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.

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.

Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

Three-dimensional image of hepatocellular carcinoma under confocal laser scanning microscope

AIM To investigate the application of confocallaser scanning microscopy(CLSM)in tumorpathology and three-dimensional( 3-D )reconstruction by CLSM in pathologic specimensof hepatocellular carcinoma(HCC).METHODS The 30μm thick sections were cutfrom the paraffin-embedded tissues of HCC,hyperplasia and normal liver,stained with DNAfluorescent probe YOYO-1 iodide and examinedby CLSM to collect optical sections of nuclei and3-D images reconstructed.RESULTS HCC displayed chaotic arrangementof carcinoma cell nuclei,marked pleomorphism,indented and irregular nuclear surface,andirregular and coarse chromatin texture.CONCLUSION The serial optical tomograms ofCLSM can be used to create 3-D reconstruction ofcancer cell nuclei.Such 3-D impressions mightbe helpful or even essential in making anaccurate diagnosis.

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.

Laser scanning fluorescence microscopic measurement of the movement of cleaving egg surface of Rana Amurensis

By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape, indicating dark stripe appeared in that place. Then the fluorescence intensity increased at the place where the botton of ∨ shape had located, and the scanning curve tuxned to ∧ shape, indicating single stripe was formed. While enhanced fluorescence appeared on the borders of ∧ shape, an M shape curve was found, show-ing double stripe occurred. During the distance between two borders of M shape incresing from 50 μm to 100μm,a fluorescence peak came to sight in the middle of the M shape, which being the cleavge furrow bottom. The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane. At that time the curve became W shape. By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage, showing the stretching of the egg surface being increased. With our[31, 33] and others[32] reports that polylysine could induce the appearance of nascent membrane and phyto-hemagglutinins could decrease or prevent the appearance of nascent membrane, we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stress lay to the outsides of cleavage furrow.

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.

Confocal endomicroscopy:Is it time to move on?

Confocal laser endomicroscopy permits in-vivo microscopy evaluation during endoscopy procedures. It can be used in all the parts of the gastrointestinal tract and includes: Esophagus,stomach,small bowel,colon,biliary tract through and endoscopic retrograde cholangiopancreatography and pancreas through needles during endoscopic ultrasound procedures. Many researches demonstrated a high correlation of results between confocal laser endomicroscopy and histopathology in the diagnosis of gastrointestinal lesions; with accuracy in about 86% to 96%. Moreover,in spite that histopathology remains the gold-standard technique for final diagnosis of any diseases; a considerable number of misdiagnosis rate could be present due to many factors such as interpretation mistakes,biopsy site inaccuracy,or number of biopsies. Theoretically; with the diagnostic accuracy rates of confocal laser endomicroscopy could help in a daily practice to improve diagnosis and treatment management of the patients. However,it is still not routinely used in the clinical practice due to many factors such as cost of the procedure,lack of codification and reimbursement in some countries,absence of standard of care indications,availability,physician imageinterpretation training,medico-legal problems,and the role of the pathologist. These limitations are relative,and solutions could be found based on new researches focused to solve these barriers.

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

Peculiar properties of morphological structures of organelle membranes were studied by fluorescent confocal microscopy. The list of objects in our experiments was represented by mitochondria, chloroplasts and vacuoles. During this study, identification of lipid microinclusions having the form of such lipid-protein structural microformations as lipid-protein microdomains, vesicles and membrane tubular structures (cytoplasmic transvacuolar strands and nanotubes) located in organelle membranes or bound up with them was conducted. Such membrane probes as laurdan, DPH, ANS and bis-ANS were used. Comparison of fluorescence intensity of these membrane probes was conducted. This investigation of the morphological properties of lipid-protein structural microformations was accompanied with analysis of 1) the phase state and 2) dynamics of microviscosity variations in the membrane elements of isolated plant cell organelles. Distributions of laurdan fluorescence generalized polarization (GP) values for the membrane on the whole and for the intensively fluorescing membrane segments were obtained. It was discovered that the microviscosity of intensively fluorescing membrane segments essentially differed from the microviscosity of the rest part of the membrane. In conclusion, some results of the study of peculiar properties of lipid-protein structural microformations related to the structure of organelle membranes and the discoveries made in this investigation are discussed.

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

Three-dimensional observation of the phase structure of high density polyethylene (HDPE)/poly(ethylene-co-butene) (PEB) blend by laser scanning confocal microscopy

In this paper,high density polyethylene (HDPE)/poly(ethylene-co-butene) (PEB) blend (50/50 wt%) was prepared through solution blending and then compression molding,and subsequently examined by laser scanning confocal microscopy (LSCM). The PEB used in this experiment was labeled with a small quantity of a fluorescein derivative to render fluorescence. The initial films showed uniform dye dis-tribution and no indication of phase separation within the resolution of optical microscopy. Sample films annealing at 140℃ followed by rapid cooling to room temperature showed obvious phase sepa-ration and bicontinuous structure. The present work indicates that by labeling one component with fluorescein derivative,LSCM can efficiently perform in situ depth profiling of polymer blends.

Study of bulk morphology of polymer latex films using laser confocal fluorescence microscopy

Unlabeled poly(methyl methacrylate) (PMMA) and polystyrene (PSt) latex particles as well as covalently fluorescent dye labeled PMMA latex particles (FPMMA) were prepared by emulsifier free emulsion polymerization and dispersion polymerization, respectively. The surface and bulk morphology of the polymer latex mixture (FPMMA/PMMA or FPMMA/PSt, weight ratio: 1/99) filmed by evaporation at room temperature was studied by laser confocal fluorescence microscopy (LCFM). It was concluded from the LCFM images that labeled PMMA domains obviously coagulated in PSt matrix not only on the surface but also in the inner layers of the latex film. Furthermore, PMMA was richer in the middle layers but poorer on the surface layer or the layers near to the substrate for the polymer latex films.

Advances in Probing Wood-Coating Interface by Microscopy: A Review

Surface coatings provide protection to wood products against weathering and other deteriorating factors, such as moisture uptake and microbial invasion. The effectiveness of coatings depends on many factors, including how well the applied coatings adhere to the wood surface. Coating adhesion to wood involves both chemical and physical interactions between the coating and wood tissues in contact, and the particular focus of this mini-review will be on the advances being made in understanding the physical aspects of the interaction by probing wood-coating interface using novel and high resolution imaging techniques, including confocal laser scanning microscopy (CLSM), SEM-backscattered electron imaging and correlative microscopy employing light, confocal and scanning electron microscopy.

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.

基于细胞微观形态定量的桃果实硬度变化差异性研究

为定量表征不同质地桃果实细胞微观形态及硬度变化的差异性,利用质构仪、多元显微成像结合计算机处理技术,追踪分析了贮藏期间不同质地桃果实(“美瑞”、“深州水蜜”及“金童5号”)果肉、果皮的硬度和细胞形态参数变化。结果表明,贮藏期间桃果肉、果皮硬度均呈现显著下降趋势,其中果肉硬度降低幅度(58.68%~78.20%)显著大于果皮硬度降低幅度(35.53%~65.19%),更适用于桃硬度软化表征。贮藏初期(贮藏1 d),桃果肉细胞形态规则、排列紧密,其中“深州水蜜”细胞截面积(A)最大(1500~33000μm 2);“金童5号”果实细胞圆度为0.70~0.90的细胞占比约为92.80%。随贮藏时间延长,“深州水蜜”细胞融合现象加剧,出现了部分巨大细胞(A>35000μm 2);“美瑞”细胞截面孔隙率呈现持续增长趋势,细胞出现皱缩现象;“金童5号”细胞截面周长增幅最小,细胞形变幅度最低。扫描电镜和透射电镜结果进一步印证了,贮藏期间溶质桃“深州水蜜”细胞结构最为疏松,细胞壁解聚严重,细胞质溶出最为明显;不溶质桃“金童5号”细胞结构相对完整,细胞质少量溶出;硬质桃“美瑞”细胞由圆形转变为椭圆形,且其结构改变程度介于溶质桃与不溶质桃之间。研究基于细胞形态的定量表征,明确不同质地桃果实硬度的差异性改变,旨在为基于质地差异的桃果实分等分级和定量表征桃果实细胞形态与硬度改变提供理论依据。

辛烯基琥珀酸木薯淀粉钠无醇酯化制备与表征

辛烯基琥珀酸淀粉钠(starch sodium octenyl succinate,SSOS)是以淀粉与辛烯基琥珀酸酐(octenyl succinic anhydride,OSA)酯化制得的食品添加剂。本研究采用OSA乳化法替代有机试剂分散OSA对木薯淀粉进行酯化制备辛烯基琥珀酸木薯淀粉钠(tapioca starch sodium octenyl succinate,TSSOS)并对其进行表征。结果表明:大豆卵磷脂(soybean phospholipid,SP)为乳化剂制备的OSA乳液体系具有较好的稳定性和分散性,SP添加量为1.00%制备的SSOS(1.00%SP-SSOS)比异丙醇分散OSA制备的SSOS(IPA-SSOP)具有更高的辛烯基琥珀酸基团(OS基团)含量、取代度(DS)以及取代效率(SE)。傅里叶红外光谱显示,1.00%SP-SSOS和IPA-SSOS均在1572 cm^(-1)和1726 cm^(-1)处出现了2个新的特征峰,其中1.00%SP-SSOS的特征峰强度更强。激光共聚焦扫描显微镜分析也证实了荧光标记的OS基团在1.00%SP-SSOS中荧光强度略强,少部分淀粉颗粒内部显示亦有OS基团,这说明利用高压均质以及SP乳化剂将OSA分散为微米级乳液再进行酯化,可以提高OSA与淀粉的接触面积从而提高酯化反应效率。综上所述,采用OSA乳化法替代有机试剂分散OSA制备SSOS的方法可行,本研究为无醇酯化反应体系应用在SSOS大规模工业化生产中提供参考依据。

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.

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.