不同细胞培养皿对激光共聚焦显微镜成像效果的影响

目的 探讨3种不同玻片厚度的激光共聚焦专用细胞培养皿制备的细胞样品在激光共聚焦显微镜(CLSM)下采集的荧光染色图像差异.方法通过活细胞及固定后细胞荧光染色实验,采用激光共聚焦显微镜观察细胞形态及荧光亮度,检测不同条件下成像的平均荧光强度,考察3种厚度玻片(0.085～0.13、0.13～0.16、0.16～0.19m...     

激光共聚焦显微镜在肿瘤研究方面的应用

激光共聚焦显微镜(LSCM)是当今世界上最先进的分子细胞生物学分析仪器。它在荧光成像基础上加装激光扫描装置，利用计算机进行图像处理，利用紫外光激发荧光探针，从而得到细胞或组织内部微细结构的荧光图像。目前在形态学、分子细胞生物学、神经科学、药理学、遗传学都有广泛地应用，在肿瘤研究中更是成为新一代强有力的研究工具。     

激光扫描共聚焦显微镜双通道图像存取问题的探讨

近年来，激光扫描共聚焦显微镜(1aser scanning confocal microscope，LSCM)越来越多地应用到细胞生物、生物医学及其它各种研究工作中。随着激光、视频和计算机技术的快速发展，LSCM日趋显示出其较普通光学显微镜和电镜所独具的优越性。尤其是在细胞二维立体结构图像的重建、活细胞内离子含量的定性、定量检测等方面。我研究室所使用的IEICA LSCM可进行多通道扫描，即可用多个PMT检测不同的荧光探针，在这一应用中，值得注意的是图像存取中的一些问题。     

脑皮层功能活动与病理状态光学检测方法研究进展

光学成像方法具备高时间、空间分辨率和多参数的信息获取能力，在神经科学研究中发挥着重要的作用，受到广泛关注。简要介绍和评述了用于脑皮层功能检测的主要光学成像方法，并重点讨论了其中两种方法——内源光信号成像与激光散斑成像的基本原理、技术进展，以及在脑功能活动与病理状态检测中的应用。     

激光共聚焦显微镜在生物医学研究中的应用

共聚焦显微镜是显微镜制作技术、光电技术、计算机技术相结合的产物 ,是现代化的光学显微镜 ,它对普通光镜做了多方面的技术改进 :用激光做光源并采用了共聚焦技术消除了透镜的色差和球差 ;运用点扫描技术 ,使标本上每一点的图像避免了相邻点的衍射光和散射光的干扰 ;结果用计算机处理 ;因而其图像高度清晰且数字化。激光共聚焦显微镜不仅可以观察活细胞、活组织的动态代谢过程 ,而且可以获得三维图像 ,与其他的生物学技术相配合 ,几乎可定性、定量定位地检测组织细胞内的任何一种生化成分。     

肿瘤相关线粒体蛋白12对肝癌细胞凋亡的抑制作用

目的： 探讨肿瘤相关线粒体蛋白12 （TAMP12）对肿瘤细胞凋亡的抑制作用。方法: （1）构建重组逆转录病毒表达载体并转染包装细胞PA317,将获得的病毒颗粒感染TAMP12低表达的肝癌细胞株HepG2。应用RT-PCR检测TAMP12 mRNA表达变化;激光扫描共聚焦显微镜（CLSM）观察双色荧光标记蛋白的亚细胞定位和定量表达。（2）应用Hoechst 33258染色和FACS检测5－氟尿嘧啶（5-FU）处理后对诱导HepG2细胞凋亡的影响。结果:（1）CLSM检测显示TAMP12主要表达于HepG2细胞线粒体中。转染细胞中TAMP12基因和蛋白呈稳定高表达。（2）5-FU诱导细胞发生凋亡后,转染细胞的核形态较为完整,但对照细胞的染色质发生凝聚、边缘化、核固缩;且FACS检测显示转染细胞的凋亡率显著低于对照细胞（P<0.05或P<0.01）。结论: TAMP12具有抑制肝癌细胞凋亡的作用。     

一种荧光碳量子点的绿色合成方法及其应用研究

本研究探索一种可用于生物医学成像的绿色、高效的荧光碳量子点合成方法,以天然淀粉和水为原料,采用水热法合成碳量子点(carbon quantum dots, CQDs)。利用透射电镜和原子力显微镜表征CQDs形貌;紫外光谱、拉曼光谱等方法表征其光学性能,共聚焦显微镜检测其在金黄色葡萄球菌和肺癌A549细胞中的生物成像能力,采用CCK-8法和体内包埋实验验证CQDs的生物安全性。合成的CQDs粒径分布窄,平均粒径为2.3nm,具有良好的水分散性和较低的生物毒性。共聚焦荧光显微镜观察证实CQDs可对细胞和细菌进行成像。体内及体外安全性实验证实该材料具有良好的生物安全性。该CQDs的制备方法简单、成本低廉、生物相容性好,具有良好的生物成像功能,在生物医学等领域具有潜在的应用价值。     

共聚焦激光扫描显微镜在病理学中的应用(摘要)

共聚焦激光扫描显微镜在病理学中的应用（摘要）刘健（第四军医大学中心实验室，西安７１００３２）共聚焦激光扫描显微镜系统（ＣＬＳＭ）是将光学显微镜技术、激光技术和计算机图像处理技术结合在一起的高技术设备。其主要性能和优点是：（１）分辨率比普通光学显微镜高...     

应用激光扫描共聚焦显微镜体外观察结肠癌细胞的定向转移

目的应用激光扫描共聚焦显微镜体外观察结肠癌细胞的定向转移,探讨一种激光扫描共聚焦显微镜观察肿瘤细胞体外转移的方法。方法以肝转移性低分化结肠腺癌lovo细胞株为模型,采用激光扫描共聚焦显微镜结合Transwell小室模型在体外观察了lovo细胞在趋化因子自细胞介素8作用下的侵袭和迁移的特征。结果在白细胞介素8作用下lovo细胞侵袭能力增强,呈现出阿米巴样的变形迁移方式。提示在激光扫描共聚焦显微镜下观察到的结肠腺癌lovo细胞的转移呈现一种类白细胞趋化的特征。结论激光扫描共聚焦显微镜是研究肿瘤细胞转移的一个新的研究工具。     

共聚焦激光扫描显微镜技术及相关荧光探针在细胞器研究中的应用

共聚焦激光扫描显微镜 (ｃｏｎｆｏｃａｌｌａｓｅｒｓｃａｎｎｉｎｇｍｉｃｒｏｓｃｏｐｅ,ＣＬＳＭ)是上世纪 80年代发展起来的先进的分子细胞生物学分析仪器。它在荧光显微镜成像基础上加装了激光扫描装置 ,利用计算机进行图像处理 ,把光学成像的分辨率提高了 30 %～ 4 0 % ,是光学显微镜发展史上的重大突破。它通过其独特的成像原理和技术[1] ,使用紫外线或可见光激发荧光探针 ,不仅可观察固定的细胞、组织切片 ,还可以对活细胞的结构、分子、离子等进行实时动态观察和检测 ,在亚细胞水平上观察诸如Ｃａ2 + ,ｐＨ值 ,膜电位等生理信号及…     

Assembled alginate/chitosan nanotubes for biological application

Biodegradable nanotubes were fabricated through the layer-by-layer (LbL) assembly technique of alternate adsorption of alginate (ALG) and chitosan (CHI) onto the inner pores of polycarbonate template with the subsequent removal of the template. The assembled materials present good film formation ability. The thickness of nanotubes wall can be controlled by changing the assembled layers. The assembled tubular structure was verified by the confocal laser scanning microscope (CLSM) using fluorescent-labeled ALG as well as the measurements of scan electron microscope (SEM) and transmission electron microscopy (TEM). Atomic force microscopy (AFM) images confirm the biodegradable feature of the assembled nanotubes as they are immersed in the pancreatin. Confocal microscopy images show that the assembled ALG/CHI nanotubes can be internalized into the cancer cell readily. The cell viability experiment proves the low cytotoxicity of ALG/CHI nanotubes. The final assembled nanotubes have presented good biodegradability and low cytotoxicity.     

激光共聚焦显微术在检测脑胶质瘤蛋白共表达中的应用

目的 了解ephrinB2及其受体EphB4蛋白在脑胶质瘤中的表达规律，并评价激光共聚焦显微术在检测脑胶质瘤组织和细胞蛋白共表达中的应用性。方法 利用双标免疫荧光分别检测EphB4/ephrinB2蛋白与GFAP或CD34蛋白在35例脑胶质瘤新鲜标本及人脑胶质瘤细胞系CHG-5、SHG-44中的共表达状况，以Leica SP2激光共聚焦显微镜观察、摄像并分析。结果 EphB4或ephrinB2蛋白与CD34蛋白可共表达于部分间质血管，EphB4和ephrinB2在肿瘤血管的表达主要定位于血管内皮细胞。在瘤组织内肿瘤细胞及两种细胞系，亦可见EphB4或ephrinB2蛋白与GFAP的共表达。SHG-44细胞中EphB4或ephrinB2红色荧光强度强于CHG-5细胞，而CHG-5细胞GFAP绿色荧光强度较之SHG-44细胞则明显增强。结论 (1)脑胶质瘤间质可能存在不同免疫表型的血管内皮，提示肿瘤血管的不同属性；(2)EphB4/ephrinB2蛋白表达可能与肿瘤细胞的分化程度有关；(3)双标免疫组化结合激光共聚焦显微术是一种可用于观测肿瘤蛋白共表达的良好方法。     

New approach for static bone histomorphometry: confocal laser scanning microscopy of maxillo-facial normal bone

To define the value of confocal laser scanning microscopy (CLSM) as a practical method for a qualitative and quantitative analysis of hard tissue, the authors have analyzed normal maxillo-facial bone. They obtained and analyzed 58 bone samples from 28 patients who underwent to implant surgery. All the samples presented intense autofluorescence primarily ascribed to collagen. Variable degrees of autofluorescence have been identified between osteones and interosteonic bone. CLSM allowed improved tissue imaging, bidimensional pictures with better resolution at cellular level, and, in particular, the possibility of different histomorphometric evaluation. The application of CLSM to bone histomorphometry represents a new and never described technique, which might produce many insights in the study of normal and pathological bone.     

Confocal laser scanning microscopy as a tool for imaging cancellous bone

Understanding the bimodal structure of cancellous bone is important for tissue engineering in order to more accurately fabricate scaffolds to promote bone ingrowth and vascularization in newly forming bone. In this study, confocal laser scanning microscopy (CLSM) was used to create detailed images of the bimodally porous intertrabecular space of defatted and deproteinized cancellous canine bone taken from the epiphysis of the humerus. The bimodal pore structure was imaged using both reflective and fluorescent modes in CLSM, resulting in four different, but complementary image types: (1) a Z-stack overlay, (2) a phi-Z scan, (3) a topographical map, and (4) a contour map. Submerging the bone in rhodamine B dye prior to fluorescent imaging enhanced the pore surface details, giving a more accurate pore size measurement. The average macropore diameter was found to be 260 +/- 97 microm while the average micropore diameter was 13 +/- 10 microm. When compared with common techniques, including microcomputed tomography, magnetic resonance imaging, scanning electron microscopy, and environmental scanning electron microscopy, for imaging cancellous bone, CLSM was found to be an effective tool, given its ability to nondestructively image the surface and near-surface pore structure.     

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model.     

Progenitor endothelial cells on vascular grafts: an ultrastructural study

The morphology of progenitor endothelial cells on vascular graft surfaces is addressed in this report. Such cells were seen to attach to intima-expressed CD34 and Flk-1 antigen and showed positive 5-bromo-2-deoxyuridine (BrdU) uptake. We examined CD34 and Flk-1 antigen-expressing endothelial progenitor cells three-dimensionally using confocal laser scanning microscopy (CLSM). Under detailed CLSM observation, through an ameboid-form cell, these progenitor endothelial cells changed from a globular to a flattened form. We also investigated these morphological changes using scanning electron microscopy. From these results, progenitor endothelial cells were observed not only near the advancing edge of endothelium, but also around the developing intimal site. Their form also changed from globular to flattened as observed in the CLSM results. These morphological changes were seen more frequently near the advancing edge and around the developing intimal site. They attached directly to vascular prosthesis fibers and likewise covered the graft luminal surface. Progenitor endothelial cells in any form had a common surface structure. We conclude from our results that progenitor endothelial cells can attach to graft fibers directly without clotting and directly cover the graft luminal surfaces.     

大鼠肝硬化门腔静脉分流术后运动皮层和脊髓一氧化氮合酶阳性神经元的变化

目的 研究大鼠肝硬化门腔静脉分流术后运动皮层和脊髓一氧化氮合酶(NOS)阳性神经元的变化。方法 采用NADPH-d黄递酶组织化学法以及NOS荧光免疫组织化学技术结合激光共聚焦扫描显微镜方法。结果 肝硬化门腔静脉分流术后运动皮层NOS和nNOS阳性细胞显著减少；脊髓NOS阳性细胞改变不明显。结论 大鼠肝硬化门腔静脉分流可引起大脑皮层细胞的改变，NO可能参与了肝硬化门腔静脉分流术后对中枢神经系统的损害。     

Nanotopographical guidance of C6 glioma cell alignment and oriented growth

The surface properties of the extracellular matrix play vital roles in cellular behavior such as adhesion, spreading, migration, proliferation and differentiation. While cell attachment and adhesion onto surfaces are mainly mediated by surface molecular interaction, cell morphology and orientation are significantly affected by the topographical cues of the substrate. We reported here the alignment of C6 glioma cells on polystyrene (PS) substrate containing periodic nanotopography. The ridge/groove type structures (210 nm in periodicity, and 30-40 nm in depth) were generated on polystyrene surface using Nd:YAG polarized laser radiation at 266 nm. The cultured cells were shown to align strictly along the direction of the ridges/grooves. And there were distinctive features such as elongated morphology and asymmetrical cell surface extensions, revealed by confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The results indicated that ordered and continuous nanostructures on substrates can pattern cell, and guide cell alignment and oriented growth along definite directions. The possible mechanism and significance of these observations were also discussed.     

A novel study on the mechanisms of drug release in PLGA-mPEG microspheres with fluorescent drug

The purpose of this research was to proof the microspheres release mechanism by a novel method-detecting and comparing the drugs fluorescent changes on the microspheres surface. Fluorescein sodium (FS, 0.4 kDa) and fluorescein isothiocyanate-bovine serum albumin (FITC-BSA, 66.8 kDa) were employed as model drugs. FS and FITC-BSA were encapsulated into PLGA-mPEG microspheres through double emulsion evaporation method, and the drug-loaded microspheres in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, confocal laser scanning microscopy (CLSM), BCA assay kit, and UV–vis spectrophotometry. FS-loaded microspheres revealed a severe initial burst release, followed by a sustained release, and we could observe a bright fluorescent on the microspheres surface during the early release period under the CLSM. The bright fluorescent gradually faded out in the later period as only 1~2% FS was remained after 14 days release. FITC-BSA-loaded microspheres revealed a typical tri-phase release profile, and we observed a weak fluorescent on the microspheres surface after the initial burst release, and the fluorescent came bright again after an obvious erosion appeared on the microspheres surface. In the later release stage, the fluorescent gradually faded out as the fast release of FITC-BSA.