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荧光显微成像技术的生物医学应用离不开荧光染料的设计与开发。有机小分子荧光染料因其易于修饰、生物相容性好、光物理性质优异等特点,在细胞生物成像领域受到了广泛关注。随着超分辨荧光显微镜的发展和技术的进步,使得荧光显微成像突破了光学衍射极限,可以获得更为精准的生物分子学信息,观察纳米尺度下亚细胞器之间的相互作用。根据不同的成像原理,科学家开发出了单分子定位成像技术、受激辐射损耗成像技术、结构光照明技术等超分辨荧光显微技术。这些技术在细胞荧光显微成像领域的应用与发展,同时对有机小分子荧光染料的设计与开发提出了新要求。本文介绍了主流超分辨荧光显微技术的原理,总结已发表的超分辨荧光显微成像荧光染料的结构和光物理性质特点,归纳了其设计要求,旨在为新型荧光染料的设计提供参考。 相似文献
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选用天然多糖中唯一的碱性多糖——壳聚糖作为稳定剂和包裹剂,成功合成了水溶性的Ag In S2量子点/低分子量壳聚糖纳米复合材料(Ag In S2/LCSMS)。利用透射电子显微镜(TEM)、FT-IR傅里叶红外光谱仪、紫外吸收光谱、荧光分光光度计等表征手段对纳米复合材料的形貌、化学组成及光学性质进行了研究。结果表明,Ag In S2/LCSMS纳米复合材料的粒径约为5~6 nm,在水相中仍具有较稳定的发光。之后,对Ag In S2/LCSMS纳米复合材料的生物相容性进行了研究,对比Ag In S2/LCSMS纳米复合材料与Ag In S2量子点的细胞活性测试结果发现,Ag In S2/LCSMS纳米复合材料的细胞活性比Ag In S2量子点有了明显的提高,说明通过低分子量壳聚糖的包裹可以明显提高纳米材料的生物相容性。因此,这类具有较好水溶性和生物相容性的荧光Ag In S2/LCSMS纳米复合材料可作为优良的生物荧光标记材料在生物医学检验、细胞以及活体成像研究中有广泛的应用前景。 相似文献
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近年来,基于碳点与适配体构建的荧光生物传感器引起了人们强烈的兴趣.与传统荧光染料、半导体量子点及稀土荧光材料相比,以碳点为荧光基元的生物传感器荧光稳定性更好,毒性更低,价格更便宜,在环境监测、生物成像及生物医疗方面有着广泛的应用前景.本文对基于碳点与适配体构建的荧光生物传感器发展现状进行了系统的总结,包括碳点与适配体介... 相似文献
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单分子荧光检测在生命科学中的应用 总被引:2,自引:0,他引:2
文章对单分子荧光检测在分子马达、离子通道、信号分子、蛋白折叠、蛋白构象变化动力学、酶活性反应、细胞过程实时观察等生命科学领域中的应用进行了介绍.这些研究结果表明,单分子荧光检测在研究生物大分子的活动规律与机制方面不但有着无法替代的优越性,而且有着广阔的发展空间. 相似文献
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细胞是动植物结构和生命活动的基本单位。细胞过程的一个重要特点就是其生化组分在时空调控上的相互作用关系。然而,利用传统的生化方法(如酵母双杂交系统、pull-down系统等)很难在空间上评估活细胞内分子间的相互作用。光学技术的快速发展,为研究活细胞中生物分子的时空动态提供了新的遗传研究工具,其中荧光共振能量转移-荧光寿命显微成像(FRET-FLIM)技术在实时探测分析活细胞中生物大分子构象变化和分子间动态相互作用过程具有独特的优势,如:实现对活细胞的实时“可视化”研究,同时具有高时空分辨率;检测更加灵敏、结果可信度高;且基于简易的数学运算完成简单快捷的分析程序。介绍FRET-FLIM技术的理论背景知识,对比了该技术与传统蛋白相互作用技术研究的利弊,同时归纳了其在蛋白相互作用、细胞生物学和疾病诊断等方面的最新应用研究进展,最后总结和讨论了FRET-FLIM技术的未来发展趋势,以期能够为揭示活细胞的结构和细胞过程相关研究提供新的见解。 相似文献
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Lira RB Cavalcanti MB Seabra MA Silva DC Amaral AJ Santos BS Fontes A 《Micron (Oxford, England : 1993)》2012,43(5):621-626
In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells. 相似文献
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通过脂质体方法成功地将三辛基氧化膦(TOPO)包覆的CdSe发光量子点从非极性有机溶剂转移到生物相容性的水溶液中.分别通过透射电镜(TEM)、荧光Mapping图像,以及光致发光(PL)光谱进行表征.TEM照片显示制备的CdSe核量子点为球形,具有良好的单分散特性,平均粒径约为3nm.CdSe-脂质体复合体的平均尺寸大约20nm,TEM清楚地显示了CdSe量子点被诱捕在脂质体中.荧光Mapping显示了CdSe-脂质体复合体的发光强度分布.脂质体方法转移TOPO包覆的CdSe量子点,借助了磷脂的双分子链与CdSe表面的TOPO配体之间的疏水相互作用,在CdSe的第一配体层外部形成第二配体层,保留了CdSe的存在环境,光致发光光谱表明,量子点-脂质复合体基本保持了CdSe核量子点的发射效率. 相似文献
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A. V. Burenin 《Optics and Spectroscopy》2017,122(1):1-7
Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the “gold standard” of immunohistochemical diagnostics. The developed nanoprobes are expected to find wide application in high-efficiency imaging of tumor and multiparameter diagnostics. 相似文献
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Cytotoxicity and fluorescence studies of silica-coated CdSe quantum dots for bioimaging applications
Muthunayagam Vibin Ramachandran Vinayakan Annie John Vijayamma Raji Chellappan S. Rejiya Naresh S. Vinesh Annie Abraham 《Journal of nanoparticle research》2011,13(6):2587-2596
The toxicological effects of silica-coated CdSe quantum dots (QDs) were investigated systematically on human cervical cancer
cell line. Trioctylphosphine oxide capped CdSe QDs were synthesized and rendered water soluble by overcoating with silica,
using aminopropyl silane as silica precursor. The cytotoxicity studies were conducted by exposing cells to freshly synthesized
QDs as a function of time (0–72 h) and concentration up to micromolar level by Lactate dehydrogenase assay, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide] assay, Neutral red cell viability assay, Trypan blue dye exclusion method and morphological examination of cells
using phase contrast microscope. The in vitro analysis results showed that the silica-coated CdSe QDs were nontoxic even at
higher loadings. Subsequently the in vivo fluorescence was also demonstrated by intravenous administration of the QDs in Swiss
albino mice. The fluorescence images in the cryosections of tissues depicted strong luminescence property of silica-coated
QDs under biological conditions. These results confirmed the role of these luminescent materials in biological labeling and
imaging applications. 相似文献
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Quantum dots (QDs), as novel fluorescence probes, have shown a great potential for bio-molecular labeling and cellular imaging.
To stain cellular targets, the sufficient intracellular delivery of QDs is required. In this work the tat, a typical membrane-permeable
carrier peptide, was conjugated with thiol-capped CdTe QDs to form CdTe Tat-QDs, and the intracellular deliveries of CdTe
QDs or CdTe Tat-QDs were compared in human hepatocellular carcinoma (QGY) cells and human breast cancer (MCF7) cells in vitro
by means of confocal laser scanning microscopy. Added into the cell dishes, both QDs and Tat-QDs adhered to the outer leaflet
of the plasma membrane of cells within a few minutes, but the binding amount of Tat-QDs was obviously higher than that of
QDs. Then both QDs and Tat-QDs can penetrate into cells, and their cellular contents increased with incubation time but both
saturated after 3 hours incubation. However the cellular levels of Tat-QDs were higher than those of QDs, with the ratio of
2.1 (±0.3) times in QGY cells and 1.5 (±0.2) times in MCF7 cells, demonstrating the enhancing effect of Tat conjugation on
the intracellular delivery of QDs. 相似文献
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Sébastien Bau Denis Bémer Florence Grippari Jean-Christophe Appert-Collin Dominique Thomas 《Journal of nanoparticle research》2014,16(10):1-13
The cysteamine-modified hyaluronic acid (HA) polymer was employed to coat quantum dots (QDs) through a convenient one-step reverse micelle method, with the final QDs hydrodynamic size of around 22.6 nm. The HA coating renders the QDs with very good stability in PBS for more than 140 days and resistant to large pH range of 2–12. Besides, the HA-coated QDs also show excellent fluorescence stability in BSA-containing cell culture medium. In addition, the cell culture assay indicates no significant cytotoxicity for MD-MB-231 breast cancer cells, and its targeting ability to cancer receptor CD44 has been demonstrated on two breast cancer cell lines. The targeting mechanism was further proved by the HA competition experiment. This work has established a new approach to help solve the stability and toxicity problems of QDs, and moreover render the QDs cancer targeting property. The current results indicate that the HA polymer-coated QDs hold the potential application for both in vitro and in vivo cancer imaging researches. 相似文献
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Although water soluble thiol-capped quantum dots (QDs) have been widely used as photoluminescence (PL) probes in various applications, the negative charges on thiol terminals limit the cell uptake hindering their applications in cell imaging. The commercial liposome complex (Sofast®) was used to encapsulate these QDs forming the liposome vesicles with the loading efficiency as high as about 95%. The cell uptakes of unencapsulated QDs and QD loaded liposome vesicles were comparatively studied by a laser scanning confocal microscope. We found that QD loaded liposome vesicles can effectively enhance the intracellular delivery of QDs in three cell lines (human osteosarcoma cell line (U2OS); human cervical carcinoma cell line (Hela); human embryonic kidney cell line (293 T)). The photobleaching of encapsulated QDs in cells was also reduced comparing with that of unencapsulated QDs, measured by the PL decay of cellular QDs with a continuous laser irradiation in the microscope. The flow cytometric measurements further showed that the enhancing ratios of encapsulated QDs on cell uptake are about 4–8 times in 293 T and Hela cells. These results suggest that the cationic liposome encapsulation is an effective modality to enhance the intracellular delivery of thiol-capped QDs. 相似文献
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Sundararajan Parani Giridharan Bupesh Elayaperumal Manikandan Kannaiyan Pandian Oluwatobi Samuel Oluwafemi 《Journal of nanoparticle research》2016,18(11):347
Water-soluble, mercaptosuccinic acid (MSA)-capped CdTe/CdS/ZnS core/double shell quantum dots (QDs) were prepared by successive growth of CdS and ZnS shells on the as-synthesized CdTe/CdSthin core/shell quantum dots. The formation of core/double shell structured QDs was investigated by ultraviolet-visible (UV–Vis) absorption and photoluminescence (PL) spectroscopy, PL decay studies, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The core/double shell QDs exhibited good photoluminescence quantum yield (PLQY) which is 70% higher than that of the parent core/shell QDs, and they are stable for months. The average particle size of the core/double shell QDs was ~3 nm as calculated from the transmission electron microscope (TEM) images. The cytotoxicity of the QDs was evaluated on a variety of cancer cells such as HeLa, MCF-7, A549, and normal Vero cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay. The results showed that core/double shell QDs were less toxic to the cells when compared to the parent core/shell QDs. MCF-7 cells showed proliferation on incubation with QDs, and this is attributed to the metalloestrogenic activity of cadmium ions released from QDs. The core/double shell CdTe/CdS/ZnS (CSS) QDs were conjugated with transferrin and successfully employed for the biolabeling and fluorescent imaging of HeLa cells. These core/double shell QDs are highly promising fluorescent probe for cancer cell labeling and imaging applications. 相似文献
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Yang Rong Yao Wan Zhen Huang Huan Zhou Yi Fan Zheng Xu Chun Song 《Journal of nanoparticle research》2014,16(6):1-9
In comparison with conventional organic dyes, quantum dots (QDs) have unique optical and electronic properties, which provide QDs with a wide scope of prospective application in biology and biomedicine. However, the toxicity of QDs and the fluorescence intensity of labeled bacteria must precede their application in bacterial imaging and tracing in vivo. Here, we show that treatment with CaCl2 significantly improved bacterial labeling efficiency of CdSe/ZnS QDs with the CdSe core size of ~3.1 nm (relative fluorescence unit (RFU) value and ratio of fluorescent E. coli) with rising CdSe/ZnS QDs concentration in a concentration-dependent manner. At 12.5 nmol/L CdSe/ZnS QDs concentration, labeled Escherichia coli (E. coli) DH5α appeared as short rod-shaped and luminescent with normal size, and the survival rate and ultrastructure did not change in comparison to the control. But the ratio of fluorescent bacteria and RFU were very low. However, the survival rate of transformed E. coli was significantly inhibited by high CdSe/ZnS QDs concentrations (≥25 nmol/L). Moreover, internalization of CdSe/ZnS QDs resulted in ultrastructure damage of transformed E. coli in a concentration-dependent manner (≥25 nmol/L). Therefore, CdSe/ZnS QDs may not suitable for tracing of bacteria in vivo. Moreover, our study also revealed that colony-forming capability assay and transmission electron microscopy could be used to comprehensively evaluate the toxicity of QDs on labeled bacteria. Our findings do provide a new direction toward the improvement and modification of QDs for use in imaging and tracing studies in vivo. 相似文献
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Trioctylphosphine oxide capped cadmium selenide quantum dots, synthesized in organic media were rendered water soluble by
silica overcoating. Silanisation was done by a simple reverse microemulsion method using aminopropyl silane as the silica
precursor. Further, the strong photoluminescence of the silica-coated CdSe quantum dots has been utilized to visualize rabbit
adipose tissue-derived mesenchymal stem cells (RADMSCs) and Daltons lymphoma ascites (DLA) cancerous cells in vitro. Subsequently
the in vivo fluorescence behaviours of QDs in the tissues were also demonstrated by intravenous administration of the QDs
in Swiss albino mice. The fluorescence microscopic images in the stem cells, cancer cells and semi-thin sections of mice organs
proved the strong luminescence property of silica-coated quantum dots under biological systems. These results establish silica-coated
CdSe QDs as extremely useful tools for molecular imaging and cell tracking to study the cell division and metastasis of cancer
and other diseases. 相似文献