Selecting fluorochrome conjugates for maximum sensitivity
Selecting fluorochrome conjugates for maximum sensitivity
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DOI：
10.1002/cyto.a.20092
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发表时间：
2004-12-01
期刊：
影响因子：
3.7
通讯作者：
Trotter, J
中科院分区：
文献类型：
--
作者：
Maecker, HT;Frey, T;Trotter, J
大多数流式细胞术用户都知道，为了最大限度地提高染色较暗的抗体的灵敏度，应为该缀合物选择明亮的荧光染料，例如藻红蛋白 (PE)。人们常常不太清楚光谱重叠在确定给定荧光通道的灵敏度中所起的作用。随着多色流式细胞术（最多 12 种颜色）(1) 的出现以及数字电子和采集后补偿的引入，这已成为一个更大的问题，这可能会影响分辨率，如下所述。在本文中，除了指导缀合物选择的一般规则外，还强调了选择次优缀合物组合的陷阱。我们从基本原理开始描述光谱重叠如何影响给定荧光通道中观察到的背景。
Most users of flow cytometry are aware that, to maximize sensitivity for an antibody whose staining is dim, a bright fluorochrome such as phycoerythrin (PE) should be selected for that conjugate. What is often less well appreciated is the role that spectral overlap plays in determining the sensitivity of a given fluorescence channel. This has become more of an issue with the advent of polychromatic flow cytometry (up to 12 colors)(1) and with the introduction of digital electronics and postacquisition compensation, which can affect resolution, as described below. In this article, the pitfalls of selecting suboptimal conjugate combinations are highlighted, in addition to general rules that should guide conjugate selection. We start from first principles to describe how spectral overlap contributes to the observed background in a given fluorescence channel.