Choice of Illumination System & Fluorophore for Multiplex Immunofluorescenceon FFPE Tissue Sections
Choice of Illumination System & Fluorophore for Multiplex Immunofluorescenceon FFPE Tissue Sections
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DOI：
10.1371/journal.pone.0162419
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发表时间：
2016-09-15
期刊：
影响因子：
3.7
通讯作者：
Bellamy, Christopher O. C.
中科院分区：
文献类型：
--
作者：
Prost, Sandrine;Kishen, Ria E. B.;Bellamy, Christopher O. C.
最近可用的新染料和替代光源，以促进复杂的组织免疫荧光研究，如多重标记尚未匹配的报告，严格评估这些新工具的考虑和相对优势，特别是在组合。产品信息通常仅限于旧荧光团（FITC，TRITC和相应的Alexa染料家族）使用的波长。因此，诸如量子点的新型试剂没有被广泛地理解或使用，尽管具有非常有利的性质，包括极亮的发射、稳定性和在激发波长下潜在地减少的组织自发荧光。使用光谱分析，我们在这里报告一个详细的关键评价和比较评价不同的光源和荧光团在多重免疫荧光的临床活检切片。比较包括汞灯、金属卤化物和3种不同的基于LED的系统，使用7个量子点（525、565、585、605、625、705），Cy3和Cy5。我们讨论了实现准确的多重荧光定量的光源和荧光团的最佳组合相关的考虑。我们强调实际的限制和混杂因素，以定量与过滤器为基础的方法。
The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.