Expanding the multicolor capabilities of basic confocal microscopes by employing red and near-infrared quantum dot conjugates.
Expanding the multicolor capabilities of basic confocal microscopes by employing red and near-infrared quantum dot conjugates.
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DOI：
10.1186/1472-6750-9-49
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发表时间：
2009-05-22
影响因子：
3.5
通讯作者：
Schaefer BC
中科院分区：
文献类型：
--
作者：
Kingeter LM;Schaefer BC
共聚焦显微镜是细胞生物学中广泛使用的方法，通常用于在细胞和亚细胞水平上研究生物组织。大多数基本的共聚焦显微镜配备干净的区别不超过四个荧光团在一个给定的样品，限制了这种方法的效用，共定位，共表达，和其他多参数分析。在这项研究中，我们评估了使用红色和近红外发射量子点染色试剂，以扩大基本的共聚焦显微镜的多参数能力。我们修改了三激光蔡司帕斯卡共聚焦显微镜，通过增加两个带通滤波器和一个长通滤波器，用于检测三种不同的红色到近红外量子点共轭物。然后，我们进行了直接比较有机染料和量子点标记的检测试剂的亚细胞结构的检测。我们发现，染色的质量通常是不可区分的，虽然量子点试剂确实有一定的限制，相对于有机染料共轭物。使用改进的Pascal系统，三个量子点缀合物，两个有机染料缀合物，和一个荧光蛋白，我们证明了在一个单一的样品中的六个不同的荧光标记的清洁歧视。我们的数据表明，几乎任何基本的共聚焦显微镜都可以通过简单地添加适当的发射滤波器来进行修改，从而可以检测红色和近红外量子点共轭物。此外，基于量子点和有机染料的二级试剂可以成功地结合在复杂的细胞内染色实验中。基本共焦仪器的多参数能力的实质性扩展可以通过与仪器更换或用附加激光器升级相比最小的财务投资来实现。
Confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. Most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. In this study, we evaluated the use of red and near-infrared emitting quantum dot staining reagents to expand the multi-parameter capabilities of basic confocal microscopes. We modified a three-laser Zeiss Pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates. We then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. We found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. Using the modified Pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample. Our data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. Additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. Substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers.
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影响因子：
56.9
作者：
Su, H;Bidère, N;Lenardo, M
通讯作者：
Lenardo, M
影响因子：
7.5
作者：
Dustin, Michael L.
通讯作者：
Dustin, Michael L.
DOI：
10.1073/pnas.0307858100
发表时间：
2004-01-27
影响因子：
11.1
作者：
Schaefer, BC;Kappler, JW;Marrack, P
通讯作者：
Marrack, P
影响因子：
14
作者：
Pinaud, F;Michalet, X;Weiss, S
通讯作者：
Weiss, S
影响因子：
2.9
作者：
Schaefer, BC;Mitchell, TC;Marrack, P
通讯作者：
Marrack, P