活体动物体内光学成像技术的研究进展

生物发光和荧光成像作为近年来新兴的活体动物体内光学成像技术,以其操作简便及直观性成为研究小动物活体成像的一种理想方法,在生命科学研究中得以不断发展。利用这种成像技术,可以直接实时观察标记的基因及细胞在活体动物体内的活动及反应。利用光学标记的转基因动物模型可以研究疾病的发生发展过程,进行药物研究及筛选等。本文综述了现有活体动物体内光学成像技术的原理、应用领域及发展前景,比较了生物发光与几种荧光技术的不同特点和应用。     

活体动物体内光学成像技术的研究进展及其应用

活体动物体内光学成像是利用基因改构进行内源性成像试剂或外源性成像试剂标记细胞、蛋白或DNA，从而非侵入性地报告小动物体内的特定生物学事件的技术。活体成像可以直观灵敏地监测基因的表达模式、标记和示踪细胞、探讨蛋白间的相互作用，因而这一技术被广泛地用于分析基因的表达模式、评价基因治疗效果、评估肿瘤的发生和转移、监测移植器官等。简要综述了现有活体动物体内光学成像技术的基本原理、技术进展和相关应用。     

小动物活体成像技术的应用

小动物活体成像技术在国内外得到越来越多的普及应用,极大地促进了生命科学特别是肿瘤研究的发展。本文就小动物活体成像技术的原理、标记方法和实际应用做简单介绍。     

生物光学成像技术在干细胞应用中的研究进展

干细胞目前已应用在多种疾病的治疗中,前景十分广阔,但干细胞存活、分布和迁移等具体机制仍未明确,需要通过长期有效、无副作用的干细胞示踪技术进一步研究.生物光学成像(OI)技术与干细胞相结合,操作简单、成像直观,并有高灵敏度和高特异性,可以实时、无创监测干细胞在动物活体内的生物学活动.本文就OI示踪技术的原理及其在干细胞应...     

鼠伤寒沙门菌动物模型活体成像技术的研究进展

鼠伤寒沙门菌的体内实验有利于开展食物中毒、胃肠炎、伤寒热等肠道传染病的防治。由于在活体内检测鼠伤寒沙门菌的动态变化存在瓶颈,使细菌致病机制的研究、疫苗及药物研发滞后。近年来应用小动物成像技术在活体中追踪转化了荧光素酶基因的鼠伤寒沙门菌越来越受到人们关注,综述该技术的应用现状及缺憾之处。     

小动物活体光学三维成像系统及其对乳腺癌的定量分析

乳腺癌具有高转移率。使用小动物活体成像技术对乳腺癌的生长及转移情况实时监测定量分析可以帮助了解疾病机制及进行药物研究。二维成像对光学信号的定位与定量是相对的。随着计算机技术的进步,可以实现对采集的图像进行三维重建,精准量化光学信号,获得空间分布的三维信息。IVIS Spectrum小动物活体光学三维成像系统同时具有高灵敏的生物发光、荧光、切伦科夫辐射二维成像及三维扫描重建功能,是小动物活体光学成像的顶级系统。本文对人乳腺癌细胞（MDA-MB-231）进行慢病毒感染,在体外稳定表达荧光素酶后,选取重度联合免疫缺陷（SCID）小鼠进行原位乳腺癌模型的建立,通过IVIS Spectrum小动物活体光学三维成像系统对小鼠进行生物发光二维成像,无创观测肿瘤的生长及转移情况。本文的创新点是利用生物发光成像断层扫描技术对小鼠模型进行定量三维成像,使用系统自带的算法直接进行三维重建,同时结合鲸鱼优化算法（WOA）优化后的三维卷积的深度编码器-解码器的网络模型进行重建。通过CT图像验证两者的重建效果,得到肿瘤的深度信息,实现对乳腺癌的精准定量分析。     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

三维反卷积显微成像技术浅谈

三维宽场反卷积显微成像技术是应用光学切片方法获取三维标本的二维图像序列,然后通过反卷积图像处理方法进行图像恢复,进而进行三纺重建的一种以光学技术和图像处理技术为核心的业微成橡方法。本讲述了光学切片的基本原理,给出了反卷积处理中点扩展函数的理论模型和实验测试方法,然后对现存的反卷积算法做了对比。对这一领域的发展趋势做了预测。     

活体生物发光成像技术及其在病毒感染研究中的应用

生物发光是动物活体光学成像技术之一,因其反应灵敏、操作简单、数据精确,而被广泛地应用于生命科学研究多个领域,观测活体动物体内病毒复制、肿瘤生长等生命过程.生物发光技术采用荧光素酶基因标记细胞或病毒,与外源注射的底物荧光素发生反应,在冷CCD成像系统下显像并进行数据记录、分析.本文简要介绍活体生物发光成像这一新技术的原理...     

三维反卷积显微成像技术浅谈

三维宽场反应卷积显微成像技术是应用光学切片方法获取三维标本的二维图像序列,然后通过反卷积图像处理方法进行图像恢复,进而进行三维重建的一种以光学技术和图像处理技术为核心的显微成像方法。本文讲述了光学切片的基本原理,给出了反卷积处理中点扩展函数的理论模型和实验测试方法,然后对现存的反卷积算法做了对比。最后,文章对这一领域的发展趋势作了预测。     

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.     

利用非侵入性光学成像技术监测在体肿瘤的生长

利用电穿孔技术将gfp表达质粒转染于SP2／0细胞,通过G418筛选获得稳定表达GFP的小鼠骨髓瘤细胞株SP2／0—GFP细胞。将稳定的转化细胞移植于同系BALB／c小鼠后腿、耳部皮下及腹腔成瘤,每隔一天对小鼠肿瘤成像,跟踪肿瘤的生长过程。结果表明,探测到的GFP荧光强度和发光范围与肿瘤的生长或消亡相关,从而证明可以利用GFP作为肿瘤细胞的标记,结合光学成像技术,对肿瘤生长过程实时追踪。     

New anti angiogenesis developments through electro-immunization: Optimization by in vivo optical imaging of intradermal electrogenetransfer

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called “electro-immunization”. In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.     

GFP标记的肿瘤生长和转移的整体荧光成像

Fugene 6脂质体介导pEGFP-C1转染人源肺癌细胞(SPC-A1),经G418抗性筛选和96孔板有限稀释获得稳定高表达GFP的单克隆细胞株SPC-A1-EGFP。裸鼠腹腔注射SPC-A1-EGFP细胞建立自发转移模型;裸鼠尾静脉注射SPC-A1-EGFP细胞建立实验转移模型。利用整体光学成像系统(wllole-body optical imaging system)对荷瘤鼠整体荧光成像。结果表明,整体光学成像系统可实时非侵入监测腹腔肿瘤生长和扩散过程,通过胸腔皮瓣窗chest—wall skin-flap window)可低侵入检测肺转移。该研究为在体监测原位移植瘤的自发转移和发现抗肿瘤新药物提供了良好实验平台。     

尼罗红示踪纳米制剂在荷瘤裸鼠体内活体成像的应用

目的拟将尼罗红包裹入纳米制剂中,利用其荧光特性来考察纳米制剂在荷瘤鼠体内的肿瘤靶向性与组织分布情况。方法采用CCK-8法检测尼罗红纳米乳[NRNE(O)]对H1688的细胞毒性。建立BALB/c裸鼠肺癌模型,灌胃给予尼罗红混悬液(NRS)及NRNE(O),通过小动物活体成像系统观察其在荷瘤裸鼠中荧光强度的动态分布情况。结果 NRNE(O)具有较低的细胞毒性和良好的生物相容性,并且在体内的吸收量及肿瘤部位的蓄积量均高于NRS;NR在纳米制剂中能快速、稳定、清晰地反映纳米制剂在小鼠体内的动态分布情况。结论本研究中制备的NRNE(O)的稳定性好,为尼罗红作为示踪剂应用于纳米制剂在动物体内的研究提供了重要的研究手段。     

一体化的光声-荧光显微镜用于活体肿瘤成像

报道了一种利用单一波长激发的同时产生光声和荧光信号的显微成像系统,本成像系统具有超高的成像分辨率(<6μm)。借助外源的造影剂在近红外的吸收特性,利用光声-荧光显微成像系统对活体肿瘤进行光声/荧光成像。实验结果表明,光声-荧光显微镜在早期肿瘤的成像和检测等方面具有潜在的应用价值。因此,通过研究和选择适当的双模态造影剂,该系统在不同病理模型中可以提供更准确的组织信息及生理参数。     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.