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.

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.

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.

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.

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.

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

High concentration of calcium ions in Golgi apparatus

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.

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.

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.

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。

扫描电镜原位激光加热与高温成像研究

扫描电子显微镜(SEM)原位研究材料高温组织与力学性能是近年来材料领域关注的一个重要方向,在扫描电子显微镜内进行原位加热实验可以直接揭示材料微观组织、力学性能与温度之间的关系。然而传统的电流加热方式在样品温度超过800℃时金属材料会释放出大量热电子,无法获得高质量图像。本研究开发了一种与SEM联用的激光加热台,该加热台使用激光作为加热源,可以对材料进行快速加热以及高分辨原位结构表征。本文详细介绍了激光加热台的设计原理并对加热台温度场进行有限元仿真模拟及分析,以镍基单晶高温合金为例,实现了1370℃原位高温成像,验证了该装置的主要功能。

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.

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.

Laser induced self-propagating high-temperature synthesis of TiNi alloy

TiNi alloy, especially porous TiNi, a good biocompatible material, can be made by laser induced self-propagating high-temperature synthesis (SHS). A 40-W CO2 laser was used to ignite the powders of Ti and Ni, and TiNi intermetallic compound was synthesized by SHS in a reaction kettle of stainless steel. High-speed photography, X-ray diffraction, and scanning electron microscopy were used to investigate and analyze the reaction process, phase composing, and microstructure of the product, respectively. The influence factors on the reaction process and the product were discussed. The results indicate that laser induced SHS is an efficient, energy-saving method; The phase ingredient of the product consists of TiNi, Ti2Ni, and Ni3Ti. With the increase of the preparing pressure of the sample, the reacting rate decreases; With the increase of the laser power and the preheating temperature, the reacting rate increases. Under the condition of 30℃/min, the synthesis reaction had been carried out consistently and completely.

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2+) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .

Cr含量对1000 MPa级高强钢熔敷金属组织演变的作用机制

设计开发了5种不同Cr含量的1000 MPa级高强钢埋弧焊材,采用丝极埋弧焊制备了Cr含量为0%~0.9%的熔敷金属,利用OM、SEM、TEM和CLSM等微观组织表征方法研究Cr含量对1000 MPa级高强钢熔敷金属组织演变的影响,并通过拉伸和冲击试验评估其力学性能.结果表明,1000 MPa级高强钢熔敷金属随着Cr含量提高,铁素体转变起始温度由723.3℃提高到740.2℃,贝氏体转变起始温度由470.2℃降低到458.5℃,铁素体转变温度区间扩大,高Cr熔敷金属中贝氏体转变速率较快,熔敷金属中针状铁素体和贝氏体铁素体增加,M-A组元由弥散分布逐渐呈链条状偏聚,先共析铁素体与残余奥氏体减少.熔敷金属冲击断裂形式由低Cr的韧性断裂向高Cr的准解理断裂转变,熔敷金属拉伸强度不断提高,相比于无Cr的熔敷金属,0.9%Cr的熔敷金属抗拉强度和屈服强度分别提高12.4%和17.0%,熔敷金属冲击吸收能量先提高后降低,在−40℃条件下,0.6%Cr的熔敷金属的低温韧性最高为84 J,熔敷金属Cr含量为0.6%时强韧性匹配效果最佳.

高温对不同粒径花岗岩剪切特性影响研究

在干热岩水力压裂过程中,高温会弱化热储岩体裂隙壁面强度从而表现出裂隙错动的行为特征,而这种弱化现象与黏聚力和内摩擦角的热变特性相关,因此,了解花岗岩在高温下的力学特性对于干热岩开发过程中热储岩体的稳定性评价具有重要意义。在不同高温条件下进行了粗、细颗粒花岗岩变角剪切试验(45°、55°、65°),分析了高温对不同粒径花岗岩黏聚力和内摩擦角的影响,结合扫描电镜与低场核磁共振试验,进一步研究了热力耦合作用对花岗岩微裂纹与孔隙结构演化规律的影响。研究结果表明:花岗岩的黏聚力随温度升高呈现先增加后降低的趋势,内摩擦角变化规律受温度影响较小,300℃为花岗岩黏聚力发生转化的阈值温度。在温度阈值前,粗颗粒花岗岩黏聚力增幅约为细颗粒花岗岩的5.73倍,温度强化效应对粗颗粒花岗岩影响更为显著。随着温度的升高,花岗岩微裂纹形式由晶内裂纹向沿晶裂纹发展。核磁共振T2谱曲线存在多个间断性峰值,温度升高使粗颗粒花岗岩大孔隙受到压实作用,导致弛豫效应减弱,而热力耦合作用对细颗粒花岗岩大孔隙发展影响有限。该研究结果有望为干热岩热储岩体稳定性评价提供参考。

同源四倍体水稻胚乳发育:极核融合和胚乳细胞化

利用激光扫描共聚焦显微术和塑料包埋半薄切片技术观察同源四倍体水稻极核融合及游离核进一步发育的过程 ,发现同源四倍体水稻约有 1 /3子房极核融合过程和游离核进一步发育与其二倍体原种的基本一致 ;2 /3子房出现各种异常现象 ,如极核未受精、受精异常和胚乳游离核发育滞后等。应用荧光增白剂染色对胚乳细胞化过程进行观察 ,直接观察到胚乳初生壁的出现和形成过程以及不同部位的胚乳细胞壁的形成机理。表明同源四倍体水稻与二倍体水稻胚乳的细胞化过程基本一致 ,胚乳细胞初始垂周壁来源于珠孔端胚囊壁内突向心游离生长 ,初始平周壁来源于游离核有丝分裂产生的成膜体 ,而中间胚乳细胞壁则有些来源于游离核有丝分裂产生的成膜体 ,有些是自由生长而来。同源四倍体水稻胚乳细胞化过程也出现相当比例的异常现象 ,如无细胞壁形成、细胞化不同步和产生分枝状的细胞壁等。同源四倍体水稻胚乳发育过程中出现的极核融合。