Kingeter Lara M, Schaefer Brian C
Department of Pathology, Human Immune Therapy Center, University of Virginia Health System, Charlottesville, VA 22908, USA.
BMC Biotechnol. 2009 May 22;9:49. doi: 10.1186/1472-6750-9-49.
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.
共聚焦显微镜是细胞生物学中广泛使用的一种方法，常用于在细胞和亚细胞水平研究生物组织结构。大多数基本的共聚焦显微镜在给定样本中能够清晰区分的荧光团不超过四个，这限制了该方法在共定位、共表达及其他多参数分析中的应用。在本研究中，我们评估了使用发射红色和近红外光的量子点染色试剂来扩展基本共聚焦显微镜的多参数功能。
我们通过添加两个带通滤光片和一个长通滤光片对蔡司Pascal三激光共聚焦显微镜进行了改装，以检测三种不同的从红色到近红外的量子点偶联物。然后我们对用于检测亚细胞结构的有机染料标记和量子点标记的检测试剂进行了直接比较。我们发现，尽管相对于有机染料偶联物，量子点试剂确实存在某些局限性，但染色质量通常难以区分。使用改装后的Pascal系统，我们用三种量子点偶联物、两种有机染料偶联物和一种荧光蛋白，在单个样本中实现了对六种不同荧光标记的清晰区分。
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添加合适的发射滤光片进行改装，从而能够检测红色和近红外量子点偶联物。此外，基于量子点和有机染料的二抗试剂可以成功地用于复杂的细胞内染色实验。与更换仪器或升级额外激光器相比，只需进行最少的资金投入，就可以大幅扩展基本共聚焦仪器的多参数功能。
