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.

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

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).

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.

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.

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。

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.

LSM 880 with Airyscan激光扫描共聚焦显微镜高级功能和管理

Zeiss LSM 880 with Airyscan是一款多功能、高分辨率、高效率的激光扫描共聚焦显微镜(以下称激光共聚焦显微镜)。结合使用和管理的经验,本文对激光共聚焦显微镜的原理、LSM 880 with Airyscan的高级功能及日常维护管理等内容进行详细介绍。列举了Airyscan、长时间多视野拍摄、Experiment Designer、荧光漂白后恢复(FRAP)、光谱扫描/拆分、荧光能量共振转移(FRET)、Tile Scan和Line Scan快速成像技巧等高级功能,供管理人员或同行使用者借鉴参考。

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.

苦豆子总碱二元醇脂质体制备及体外透皮特性研究

目的在制备苦豆子总碱脂质体的过程中,加入两种水相混溶渗透促进剂:乙醇和丙二醇,以获得新的、稳定的透皮给药系统,增强苦豆子总碱(SATA)的皮肤传递。方法注入法制备二元醇脂质体,并对其形态,大小,Zeta电位进行表征,包封率、囊泡稳定性和皮肤渗透特性为优化乙醇和丙二醇配比的重要考察因素,采用Franz扩散池对小鼠皮肤进行体外皮肤渗透实验,比较小鼠皮肤的累积渗透量。用激光共聚焦扫描电镜观察含有荧光素罗丹明B的苦豆子总碱二元醇脂质体在离体小鼠皮肤中的穿透深度及荧光强度。结果当乙醇∶丙二醇=7∶3(w/w)时,二元醇脂质体的稳定性最好、包封率最高(89.13±0.42)%,粒径最小(103.00±16.15)nm,透皮深度(170μm)最大和最强的荧光强度(Max FI=160 AU)。结论制备的苦豆子总碱二元醇脂质体(乙醇∶丙二醇=7∶3,w/w)是一种高效且稳定的经皮传递载体。

激光扫描共聚焦显微分析技术表征页岩亚微米级孔隙中的含油性——以准噶尔盆地芦草沟组页岩为例

亚微米级孔隙及其含油性是页岩油勘探开发所需的重要信息。为应用激光扫描共聚焦显微镜有效观测亚微米级孔隙的含油性,修改了激光扫描共聚焦显微镜主分光器和检测器的前置滤光片配置,使之能较好地进行反射光和荧光联合扫描,消除矿物所发荧光的干扰,实现了亚微米级孔隙及其含油性的精确观测。该方法在准噶尔盆地吉木萨尔凹陷芦草沟组页岩油研究中进行了应用,发现了大量的亚微米级含油孔隙;页岩油多呈不规则形状、斑块状和星点状。这套页岩的有机质成熟度Ro为0.6%~1.1%,平均约为0.9%,但轻、重质组分的分布具有显著的非均一性,不完全受成熟度控制。在有些部位,轻质组分含量及其与重质组分的比值均较高,但在成熟度相近甚至稍高的邻近部位,却存在以重质组分为主的情况。页岩层系内部的油气运移也是一个重要的控制因素。页岩中的轻质和重质组分的观测与分布规律研究,对于页岩油勘探开发十分重要,激光扫描共聚焦显微新技术可成为重要的观测手段。

激光共聚焦显微法测定面团面筋网络结构的影响因素

【目的】面团样品切片、混合染色剂浓度以及染色时间是激光共聚焦显微镜测定面团面筋网络结构的关键影响因素。本研究旨在分析这些因素对面团面筋网络结构测定结果的影响规律,进而优化出最佳的切片厚度和染色条件,为面团面筋网络结构及面制品品质高质量研究提供技术支撑。【方法】选取面筋筋力不同的两个小麦品种郑麦366(Z366)和小偃22(X22)为试验材料,磨粉制作成面团,并分别冷冻0 d和1 d;用冷冻切片机将面团分别切成10、14和20μm厚度的薄片,并配制不同浓度罗丹明B(RDB)和异硫氰酸荧光素(FITC)混合染液,分别染色1、10和20 min,分析这些因素对激光共聚焦显微法(CLSM)测定面团面筋网络结构的影响。【结果】切片厚度为10μm时,郑麦366(Z366)和小偃22(X22)面团的面筋网络结构均最清晰,且在此切片厚度下,两个品种冷冻0 d和冷冻1 d的面团面筋网络结构之间的差异均最显著(P<0.01)。染色条件研究结果表明,低浓度染色剂(0.001%+0.01%)染色时间越长,面筋蛋白网络结构呈现越清晰;高浓度染色剂(0.025%+0.25%)在染色1 min时,面筋网络结构呈现最佳,染色10 min时,面筋网络结构最差。【结论】面团切片厚度10μm,不同筋力和不同冷冻处理的面团网络结构差异最大,即面筋网络结构区分效果最明显;在此切片厚度条件下,当不考虑时间成本时,运用低浓度混合染色剂染色20 min面筋网络结构呈现效果最佳;当需要节省时间并观察淀粉颗粒细微结构时,使用高浓度染色剂染色1 min效果最好。

基于点扫描的高时空分辨荧光显微成像技术进展

激光点扫描荧光显微镜凭借高分辨率、高灵敏度、高特异性、可光学层切三维成像和动态成像等优势,已成为生命科学研究中广泛应用的重要工具.随着研究者对生命科学研究的逐渐深入,由于受光学衍射极限影响和逐点扫描探测的限制,传统点扫描共聚焦显微镜的时空分辨率已无法完全满足相关研究需求,在实际应用中面临诸多挑战.近二十年来,超分辨荧光显微成像技术取得了重要进展,研究人员发展了多种高空间分辨率、高时间分辨率的点扫描荧光显微成像技术,对于生物成像等具有重要意义.然而,有关该领域最新进展的综述论文较少,系统地讨论、总结基于点扫描的高时空分辨荧光显微成像技术的研究进展,对于其未来研究发展极为重要.本文主要从时间分辨率与空间分辨率两个维度分别介绍了各类点扫描荧光显微成像技术的基本原理及相关进展,并介绍了高时空分辨显微成像技术及应用,最后讨论展望了高时空分辨点扫描荧光显微镜的未来发展趋势和挑战.

生物浸出过程中黄铁矿表面胞外多聚物和铁离子的分布及含量变化

微生物-矿物界面是生物浸出的主要生化反应场所,界面作用机制是阐明微生物浸矿行为的关键。本文通过Acidithiobacillus caldus、Leptospirillum ferriphilum和Sulfobacillus thermosulfidooxidans三种中度嗜热混合菌对黄铁矿颗粒和黄铁矿切片分别进行了生物浸出实验和生物膜的定殖实验,采用高选择性和高灵敏性的金属荧光探针结合激光共聚焦显微镜、扫描电子显微镜和物理提取法对生物浸出过程中界面胞外聚合物(EPS)和铁离子的分布及其含量变化进行了研究。结果表明,EPS平铺在黄铁矿切片表面形成的片状生物膜主要在生物浸出的前期和中期形成,在第15天浓度达17.21 mg/g,主要成分为胞外蛋白。在生物浸出前期,黄铁矿表面主要以Fe2+(0.75 mg/g)为主,Fe3+仅有0.08 mg/g,随着浸出时间的增加,大量的Fe3+(11.97 mg/g)富集在微生物-矿物界面。

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 .