Bhakdi Sebastian C, Thaicharoen Ponpan
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
X-Zell Biotec, Bangkok 10400, Thailand.
Methods Protoc. 2018 Jun 1;1(2):20. doi: 10.3390/mps1020020.
Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell-cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry-which is capable of staining 20 or more fluorophores at the same time-is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting.
免疫荧光染色已成为病理学和生物医学科学领域识别稀有细胞、细胞间相互作用以及亚微观细胞成分的重要工具。然而，在许多实验环境中，传统的宽场荧光显微镜通常仅限于同时对三到四种荧光团进行成像，这一事实给实验带来了困扰。由于缺乏形态学信息且检测限较高，即使是能够同时对20种或更多荧光团进行染色的流式细胞术，其适用性也受到限制，尤其是在稀有细胞检测等领域。其他先进的成像方法，如共聚焦激光扫描显微镜和成像流式细胞术，可能会解决这些缺点，但反过来需要复杂的下游数据处理和高额的资金投入。在此，我们描述了一种新方法和滤光片设置，可在配备标准高压汞光源的传统宽场荧光显微镜上常规使用多达七种荧光团。对通道间串扰的量化以及对掺入白细胞中的癌细胞进行实际的七彩色成像表明，无需数字补偿校正算法。因此，我们的设置允许在标准研究或常规实验室环境中对稀有细胞群体进行详细分析、抗原共定位以及细胞形态分析。
