondria.CONCLUSION： Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca^2＋ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca^2＋ in the cell, turning on the mechanism for apoptosis.

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.

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.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.